THE MULTIPLE MOLECULAR FORMS OF …

THE MULTIPLE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN SKELETALMUSCLES: A FUNCTIONAL ANALYSIS

Josephine Yuen-Wai Lai

A dissertation submitted to Imperial College of Science and Technology in candidature for the Diploma of Imperial College and to

the University of London for the degree of Doctor of Philosophy.

Department of Biochemistry Imperial College University of London London SW7 2AZ.

August, 1985

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JOSEPHINE Y. W. LAI

THE MULTIPLE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN SKELETALMUSCLES: A FUNCTIONAL ANALYSIS

ABSTRACT

A number of molecular forms of acetylcholinesterase (AChE) from

vertebrate skeletal muscles have been identified on sucrose density

gradients. The heaviest form, H2 C, is concentrated at the neuromuscular

junction of twitch muscle fibres, while the light (L^ and L2 ) forms

predominate at the avian tonic fibre endplate. It is thought likely

that the distribution of these forms in muscles may reflect their

functions in different muscle types. However, previous studies on the

distribution of AChE forms in mammalian muscles had not been able to

show any clear relationship between their distribution, functions and

fibre-type-specific localization. Furthermore, the proportion of the H2 C

form relative to other forms is small, which does not reflect its

presumed physiological importance. H2C AChE disappears after chronic

denervation and reappears upon reinnervation, and thus is clearly under

neural control. This study aims to study the relationship between the

distribution of AChE forms and muscle fibre types, as well as the

neurotrophic regulation which may contribute to such distribution.

In marked contrast to previous reports, it was found that H2 C

AChE and a significant H^c component predominate in most mammalian and

avian twitch muscles, and which are the only forms located at the

endplate. L and M forms are confined to the extrajunctional regions, the

variations in their concentrations contribute to the overall variability

in the distribution of AChE forms. Moreover, their contribution may be

overestimated if proteolytic degradation of the H AChE was not

adequately prevented. Slow-twitch fibres contained extrajunctional H2 C

which was also observed in fast-twitch muscle subjected to continuous

low frequency stimulation. On chronic denervation, the endplate H AChE

decreased considerably, while the extrajunctional AChE forms, including

the H AChE, accounted for the overall increase in AChE activity in some

species. It is concluded therefore that endplate H AChE is maintained by

innervation and is by far the major form at the endplate, while the

extrajunctional AChE is probably regulated by muscle activity.

Monoclonal antibodies specific for avian AChE had been raised from

purified chick brain AChE. These antibodies are now used as specific

affinity ligands for the purification of the avian H2 C form.

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Preface

The work presented in this dissertation was performed between

October, 1981 and April, 1985 in the Department of Biochemistry,

Imperial College, London. All the work described herein is my own except

where otherwise indicated in the text.

I am deeply grateful to my supervisor, Professor E.A. Barnard,

F.R.S., for his encouragement and advice throughout the course of this

work. I am also indebted to Dr. William R. Randall for his technical

supervision, much inspiring discussion, and for his constructive

criticism of several chapters in this thesis. I wish also to record my

special thanks to Dr. Penelope J. Barnard, Dr. J. Jedrzejczyk, Dr. John

A. Pizzey, Dr. Rudolf Shipolini, Mr. Jonathan Jarvis, Mr. David Green,

Mr. John F.R. Cavanagh, Mr. Anthony Lai and Dr. David Lane for their

invaluable assistance and enjoyable collaboration. I also thank Miss

Alison Bartlett for typing this thesis. Finally, my gratitude goes to

all my colleagues for their friendship and generosity, who have made the

past 3 years a fruitful and memorable experience. I am also grateful to

the Croucher Foundation for generous financial support during the year

83/84.

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To my parents and to Frank

C O N T E N T S

page

Abstract nPreface ivList of abbreviations xList of figures xiList of tables xm

CHAPTER 1

GENERAL INTRODUCTION

1.1 Acetylcholinesterase 21.1.1 Enzyme properties 21.1.2 Inhibition of cholinesterases 41.1.3 Molecular structure of AChE: multiple molecular forms 51.1.4 Localisation of AChE 121.1.5 Purification of AChE 151.1.6 Immunological studies of AChE 16

1.2 Classification of skeletal muscle fibre types 161.2.1 Physiological characterisation 171.2.2 Histochemical and biochemical characterisation 181.2.3 Morphological and uItrastructuraI characterisation 191.2.4 Fibre type composition of muscles 19

1.3 Skeletal muscle AChE in health and disease 211.3.1 Biosynthesis of AChE in skeletal muscles 211.3.2 Neurotrophic regulation of AChE 231.3.3 Avian muscular dystrophy 251.3.4 This thesis 27

•

CHAPTER 2

MULTIPLE MOLECULAR FORMS OF AChE IN MAMMALIAN AND AVIAN SKELETAL MUSCLES

2.1 Introduction 302.2 Materials and Methods 36

2.2.1 Animals 362.2.2 Muscles 362.2.3 Materials 372.2.4 Precautions applied in tissue extraction 372.2.5 Microdissection 402.2.6 Sedimentation analysis 412.2.7 Enzyme assay 41

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2.3 Results 44

Section I: Effects of extraction conditions on the distribution of AChE forms

2.3.1 Protease activities in muscle extracts and the effects of 44protease inhibitors on AChE extraction

2.3.2 Extraction of AChE by rapid processing In the presence of 45protease Inhibitors

2.3.3 The effect of perfusion 532.3.4 The effect of freezing muscle tissues 53

Section II: Distribution of AChE forms in mammalian and avian skeletal muscles

2.4

2.5

2.3.5 Molecular profiles of AChE In mammalian skeletal muscles2.3.6 Molecular profiles of AChE in avian muscles2.3.7 Molecular profiles of AChE In muscles of mixed fibre type

Discussion

2.4.1 Autolysls of AChE In tissue crude extracts2.4.2 Correlation of the degradation of the H2C form with the

proteolytic activity In tissue crude extracts2.4.3 Inhibition of proteolysis stabilised the distribution of the

molecular forms of AChE2.4.4 Time-course and mechanism of AChE degradation2.4.5 H2c I s a dominant component of mammal Ian skeletal muscle ACHE2.4.6 AChE proflles In fast- and slow-twltch muscles2.4.7 AChE profiles In chicken twitch and tonic muscles2.4.8 Non-endplate AChE In type I and type II fibres2.4.9 Endplate AChE In twitch and tonic fibres

Conclusion

54636666

6670

71

72 75 75777879

80

CHAPTER 3

THE EFFECT OF CHRONIC DENERVATION ON THE ENDPLATE AND NON-ENDPLATE AChE IN HAMALIAN FAST- AND SLOW-TWITCH MUSCLES

3.1 Introduction 83

3.2 Materials and Methods 853.2.1 Animals 853.2.2 Muscles 863.2.3 Surgical procedures 863.2.4 Partial denervation 873.2.5 Muscle extract preparation 873.2.6 Measurement of endplate and non-endplate AChE 87

3.3 Results 88

3.3.1 Denervation-induced changes In AChE activities In different 88species

- V 1 1 -

3.3.2 Effects of denervation on the AChE profiles of fast and 92slow muscles

3.3.3 Differential analysis of endplate and non-endplate AChE in 100normal and denervated muscle fibres

3.4 Discussion 1003.4.1 Changes in AChE activities Induced by chronic denervation 1003.4.2 AChE in the endplates of normal and denervated muscle fibres 1053.4.3 AChE in the non-endplate regions of normal and denervated 106

muscle fibres3.4.4 Effect of partial and total denervation on the two regions of 108

semimembranosus

3.5 Conclusion 110

CHAPTER 4

CHANGES IN MOLECULAR FORMS OF AChE IN CHRONICALLY STIMULATED CHICKENMUSCLES


4.2.1 Nerve stimulation 1154.2.2 Extraction of AChE 1164.2.3 Sedimentation analysis 117

4.3 Results ii7

4.3.1 Changes in AChE activity in stimulated normal chicken PLD 117muscle

4.3.2 Separation of endplate and non-endplate forms of AChE 1224.3.3 The reversal of the effect of stimulation 1224.3.4 Changes in AChE activity in stimulated dystrophic chicken PLD 122

muscle4.3.5 Separation of endplate and non-endplate forms of AChE 1324.3.6 Changes in AChE activities in stimulated leg chicken muscles 132

4.4 Discussion 134

4.4.1 Changes of AChE in stimulated normal chicken PLD 134% 4.4.2 Changes of AChE in stimulated dystrophic chicken PLD 143

4.4.3 Changes of AChE in leg muscles of normal and dystrophic 146chickens

CHAPTER 5

MONOCLONAL ANTIBODIES TO PURIFIED CHICK BRAIN AChE


5.2.1 Materials 1515.2.2 Purification of chick brain AChE 152

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5.2.3 Enzyme assay 1545.2.4 Immunisation and cell culture 1545.2.5 Screening protocol 1565.2.6 Purification of monoclonal antibodies 1565.2.7 Gel electrophoresis and western blotting 1575.2.8 Immunohistochemistry 158

5.3 Results 158

5.3.1 Purification of chick brain AChE 1585.3.2 Characterisation of monoclonal antibodies to AChE 160

5.3 Discussion no

CHAPTER 6

GENERAL DISCUSSION 175

Appendix I: Rapid processing of skeletal muscle crude 181extracts

Appendix II: Molecular size determination of AChE by 183radiation inactivation

References 189

List of publications 209

*

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LIST OF ABBREVIATIONS

Abbreviations

ACB-1 Monoclonal antibody 1

ACB-2 Monoclonal antibody 2

ACh Acetylcholine

AChE Acetylcholinesterase

ALD Anterior latissimus dorsi

AT Anterior tibia1 is

ATPase ATP-phosphohydro1ase

BTEE Benzoyl-tyrosy 1 ethyl ester

Cat Cata1ase

Con A Concanavalin A

DFP Diisopropyl phosphorof1uoridate

DM EM Dulbecco's minimum essential medium

DTNB 5,51—dithiobis—(2-nitrobenzoic acid)

EDL Extensor digitorum longus

FITC Fluorescein isothiocyanate

3-gal 3-Galactosidase

HRP Horse radish peroxidase

ig Immunoglobulin

isoOMPA Tetraisopropy1pyrophosphoramide

LDH Lactate dehydrogenase

MAc N-methy1acr i d i n i urn

NEM N-ethyImaleimide

^ NP-40 Non i det P-40

ONPG O-nitrophenyIgalactosi de

PBS Phosphate buffered saline

PLD Posterior latissimus dorsi

PMSF PhenyImethy1su1fony1f1uoride

(pChE Pseudocholinesterase

RER Rough endoplasmic reticulum

SA Semimembranosus accessorius

SDS-PAGE Sodium dodecyl su1phate-Polyacrylamide gel electrophoresis

SP Semimembranosus proprius

SVI Vipera ammodytes venom trypsin inhibitor

TCA Trichloroacetic acid

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List of figures Rage

Fig 1.1 Schematic representation of the hydrolysis of acetylcholine.

3

Fig 1.2 Hypothetical model of the binding of acetylcholine to the active site of AChE.

3

Fig 1.3 Schematic representation of the collagen tailed form of AChE from Electrophorus.

7

Fig 1.4 Schematic representation of the quaternary structure of the asymmetric and globular forms of AChE.

9

Fig 2.1 Casein hydrolysing activity of tissue crude extracts from various species.

46

Fig 2.2 Dose dependent inhibition of casein hydrolysing activity of rat muscle extracts by bacitracin, SVI and a2 -macroglobulin.

47

Fig 2.3 Dose dependent inhibition of bovine a-chymotrypsin activity by SVI.

48

Fig 2.4 Effect of prolonged storage of guinea pig muscle crude extracts on the distribution of the molecular forms of AChE.

50

Fig 2.5 Protease inhibitors stabilise the distribution of AChE forms in muscle crude extracts.

51

Fig 2.6 Typical sucrose gradient profiles of AChE forms in some mammalian type II muscles.

55


56


58

Fig 2.9 Typical sucrose gradient profiles of AChE forms in some mammalian type I muscles.

59

Fig 2.10 Molecular forms of AChE from type I and type II muscles fibres.

61

Fig 2.11 The distribution of molecular forms of AChE in the endplate and non-endplate regions from type I and type II muscles.

62

Fig 2.12 Molecular forms of AChE in the anterior and posterior regions of the chicken adductor profundus.

65

Fig 2.13 Sucrose gradient profile of AChE in the chicken PLD muscle.

67

Fig 2.14 Sucrose gradient profiles of AChE in some muscles of mixed fibre type composition.

68

Fig 2.15 Distribution of molecular forms of AChE in the endplate and non-endplate regions of the rat diaphragm.

69

Fig 3.1 Histochemical characterisation of contralateral normal and denervated rabbit semimembranosus muscle.

89

Fig 3.2 Sucrose gradient profiles of contralateral control and chronically denervated hamster EDL.

93

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94Fig 3.3 Sucrose gradient profiles of AChE in control and denervated type II muscles.

Fig 3.4 Sucrose gradient profiles of AChE in contralateral 95control and denervated type I muscles.

Fig 3.5 Sucrose gradient profiles of AChE in the endplate 101region of control and denervated guinea pig muscle fibres.

Fig 3.6 Sucrose gradient profiles of extrajunctional AChE in 102 control and denervated guinea pig muscle fibres.

Fig 3.7 Changes in the intensity of endplate AChE staining in 103 denervated guinea pig soleus.

Fig 4.1 Sucrose gradient profiles of AChE in normal PLD after 120different periods of stimulation.

Fig 4.2 Electron micrographs of PLD muscle from a 19-week old 123normal chicken.

Fig 4.3 Sucrose gradient profiles of endplate and non-endplate 125AChE in control and 3-week stimulated normal PLD.

Fig 4.4 Sucrose gradient profiles of AChE in dystrophic PLD 130after different periods of stimulation.

Fig 4.5 Frozen cross-section of PLD muscles from a 14-week 131old dystrophic chicken.

Fig 4.6 Sucrose gradient profiles of endplate and non-endplate 133AChE in control and 5-week stimulated dystrophic PLD.

Fig 4.7 Sucrose gradient profiles of AChE in leg muscles of 137normal chicken after chronic stimulation.

Fig 4.8 Sucrose gradient profiles of AChE in leg muscles of 138dystrophic chicken after chronic stimulation.

Fig 5.1 Sucrose gradient profile of AChE from crude extract 159of one day old chick brain.

Fig 5.2 Analysis of purified chick brain AChE using SDS-PAGE. 162

Fig 5.3 SDS-PAGE of purified ACB-1 and ACB-2. 162 iFig 5.4 Dilution curves of ACB-1 and ACB-2. 165 ;

Fig 5.5 Competition of ACB-1 and ACB-2 for epitopes of AChE. 167

Fig 5.6 Reactivity of ACB-1 to avian AChE by western blotting. 168

Fig 5.7 Immunohistochemical localisation of AChE in unfixed 171chicken PLD muscle using ACB-1 and ACB-2.

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List of tables p a g e

Table 1.1 Sedimentation coefficients of the molecular forms of AChE in chicken and rat skeletal muscle.

11

Table 2.1 The fibre type composition of some mammalian and chicken skeletal muscles which had been chosen for AChE analysis.

33

Table 2.2 Combination of protease inhibitors used during AChE extraction from different tissues.

35

Table 2.3 AChE activity in some mammalian muscles. 64

Table 3.1 Relative activities of AChE in denervated rabbit, guinea pig and hamster muscles.

91

Table 3.2 Relative activities of L and H2c form in denervated rabbit, guinea pig and hamster muscles.

97

Table 3.3 Changes in total AChE and its molecular forms in the partially denervated rabbit semimembranosus.

98

Table 3.4 The relative activities (per muscle) of AChE and its 99molecular forms in the denervated guinea pig and rabbit semimembranosus.

Table 4.1 Increases in AChE concentration in the normal PLD after different periods of stimulation.

118

Table 4.2 Reversal of stimulation-induced changes in AChE after cessation of the stimulus.

127

Table 4.3 Decreases in AChE concentration in dystrophic PLD after different periods of stimulation.

128

Table 4.4 Fibre type composition of some normal and stimulated fast-twitch muscles in the lower limb of chicken.

135

Table 4.5 Increases in AChE concentration in leg muscles of normal and dystrophic chickens after different periods of stimulation.

136

Table 5.1 Purification of AChE from day old chick brain. 161

Table 5.2 Reactivity of ACB-1 and ACB-2 with <PChE and AChE from other species.

169

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CHAPTER 1

GENERAL INTRODUCTION

-1-

1.1 Acetylcholi nesterase

1.1.1 Enzyme properties

The term acetylcholinesterase (AChE; E.C. 3.1.1.7) was first

introduced by Augustinsson and Nachmansohn (1949) to describe the

particular type of esterase shown to be associated with a variety of

conducting tissues (Nachmansohn and Rothenberg, 1944; 1945) and

erythrocytes (Stedman and Stedman, 1935; Hawes, 1940), and is distinct

from the other non-specific cholinesterases or 1 pseudocholinesterases1

( V'ChE; E.C.3.1.1.8) found in serum (Stedman et al., 1932). AChE

catalyses the hydrolysis of the neurotransmitter acetylcholine (ACh)

which is schematically shown in Fig 1.1. AChE has a high affinity for

its main substrate ACh. The optimal substrate concentration has been

well-defined and substrate inhibition at high ACh concentrations has

been shown to occur (Stedman and Stedman, 1935). Structure-substrate

specificity study showed that the rate of hydrolysis decreases with

increasing length of the acyl chain, i.e. in the order: acetyl- >

propionyl- > butyryl-choline (Nachmansohn and Wilson, 1951). In

contrast, ChE exhibits a reverse order of substrate preference.

Based on results from enzyme kinetic studies, a hypothetical

model of enzyme-substrate interaction was first proposed by Nachmansohn

and Wilson (1951). It was suggested that the catalytic site consists of

a negatively charged anionic and an esteratic subsite. The anionic

subsite binds to cationic substrates or inhibitors while the esteratic

subsite, which contains a serine hydroxyl group, undergoes acylation

during a catalytic turnover (Schaffer et al., 1973). Furthermore, the

existence of peripheral anionic sites on AChE has been suggested by the

effect of certain multi-quaternary AChE antagonists on AChE activity at

low ionic strength (Changeux, 1966). Flaxedil, for example, reverses the

-2 -

o? h 3

H3C - N - c h2—c H2- ° - C - C H 3 + 2 H2 0 ;=;

CHg

FIG. 1.1 Schematic representation of the hydrolysis of

acetylcholine.

t

anionicsite

esteraficsite

h 3 c - n - c h 2 - c h 2 - o - c - o h

CH. ICH

FIG. 1.2 Hypothetical model of the binding of acetylcholine to

the active site of AChE. From Nachmansohn and Wilson, 1951.

-3-

enzyme inhibition due to decamethonium although it does not itself bind

to the catalytic anionic site occupied by the inhibitor. It is likely

therefore that allosteric influence is exerted by ligand binding at

peripheral sites (Kitz et al., 1970; Changeux, 1966).

AChE is classified as a serine hydrolase because of its many

similar characteristics to those possessed by classical serine

hydrolases such as a-chymotrypsin. AChE exhibits irreversible active-

site phosphorylation by organophosphorous compounds. Equivalent weight

determination and peptide analysis indicate that phosphorylation only

involves one serine residue. This serine residue is part of a peptide

which shows significant homology with other enzymes of this class

(Schaffer et al., 1973). Both AChE and a-chymotrypsin exhibit bell-

shaped activity versus pH relationship. The apparent pKa of 6-7 for AChE

suggests a general acid-base catalysis by imidazole (Nachmansohn and

Wilson, 1951).

1.1.2 Inhibitors of cholinesterases

'Anticholinesterases' have been commonly used for biochemical

and physiological studies of cholinesterases (for review see Silver,

1974). The inhibition by organophosphorus compounds is characteristic of

cholinesterases. These compounds form a stable phosphorylated complex

with the serine hydroxyl moiety at the esteratic site of the enzyme.

Most organophosphorus compounds, such as diisopropyl phosphorofluoridate

(DFP), react with the enzyme only at the esteratic site. Some

organophosphorus compounds, however, bind simultaneously to both the

anionic and esteratic sites, and yield a stable phosphorylated enzyme

intermediate. The reversibility of these inhibitors depend on the rate

of regeneration by the hydrolysis of the intermediate complex. Esters of

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phosphorus, e.g. echothiophate (Tammelin, 1957), substituted carbamic,

such as eserine and neostigmine (Machne and Unna, 1963), and sulfonic

acid derivatives such as phenyl methylsulfonylfluoride (PMSF), also form

stable complexes with the active sites. Another class of

anticholinesterases, which includes the quaternary compounds

decamethonium and edrophonium, interferes with substrate binding by

attaching to the anionic site (Long, 1963). A series of bis-quaternary

compounds, such as BW 284C51, also inhibit substrate binding by blocking

the anionic site (Fulton and Mogey, 1954). BW 284C51 is often used as a

selective inhibitor of AChE, while isoOMPA, an organophosphorus compound

(see earlier), is frequently used as a selective inhibitor of ^ChE.

These two inhibitors operate at different range of concentrations for

optimal selectivity (Holmstedt, 1957). The exact optimal concentrations

may vary between species and method used (Austin and Berry, 1953). For

mammalian and avian tissues, concentrations of 5 X 10"^M and 10~^m for

BW 284C51 and isoOMPA respectively were found to be most effective

(Hall, 1973; Lyles et_a]_., 1979).

1.1.3 Molecular structure of AChE : multiple molecular forms

The existence of multiple stable forms of AChE was first

demonstrated by Massoulie and Rieger (1969) in high ionic strength

extracts of fresh electric organ tissues of Electrophorus. These

molecular forms can be identified on sucrose density gradients, and show

characteristic sedimentation coefficients of 8S, 14S and 18S. Electron

microscopy of the purified enzyme shows that they have an asymmetric

structure consisting of a cluster of globular units attached to a rod

like 'tail' of approx. 50nm long (Dudai _et__al_., 1973). The globular

units, which contain the enzyme catalytic sites, are grouped into

tetramers. Each tail peptide can be joined to one, two or three of these

-5-

t e t r a m e r s ( Bon _ e t _ a l _ . , 1 9 7 6 ) . Each s u b u n i t ha s a m o l e c u l a r m ass o f a b o u t

80 kd (Duda i and S i l m a n , 1 9 7 4 ; R o s e n b e r r y and R i c h a r d s o n , 1 9 7 7 ) and t h e

t a i l p e p t i d e a b o u t 100 kd (Bon e t a l . , 1 9 7 6 ) . T h i s t a i l p e p t i d e i s

i n t e r e s t i n g i n t h a t i t c o n t a i n s a l a r g e number o f h y d r o x y p r o l i n e and

h y d r o x y l y s i n e r e s i d u e s w h ic h i s c h a r a c t e r i s t i c o f c o l l a g e n and i s

s e n s i t i v e t o c o l l a g e n a s e t r e a t m e n t i n a t e m p e r a t u r e d e p e n d e n t manner

(Bon and M a s s o u l i e , 1 9 7 8 ; A n g l i s t e r and S i l m a n , 1 9 7 8 ; R o s e n b e r r y e t a l . ,

1 9 8 0 ) , b u t i s p e p s i n r e s i s t a n t (Mays and R o s e n b e r r y , 1 9 8 1 ) ( F i g 1 . 3 ) .

T h e s e a s y m m e t r i c m o l e c u l e s a g g r e g a t e u n d e r lo w i o n i c s t r e n g t h c o n d i t i o n s

( G r a f i u s and M i l l a r , 1 9 6 5 ; Bon e t a l . , 1 9 7 8 ) . Such a g g r e g a t i o n c a n be

a b o l i s h e d by c o l l a g e n a s e o r l i m i t e d t r y p s i n t r e a t m e n t , a s w e l l a s upon

s t o r a g e , w h er e b y t h e c o l l a g e n - t a i l i s c l e a v e d , y i e l d i n g g l o b u l a r

t e t r a m e r s t h a t do n o t i n t e r a c t a t lo w s a l t c o n d i t i o n (Bon and M a s s o u l i e ,

1 9 7 8 ) . T h e s e t e t r a m e r s a r e l i n k e d t o t h e t a i l by d i s u l p h i d e bo n d s t o two

o f t h e s u b u n i t s , w h ic h form q u a t e r n a r y a s s o c i a t i o n w i t h t h e r e m a i n i n g

d i s u l p h i d e - l i n k e d d i m e r ( R o s e n b e r r y and R i c h a r d s o n , 1 9 7 7 ) , a s shown i n

F i g 1 . 3 . I t i s i m p o r t a n t t o n o t e t h a t t h e p o s i t i o n s o f t h e d i s u l p h i d e

b r i d g e s a r e o n l y h y p o t h e t i c a l . The m u l t i - s u b u n i t m o l e c u l a r f o r m s r e m a in

s t a b l e and a c t i v e e v e n a f t e r f u l l r e d u c t i o n o f t h e d i s u l p h i d e b o n d s ,

i n d i c a t i n g t h a t q u a t e r n a r y a s s o c i a t i o n s b e t w e e n t h e s u b u n i t s , and i n t h e

c a s e o f t h e a s y m m e t r i c f o r m s , b e t w e e n t h e s u b u n i t s and t h e t a i l i s

* i m p o r t a n t i n s t a b i l i s i n g t h e m u l t i m e r i c s t r u c t u r e .

The g l o b u l a r t e t r a m e r s ca n be f u r t h e r d i s s o c i a t e d i n t o d i m e r s

and monomers by l i m i t e d t r y p s i n d i g e s t (Bon and M a s s o u l i e , 1 9 7 6 ) . A

s y s t e m o f n o m e n c l a t u r e f o r t h e a s y m m e t r i c and g l o b u l a r fo rm s h a s b e e n

i n t r o d u c e d by Bon e t a l . ( 1 9 7 9 ) . The g l o b u l a r , t a i l - l e s s f o r m s and

c o l l a g e n - t a i l e d a s y m m e t r i c fo rm s w e r e d e s i g n a t e d a s G and A f o r m s

r e s p e c t i v e l y . Each o f t h e t h r e e m o l e c u l a r f o rm s i n e a c h c a t e g o r y i s

c l a s s i f i e d by a s u b s c r i p t t o i n d i c a t e t h e number o f c a t a l y t i c u n i t s

-6-

oo

FIG- 1-3 S c h e m a t i c r e p r e s e n t a t i o n o f t h e c o l l a g e n - t a i l e d form

o f AChE from E l e c t r o p h o r u s . The c i r c l e s r e p r e s e n t c a t a l y t i c

s u b u n i t s ; - S - S - , d i s u l p h i d e bonds w h ic h l i n k c a t a l y t i c s u b u n i t s

t o g e t h e r o r t o t h e c o l l a g e n t a i l . The t r i p l e h e l i c a l s t r u c t u r e

r e p r e s e n t s t h e c o l l a g e n t a i l w h ose s u b u n i t s a r e a l s o l i n k e d by

i n t e r d i s u l p h i d e b r i d g e s (n*v5). From Mays and R o s e n b e r r y , 1 9 8 1 .

- 7 -

p r e s e n t ( F i g 1 . 4 ) .

In T o r p e d o e l e c t r i c o r g a n e x t r a c t s , a p a r t from t h e c o l l a g e n

t a i l e d f o r m s , a p o p u l a t i o n o f AChE i n s e n s i t i v e t o s a l t c o n c e n t r a t i o n and

i n t e r a c t s w i t h n o n - i o n i c d e t e r g e n t s h a s b e e n i s o l a t e d ( W it z e m a n n , 1 9 8 0 ;

M a s s o u l i e and B o n , 1 9 8 2 ; V i r a t e l l e and B e r n h a r d , 1 9 8 0 ; L e e e t a l . ,

1 9 8 2 ) . The c a t a l y t i c s u b u n i t s i n t h i s po ol o f d i m e r i c and t e t r a m e r i c

f o r m s a r e s e n s i t i v e t o p r o n a s e o r p r o t e i n a s e K d i g e s t i o n w h ic h a b o l i s h e d

t h e i r i n t e r a c t i o n w i t h d e t e r g e n t s (L i and Bon , 1 9 8 3 ) .

A number o f m o l e c u l a r f o r m s o f AChE w ere a l s o f o u n d i n a v a r i e t y

o f mammalian t i s s u e s , i n c l u d i n g human e r y t h r o c y t e membrane ( O t t e t a l . ,

1 9 7 5 ; 1 9 8 2 ) and m u s c l e s ( C a r s o n e t a l . , 1 9 7 9 ) , b o v i n e e r y t h r o c y t e

membrane (B erm an , 1 9 7 3 ) and serum ( R a l s t o n e t a l . , 1 9 8 5 ) , r a t d ia p h r a g m

( H a l l , 1 9 7 3 ; Y ou nk in e t a l . , 1 9 8 2 ) , v e r t e b r a t e h i n d - l i m b m u s c l e s

( G r o s w a l d and D e t t b a r n , 1 9 8 3 a ; G i s i g e r and S t e p h e n s , 1 9 8 3 ; B a r n a r d et_

a l . , 1 9 8 4 a ; L y l e s e t a l . , 1 9 8 2 ; L y l e s and B a r n a r d , 1 9 8 0 ; B a r n a r d e t a l . ,

1 9 8 4 b ; Rotund o and Fam brough, 1 9 7 9 ) , n e u r a l t i s s u e s ( F e r n a n d e z e t a l . ,

1 9 7 9 ; Bon e t a l . , 1 9 7 9 ; Sung and R u f f , 1 9 8 3 ; G r a s s i e t a l . , 1 9 8 2 ) ,

c a r d i a c and smooth m u s c l e s (S k au and B r i m i j o i n , 1 9 8 0 ) , and c h i c k e n

p la s m a ( L y l e s e t a l . , 1 9 8 0 ) . T h e s e AChE fo rm s a r e a l s o s y n t h e s i z e d by

c u l t u r e d m u s c l e c e l l s ( S u g i y a m a , 1 9 7 7 ; R e i g e r e t a l . , 1 9 8 0 ; I n e s t r o s a e t

a l . , 1 9 8 2 ; B u l g e r e t a l . , 1 9 8 2 ) . They a r e t h o u g h t t o be a n a l o g o u s t o

• t h o s e i n E l e c t r o p h o r u s o r T o r p e d o AChE (Bon e t a l . , 1 9 7 9 ; V i g n y e t a l . ,

1 9 7 9 ) , s i n c e a number o f l o w s a l t i n s o l u b l e f o r m s r e s e m b l e t h e

a s y m m e t r i c f o rm s o f E l e c t r o p h o r u s , and t h e low s a l t s o l u b l e fo r m s may

r e p r e s e n t t h e v a r i o u s g l o b u l a r f o rm s w h ic h h a v e b e e n d e f i n e d . On t h e

o t h e r h a n d , i n v i e w o f t h e l i m i t e d i n f o r m a t i o n a v a i l a b l e on t h e

m o l e c u l a r s t r u c t u r e and c o m p o s i t i o n o f t h e s e m o l e c u l a r f o r m s i n

mammalian and a v i a n t i s s u e s , an e m p i r i c a l s y s t e m o f n o m e n c l a t u r e b a s e d

on s e d i m e n t a t i o n c h a r a c t e r i s t i c s o f t h e v a r i o u s m o l e c u l a r fo r m s h a s b e e n

-8-

ooe P n QjO OO

O p

\ /

ooQO oo og4 g2 Gi

FIG- 1.4 S c h e m a t i c r e p r e s e n t a t i o n o f t h e q u a t e r n a r y s t r u c t u r e

o f t h e a s y m m e t r i c (A) and g l o b u l a r (G) fo rm s o f AChE, a s

e s t a b l i s h e d f o r E l e c t r o p h o r u s . The c i r c l e s r e p r e s e n t i n d i v i d u a l

c a t a l y t i c s u b u n i t ; s h o r t l i n e s w h ic h j o i n t h e c i r c l e s r e p r e s e n t

t h e i n t e r s u b u n i t d i s u l p h i d e b r i d g e s . The c o l l a g e n t a i l i s

s y m b o l i s e d by t r i p l e l i n e s . The g l o b u l a r G4 form c o r r e s p o n d s t o

t h e a u t o l y t i c p r o d u c t , and h e n c e r e t a i n s a n o n - c o l l a g e n i c p e p t i d e

from t h e t a i l . T h i s s i m p l i f i e d r e p r e s e n t a t i o n o f t h e m o l e c u l a r

form s d o e s n o t i n c l u d e d i f f e r e n c e s t h a t may e x i s t f o r e a c h

q u a t e r n a r y s t r u c t u r e i n t e r m s o f i n t e r s u b u n i t d i s u l p h i d e b o n d s ,

g l y c o s i d i c r e s i d u e s , i n t r a s u b u n i t c l e a v a g e s , p r e s e n c e o f b i n d i n g

do m ain s o r n o n - c a t a l y t i c s u b u n i t s . A d a p te d from M a s s o u l i e and

Bon, 1 9 8 2 .

- 9 -

p r o p o s e d by B a r n a r d and c o w o r k e r s ( B a r n a r d e t a l . , 1 9 8 2 ; 1 9 8 4 b ) . The

v a r i o u s m o l e c u l a r f o rm s w e r e g i v e n t h e i n i t i a l s L, M o r H d e s i g n a t i n g

l i g h t , medium and h e a v y f o r m s r e s p e c t i v e l y . T h e s e t h r e e g r o u p s w e r e

f u r t h e r s u b d i v i d e d i n t o , 1-2, M, Mc , Hl c and H2 C , w h er e t h e numbers i n

t h e s u f f i x d e n o t e t h e s u b s p e c i e s w i t h i n e a c h g r o u p , and t h e l e t t e r c

d e n o t e s t h e p r e s e n c e o f a c o l l a g e n - l i k e t a i l p e p t i d e . B e c a u s e t h i s

s y s t e m o f n o m e n c l a t u r e h as t h e a d v a n t a g e s o f a d e q u a t e l y d e s c r i b i n g e v e r y

m o l e c u l a r form f o u n d t o - d a t e and d o e s n o t make any p r e s u p p o s i t i o n

c o n c e r n i n g t h e m o l e c u l a r c o m p o s i t i o n s o f t h e v a r i o u s m o l e c u l a r f o r m s , i t

w i l l be u s e d t h r o u g h o u t t h e p r e s e n t w o r k .

The c h a r a c t e r i s a t i o n o f t h e a m p h i p a t h i c f o r m s o f AChE from human

e r y t h r o c y t e membranes ( B r o d b e c k e t a l . , 1 9 8 4 ; R o s e n b e r r y and S c o g g i n ,

1 9 8 4 ; D u t t a - C h o u d h u r y and R o s e n b e r r y , 1 9 8 4 ) i l l u s t r a t e t h e a p p a r e n t

d i v e r s i t y o f t h e m o l e c u l a r s t r u c t u r e s w i t h i n t h e m o l e c u l a r f o r m s .

M o l e c u l a r fo r m s w h ic h h a v e i d e n t i c a l s e d i m e n t a t i o n p a r a m e t e r s c a n be

d i f f e r e n t s t r u c t u r a l l y and p h y s i o c h e m i c a l l y . F o r e x a m p l e , f u r t h e r

d e s i g n a t i o n i s c l e a r l y n e e d e d t o d i s t i n g u i s h h y d r o p h o b i c and h y d r o p h i l i c

f o rm s o f AChE, e . g . l_2 h may be u s e d t o d e s c r i b e t h e h y d r o p h o b i c ( h ) ,

d i m e r i c n a t u r e o f human e r y t h r o c y t e AChE. The v a r i o u s m o l e c u l a r f o rm s

may a l s o d i f f e r i n t h e i r c a r b o h y d r a t e c o n t e n t , w h ic h i s e v i d e n t from

t h e i r d i f f e r e n t l e c t i n b i n d i n g c h a r a c t e r i s t i c s ( R o t u n d o , 1 9 8 4 ) . D e s p i t e

t h i s m o l e c u l a r h e t e r o g e n e i t y , t h e v a r i o u s f o rm s o f AChE w i t h i n e a c h

g r o u p e x h i b i t e s s e n t i a l l y i d e n t i c a l c a t a l y t i c p r o p e r t i e s ( B a r n e t t and

R o s e n b e r r y , 1 9 7 9 ) . The s e d i m e n t a t i o n c o e f f i c i e n t s o f t h e v a r i o u s f o rm s

o f AChE i n mammalian and a v i a n t i s s u e s a r e su m m ar ized i n T a b l e 1 . 1 .

The v a r i o u s AChE fo r m s may be m o d i f i e d o r d i s s o c i a t e d by

p r o t e o l y t i c d e g r a d a t i o n . The c o l l a g e n - t a i l e d f o r m s , i n p a r t i c u l a r , a r e

s u s c e p t i b l e t o a u t o l y s i s upon s t o r a g e ( S i l m a n e t a l . , 1 9 7 8 ) . On t h e

o t h e r h a n d , a s s o c i a t i o n o f t h e l o w e r m o l e c u l a r w e i g h t f o rm s i n t o h i g h

-10-

Table 1.1 Sedimentation coefficients of the molecular fomjs of

AChE in chicken and rat skeletal muscle.

Species ' -Molecular form

L1 L2 M Hlc HZc

Chicken 4 6.5 11.5 14.8 20

Rat 3.5 6 9.9 13 16.7

(Bon, Vigr\y and Massoulie, 1979)

m o l e c u l a r w e i g h t a g g r e g a t e s was o b s e r v e d i n s t o r e d m ous e b r a i n e x t r a c t s

(Adamson e t a l . , 1 9 7 5 ) . S i m i l a r a g g r e g a t i o n o f t h e G f o r m s was o b s e r v e d

i n b o v i n e c a u d a t e n u c l e u s e x t r a c t s ( Hoi 1u n g e r and N i k l a s s o n , 1 9 7 3 ) and

c o u l d b e p r e v e n t e d by d e t e r g e n t ( G r a s s i e t a l . , 1 9 8 2 ) . Such i n v i t r o

i n t e r c o n v e r s i o n o f t h e m o l e c u l a r f o rm s u n d e r m in e s e f f o r t s t o s t u d y t h e

d i s t r i b u t i o n o f t h e s e f o r m s . I t a l s o makes more d i f f i c u l t t h e

c o r r e l a t i o n b e t w e e n c h a n g e s i n t h e d i s t r i b u t i o n o f t h e m o l e c u l a r f o rm s

and v a r i o u s p a t h o l o g i c a l s t a t e s , s u c h a s d e n e r v a t i o n i n d u c e d a t r o p h y

( T h e s l e f f , 1 9 7 4 ; B acou e t a l . , 1 9 8 2 ) and m u s c u l a r d y s t r o p h y ( G i s i g e r and

S t e p h e n , 1 9 8 3 ; L y l e s e t a l . , 1 9 8 2 ) .

1.1.4 Localisation of AChE

The s t r u c t u r a l h e t e r o g e n e i t y o f AChE i n a w i d e v a r i e t y o f

t i s s u e s i s now c o n s i d e r e d t o be r e l a t e d t o t h e i r d i s t r i b u t i o n and

s p e c i f i c l o c a l i z a t i o n i n t h e s e t i s s u e s ( f o r r e v i e w s e e R o s e n b e r r y ,

1 9 8 2 ) . The a m p h i p a t h i c f o r m s o f AChE from human e r y t h r o c y t e s , human

b r a i n and T o r p e d o e l e c t r i c o r g a n i n t e r a c t s t r o n g l y w i t h d e t e r g e n t

m i c e l l e s , and r e q u i r e d e t e r g e n t t o m a i n t a i n t h e i r c a t a l y t i c a c t i v i t y

( R o m e r - L u t h i e t a l . , 1 9 8 0 ; B r o d b ec k and O t t , 1 9 8 4 ) . In human e r y t h r o

c y t e s , a p a p a i n s e n s i t i v e , h y d r o p h o b i c p e p t i d e f r a g m e n t i s t h o u g h t t o be

r e s p o n s i b l e f o r t h e i n t e r a c t i o n o f AChE d i m e r w i t h l i p o s o m e s and i s

l i k e l y t o a s s o c i a t e w i t h t h e e r y t h r o c y t e membrane ( D u t t a - C h o u d h u r y and

R o s e n b e r r y , 1 9 8 4 ) . The a m p h i p a t h i c f o rm s o f AChE i n n e u r a l t i s s u e s , on

t h e o t h e r h a n d , a p p e a r s t o be a t e t r a m e r (M fo rm ) ( R a k o n c z a y e t a l . ,

1 9 8 1 ; S o r e n s e n e t a l . , 1 9 8 2 ; G r a s s i e t a l . , 1 9 8 2 ; R o t u n d o , 1 9 8 4 ) w h ic h

may be c o n v e r t e d t o a l o w s a l t s o l u b l e form by p r o n a s e o r p r o t e i n a s e K

t r e a t m e n t (L i and B o n , 1 9 8 3 ) . A d e t e r g e n t s o l u b l e L^ form h a s b e e n

o b s e r v e d i n a n e u r o b l a s t o m a s y m p a t h e t i c g a n g l i o n h y b r i d c e l l l i n e (T 2 8 )

( L a z a r and V i g n y , 1 9 8 0 ) . I t a p p e a r s t h a t t h e s e d e t e r g e n t s o l u b l e ,

g l o b u l a r AChE f o r m s i n n e u r a l t i s s u e s may h a v e d i f f e r e n t i n t r a c e l l u l a r

- 12-

and e x t r a c e l l u l a r l o c a l i s a t i o n , a s o b s e r v e d by t h e i r d i f f e r e n t i a l

s u s c e p t i b i l i t y t o i n h i b i t i o n by h y d r o p h i l i c i n h i b i t o r s ( T a y l o r e t a l . ,

1 9 8 1 ; L a z a r and V i g n y , 1 9 8 0 ) . I n t r a c e l l u l a r l y , t h e g l o b u l a r f o rm s a r e

a s s o c i a t e d w i t h t h e e n d o p l a s m i c r e t i c u l u m d u r i n g a x o n a l t r a n s p o r t

( M a s s o u l i e and B o n , 1 9 8 2 ; Couraud and Di G i a m b e r a r d i n o , 1 9 8 0 ) .

E x t r a c e l l u l a r l y , t h e y may be a s s o c i a t e d w i t h t h e p la s m a m embrane , a s

d e m o n s t r a t e d h i s t o c h e m i c a l l y i n m o t o r n e u r o n s ( L e w i s and S h u t e , 1 9 6 9 ) .

The lo w s a l t s o l u b l e , d e t e r g e n t i n s e n s i t i v e , g l o b u l a r fo r m s o f

AChE a r e m o st a b u n d a n t i n t i s s u e f l u i d s su c h a s serum ( G i s i g e r and

V i g n y , 1 9 7 7 ; Hodgson and Chubb, 1 9 8 3 ) o r p la sm a ( L y l e s e t a l . , 198 0 and

u n p u b l i s h e d r e s u l t s ) and c e r e b r a l s p i n a l f l u i d (Chubb e t a l . , 1 9 7 6 ) .

They a r i s e p r o b a b l y from t h e s e c r e t i o n o f n e u r a l ( S k a u and B r i m i j o i n ,

1 9 7 8 ; Chubb e t a l . , 1 9 7 6 ; G i s i g e r and V i g n y , 1 9 7 9 ) and m u s c l e c e l l s

( W i l s o n e t a l . , 1 9 7 3 ; B u l g e r e t a l . , 1 9 8 2 ; R ot u n d o and Fambroug h,

1 9 8 0 b ) . T h e s e f o r m s a l s o c o m p r i s e o f a f r a c t i o n o f t h e g l o b u l a r f o rm s i n

t h e r a t d ia p h r a g m ( Y o u n k in e t a l . , 1 9 8 2 ) and n e u r a l t i s s u e s ( L a z a r and

V i g n y , 1 9 8 0 ) . S e l e c t i v e i n h i b i t i o n o f e x t e r n a l l y l o c a t e d AChE f o r m s

sh ow s t h a t t h e s e lo w s a l t s o l u b l e form s a r e l o c a t e d l a r g e l y

i n t r a c e l l u l a r l y and a r e p r o b a b l y c o n f i n e d t o t h e lumen o f t h e

e n d o p l a s m i c r e t i c u l u m ( K r e u t z b e r g e t a l . , 1 9 7 3 ) . In m u s c l e c u l t u r e s ,

t h e y r e p r e s e n t a d i s t i n c t p o o l o f enzyme w i t h h i g h t u r n o v e r r a t e ,

• d e s t i n e d f o r s e c r e t i o n and d o e s n o t fo rm any m e m b r a n e - a s s o c i a t e d

i n t e r m e d i a t e ( R o t u n d o and Fambrough, 1 9 8 0 a ; 1 9 8 0 b ) .

The c o l l a g e n - t a i l e d , a s y m m e t r i c f o r m s o f AChE h a v e a t t r a c t e d

much i n t e r e s t b e c a u s e t h e y ca n be i s o l a t e d i n h i g h c o n c e n t r a t i o n from

e e l e l e c t r i c o r g a n (N ach m an so h n , 1 9 5 9 ) and a r e l o c a l i s e d a t t h e

n e u r o m u s c u l a r j u n c t i o n o f s k e l e t a l m u s c l e s ( H a l l , 1 9 7 3 ; Bon e t a l . ,

1 9 7 9 ; J e d r z e j c z y k e t a l . , 1 9 8 1 ) . I t se em s l i k e l y t h a t t h e s e f o r m s a r e

- 1 3 -

c l o s e l y a s s o c i a t e d w i t h t h e s y n a p t i c f u n c t i o n o f AChE. C o l l a g e n - t a i l e d

f o r m s a r e a l s o f o u n d i n t h e a t r i a o f r a t h e a r t (S k au and B r i m i j o i n ,

1 9 8 0 ) , and i n n e u r a l t i s s u e s ( G i s i g e r e t a l . , 1 9 7 8 ; F e r n a n d e z e t a l . ,

1 9 7 9 ; Skau and B r i m i j o i n , 1 9 8 0 ; G r a s s i e t a l . , 1 9 8 1 ) ( s e e a l s o C h a p t e r

5 ) . The l o c a l i s a t i o n o f t h e a s y m m e t r i c fo rm s i n t i s s u e s o t h e r t h a n

s k e l e t a l m u s c l e s and e l e c t r i c o r g a n h a s n o t b e e n s t u d i e d i n d e t a i l .

H o w e v e r , i t i s known t h a t i n m o to r n e u r o n e s t h e y a r e t r a n s p o r t e d by f a s t

a x o n a l f l o w and m us t t h e r e f o r e be i n t r a c e l l u l a r l y l o c a t e d (Di

G i a m b e r a r d i n o and C o u ra u d , 1 9 7 8 ) . The H fo rm s c a n a l s o b e f o u n d

e x t r a j u n c t i o n a l l y i n human i n t e r c o s t a l m u s c l e s ( C a r s o n e t a l . , 1 9 7 9 ) and

n e o n a t a l r a t s ( S k e l e t j and B r z i n , 1 9 8 0 ) .

T h e r e i s now a g r e a t d e a l o f e v i d e n c e , i n a d d i t i o n t o t h e i r

s o l u b i l i s a t i o n p r o p e r t i e s , t h a t t h e c o l l a g e n - t a i l e d fo rm s a r e a s s o c i a t e d

w i t h t h e b a s a l l a m i n a a t t h e m o t o r e n d p l a t e s ( f o r r e v i e w s s e e M a s s o u l i e

and Bon , 1 9 8 2 ; B r i m i j o i n , 1 9 8 3 ) . The H f o rm s from E l e c t r o p h o r u s

i n t e r a c t s s t r o n g l y w i t h a h e p a r a n - s u l p h a t e p r o t e o g l y c a n - l i k e compound

v i a t h e c o l l a g e n - t a i l u n i t ( V i g n y e t a l . , 1 9 8 2 ; Br andan and I n e s t r o s a ,

1 9 8 4 ) . H o m o g e n i s a t i o n o f r a t m u s c l e e n d p l a t e s w i t h h e p a r i n

p r e f e r e n t i a l l y s o l u b i l i s e s t h e a s y m m e t r i c form s o f AChE ( T o r r e s and

I n e s t r o s a , 1 9 8 3 ) . A r a t p h eoch rom ocy to m a d e r i v e d c e l l l i n e w h ic h l a c k s a

h e p a r a n on t h e c e l l s u r f a c e c o n t a i n s much l e s s e x t r a c e l 1 u l a r l y l o c a t e d

• a s y m m e t r i c form ( I n e s t r o s a e t a l . , 1 9 8 5 ) . H ow ev er , G r a s s i e t a l . ( 1 9 8 3 )

d i d n o t o b s e r v e any s i g n i f i c a n t i n t e r a c t i o n b e t w e e n t h e a s y m m e t r i c form

and v a r i o u s m a t r i x p r o t e i n s . F o c a l AChE s t a i n i n g c a n s t i l l be

d e m o n s t r a t e d h i s t o c h e m i c a l l y (McMahan e t a l . , 1 9 7 8 ) and

i m m u n o c y t o c h e m i c a l l y ( S a n e s and H a l l , 1 9 7 9 ) a f t e r t h e m o t o r axon and t h e

m u s c l e f i b r e h a v e b o t h b e e n r e m o v e d . S e v e r a l o b s e r v a t i o n s s u g g e s t t h e

p r e s e n c e o f AChE o t h e r t h a n t h e a s y m m e t r i c fo rm s a t t h e m o to r e n d p l a t e .

S k e t e l j and B r z i n ( 1 9 7 9 ) f o u n d t h a t c o l l a g e n a s e r e l e a s e d l e s s AChE from

- 1 4 -

t h e e n d p l a t e t h a n p a p a i n , w h ic h a l s o m o d i f i e s membrane bound f o r m s , a s

m e n t i o n e d a b o v e . G l o b u l a r f o r m s w e r e f o u n d t o b e p r e d o m i n a n t a t t h e

e n d p l a t e s i t e i n r a t h i n d - l i m b m u s c l e s ( V i g n y e t a l . , 1 9 7 6 ; F e r n a n d e z e t

a l . , 1 9 7 9 ; Y ou n k in e t a l . , 1 9 8 2 ) and c h i c k e n t o n i c m u s c l e s ( L y l e s and

B a r n a r d , 1 9 8 0 ) .

1 . 1 . 5 P u r i f i c a t i o n o f ACHE

The i s o l a t i o n and p u r i f i c a t i o n o f AChE a r e e s s e n t i a l f o r t h e

s t r u c t u r a l and k i n e t i c d e t e r m i n a t i o n o f t h e e n z y m e . P u r i f i c a t i o n

t e c h n i q u e s h a v e b e e n g r e a t l y im p r o v e d by t h e a p p l i c a t i o n o f a f f i n i t y

c h r o m a t o g r a p h y . A number o f a c r i d i n i u m o r t r i m e t h y l ammonium b a s e d

s y n t h e t i c a f f i n i t y l i g a n d s h a v e b e e n e m p lo y e d ( M a s s o u l i e e t a l . , 1 9 7 6 ;

V a l l e t t e e t a l . , 1 9 8 3 ) . T h e s e l i g a n d s b i n d t o t h e a n i o n i c s i t e o f AChE

v i a t h e ammonium and w h ic h ca n be s u b s e q u e n t l y d i s p l a c e d by AChE

i n h i b i t o r s s u c h a s d e c a m e t h o n i u m . The c h o i c e o f a p a r t i c u l a r a f f i n i t y

l i g a n d g e n e r a l l y d e p e n d s on t h e s o u r c e o f t h e enzyme and t h e c o n d i t i o n s

r e q u i r e d f o r i t s s o l u b i l i s a t i o n . The l e n g t h o f t h e s p a c e r arm and t h e

c o n c e n t r a t i o n o f t h e a f f i n i t y l i g a n d a r e a l s o c r i t i c a l on t h e e f f i c i e n c y

o f t h e l i g a n d ( V a l l e t t e e t a l . , 1 9 8 3 ) . A f f i n i t y c h r o m a t o g r a p h y h a s b e e n

u s e d i n t h e p u r i f i c a t i o n o f AChE from e e l (Dud a i and S i l m a n , 1 9 7 4 ;

R o s e n b e r r y e t a l . , 1 9 8 0 ) and T o r p e d o e l e c t r i c o r g a n ( H o p f f e t a l . , 1 9 7 3 ;

V i r a t e l l e and B e r n h a r d , 1 9 8 0 ; Le e e t a l . , 1 9 8 2 ; Lwebuga-Mukasa e t a l . ,

1 9 7 6 ) , human e r y t h r o c y t e membrane ( O t t e t a l . , 1 9 7 5 ; Berman and Y ou n g ,

1 9 7 1 ; R o s e n b e r r y and S c o g g i n , 1 9 8 4 ) , c h i c k b r a i n ( R o t u n d o , 1 9 8 4 ) ,

mammalian b r a i n ( R a c k o n c z a y e t a l . , 1 9 8 1 ; R e a v i l l and P lumm er, 1 9 7 8 ;

S o r e n s e n e t a l . , 1 9 8 2 ; Chan e t a l . , 1 9 7 2 ; Yamamura e t a l . , 1 9 7 3 ) and

s n a k e venom (Raba e t a l . , 1 9 7 9 ) . P u r i f i c a t i o n o f b o v i n e serum AChE u s i n g

e d r o p h o n i u m - l i n k e d S e p h a r o s e h a s a l s o b e e n a c h i e v e d (H o d g so n and Chubb,

1 9 8 3 ) .

- 1 5 -

1.1.6 Immunological study of ACHE

The a p p l i c a t i o n o f i m m u n o l o g i c a l t e c h n i q u e s t o t h e s t u d y o f AChE

h a s made p o s s i b l e a number o f a d v a n c e s i n t h e u n d e r s t a n d i n g o f t h e

e n z y m e . F i r s t l y , AChE d o e s n o t s h a r e common a n t i g e n i c d e t e r m i n a n t s w i t h

/ C h E , a s shown by many s t u d i e s , s u g g e s t i n g t h a t t h e tw o e n z y m e s a r e

l i k e l y t o be d i f f e r e n t g e n e p r o d u c t s ( B r i m i j o i n j e t _ a l _ . , 1 9 8 3 ; R o t u n d o ,

1 9 8 4 b ; M i n t z and B r i m i j o i n , 1 9 8 5 b ) . S e c o n d l y , a n t i b o d i e s t o p u r i f i e d r a t

b r a i n AChE b i n d a l l t h e m o l e c u l a r f o rm s w i t h s i m i l a r a f f i n i t i e s (Marsh

e t a l . , 1 9 8 4 ) . In c o n t r a s t , p r e f e r e n t i a l b i n d i n g o f p o l y c l o n a l

a n t i b o d i e s t o d e t e g e n t s o l u b l e , b u t n o t t o i n s o l u b l e AChE, h a s b e e n

o b s e r v e d i n r a t b r a i n ( Z a n e t t a e t a l . , 1 9 8 1 ) and human c a u d a t e n u c l e u s

( S o r e n s e n e t a l . , 1 9 8 2 ) . L i k e w i s e , D o c t o r e t a l . ( 1 9 8 3 ) r e p o r t e d

m o n o c l o n a l a n t i b o d i e s w h ic h a r e a b l e t o d i s t i n g u i s h b e t w e e n a s y m m e t r i c

and g l o b u l a r f o r m s . T h i r d l y , t h e ho m o lo g y b e t w e e n AChE from d i f f e r e n t

s p e c i e s c a n a l s o be s t u d i e d u s i n g m o n o c l o n a l a n t i b o d i e s ( B r i m i j o i n e t

a l . , 1 9 8 3 ; Fambrough e t a l . , 1 9 8 2 ; R o t u n d o , 1 9 8 4 b ; M in t z and B r i m i j o i n ,

1 9 8 5 b ) . T h e s e s t u d i e s show t h a t , i n g e n e r a l , l i t t l e c r o s s - r e a c t i v i t y i s

s e e n b e t w e e n AChE from d i f f e r e n t c l a s s e s o f a n i m a l s , w h i l e more

s u b s t a n t i a l c r o s s - r e a c t i v i t y o c c u r s among d i f f e r e n t o r d e r s o f t h e same

c l a s s . T h i s s u g g e s t s t h e l i k e l i h o o d o f e v o l u t i o n a r y d i s t i n c t i o n s b e t w e e n

t h e s e s p e c i e s o f AChE. F i n a l l y , by e x p l o i t i n g t h e c r o s s - r e a c t i v i t y o f

m o n o c l o n a l a n t i b o d i e s , i m m u n o a f f i n i t y p u r i f i c a t i o n o f r a b b i t b r a i n AChE

h a s b e e n a c h i e v e d u s i n g m o n o c l o n a l a n t i b o d i e s r a i s e d a g a i n s t p u r i f i e d

human e r y t h r o c y t e AChE ( M i n t z and B r i m i j o i n , 1 9 8 5 a ) .

1.2 Classification of skeletal muscle fibre types

S k e l e t a l m u s c l e i s a h e t e r o g e n e o u s t i s s u e due t o a) t h e

e x i s t e n c e o f a s p e c t r u m o f f i b r e s and b) t h e v a r i a b l e p a t t e r n o f t h e i r

a s s e m b l y . S e v e r a l t y p e s o f m u s c l e f i b r e s c a n be d i s t i n g u i s h e d on t h e

-16-

b a s i s o f t h e i r p h y s i o l o g i c a l , h i s t o c h e m i c a l , b i o c h e m i c a l and u l t r a -

s t r u c t u r a l p r o p e r t i e s (V r b o v a e t a l . , 1 9 7 8 ) . T h e s e m u s c l e f i b r e t y p e s

h a v e b e e n c l a s s i f i e d i n t o a number o f c a t e g o r i e s i n mammalian m u s c l e s

( B r o o k e and K a i s e r , 1 9 7 0 ; B a r n a r d e t a l . , 1 9 7 0 ) . A c l a s s i f i c a t i o n s y s t e m

r e l a t e d t o t h a t f o r t h e mammalian s y s t e m h a s a l s o b e e n d e s c r i b e d f o r

a v i a n m u s c l e s (Ashmore and D o e r r , 1 9 7 1 ; B a r n a r d e t a l . , 1 9 8 2 ) .

1.2.1 Physiological characterisation

S k e l e t a l m u s c l e s c a n b e d i v i d e d i n t o two t y p e s , v i z . t w i t c h and

t o n i c m u s c l e s on t h e b a s i s o f t h e i r c o n t r a c t i l e p r o p e r t i e s . T w i t c h

m u s c l e s a r e com p osed o f f a s t - and s l o w - t w i t c h f i b r e s . A c h a r a c t e r i s t i c s

o f t h e s e f i b r e s i s t h e i r a b i l i t y t o p r o p a g a t e a c t i o n p o t e n t i a l s and t o

r e s p o n d w i t h an a l l - o r - n o n e t w i t c h . Both t y p e s o f f i b r e s a r e i n n e r v a t e d

by an en p l a q u e t y p e o f n e r v e e n d i n g s . Th ey c a n be d i s t i n g u i s h e d h o w e v e r

by t h e i r c o n t r a c t i o n - r e l a x a t i o n and f a t i g u a b i 1 i t y c h a r a c t e r i s t i c s ( B u r k e

e t a l . , 1 9 7 1 ; C l o s e , 1 9 7 2 ) .

T o n i c m u s c l e s c o n t a i n f i b r e s w h ic h a r e m u l t i p i e l y i n n e r v a t e d

w i t h en g r a p p e t y p e o f n e r v e e n d i n g s . They r e s p o n d t o s u s t a i n e d s t i m u l i

w i t h s l o w , t o n i c c o n t r a c t i o n and u s u a l l y do n o t e x h i b i t an a c t i o n

p o t e n t i a l . T o n i c f i b r e s a r e r a r e i n mammals, and ca n o n l y b e f o u n d i n

s p e c i a l i s e d m u s c l e s s u c h a s t h e e x t r a o c u l a r and o e s o p h a g e a l m u s c l e s

( Y e l l i n , 1 9 6 9 ; H e s s , 1 9 7 0 ) .

T h e r e a r e a l s o c o n s i d e r a b l e d i f f e r e n c e s i n t h e c h a r a c t e r i s t i c s

o f n e u r o m u s c u l a r t r a n s m i s s i o n b e t w e e n d i f f e r e n t t y p e s o f f i b r e s

( M a g a z a n i k , 1 9 7 9 ; 1 9 8 4 ; McArdle and A l b u q u e r q u e , 1 9 7 3 ) . In mammals ,

f a s t - t w i t c h m u s c l e f i b r e s r e c e i v e a h i g h e r f r e q u e n c y o f m o t o n e u r o n

d i s c h a r g e ( E c c l e s e t a l . , 1 9 5 8 ) , show a h i g h e r f r e q u e n c y o f e n d p l a t e

p o t e n t i a l (M cA rdle and A l b u q u e r q u e , 1 9 7 3 ) and h a v e a h i g h e r q u a n t a l

c o n t e n t p e r e n d p l a t e p o t e n t i a l ( T o n g e , 1 9 7 4 ) t h a n t h o s e o f t h e s l o w -

- 1 7 -

t w i t c h f i b r e s . F u r t h e r m o r e , a c e t y l c h o l i n e (ACh) s e n s i t i v i t y c a n be

d e t e c t e d a l o n g t h e e n t i r e l e n g t h o f t h e s l o w - t w i t c h f i b r e s ( M i l e d i and

Z e l e n a , 1 9 6 6 ) w h i l e s e n s i t i v i t y t o ACh i s r e s t r i c t e d t o t h e e n d p l a t e

r e g i o n i n t h e f a s t - t w i t c h f i b r e s . The s l o w - t w i t c h m u s c l e s may a l s o

c o n t a i n l e s s ACh r e c e p t o r s ( S t e r z e t a l . , 1 9 8 3 ) and AChE ( M a g a z a n ik e t

a l , , 1 9 8 4 ) a t t h e e n d p l a t e r e g i o n .

1.2.2 Histochemical and biochemical characterisation

The h i s t o c h e m i c a l t e c h n i q u e s u s e d t o d i s t i n g u i s h t h e t y p e s o f

m u s c l e f i b r e s a r e b a s e d on t h e i d e n t i f i c a t i o n o f t h e m e t a b o l i c and

c o n t r a c t i l e e l e m e n t s o f m u s c l e f i b r e s ( D u b o w i t z and B r o o k , 1 9 7 3 ) .

A n a e r o b i c f i b r e s a r e c h a r a c t e r i s e d by a h i g h l e v e l o f enzyme m a r k e r s f o r

g l y c o l y t i c m e t a b o l i s m , s u c h a s p h o s p h o r y l a s e , l a c t a t e d e h y d r o g e n a s e and

a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e . The g l y c o g e n c o n t e n t o f t h e s e f i b r e s

a r e a l s o more a b u n d a n t . T h e i r a e r o b i c c a p a c i t y , on t h e o t h e r h a n d , i s

v e r y l i m i t e d a s i l l u s t r a t e d by lo w s u c c i n a t e d e h y d r o g e n a s e a s w e l l a s

lo w c y t o c h r o m e and m y o g l o b u l i n c o n c e n t r a t i o n s . A e r o b i c f i b r e s , i n

c o n t r a s t , h a v e a h i g h l e v e l o f e n zy m es f o r o x i d a t i v e m e t a b o l i s m and a

l o w l e v e l o f e n zy m es f o r g l y c o l y s i s .

I t was f o u n d t h a t t h e s p e e d o f c o n t r a c t i o n o f s k e l e t a l m u s c l e s

c o r r e l a t e d w i t h t h e a d e n o s i n e t r i p h o s p h a t a s e (A T P a se ) a c t i v i t y ( B a r a n y ,

1 9 6 7 ) and i t s s t a b i l i t y i n d i f f e r e n t pH m e d ia (Baran.y e t a l . , 1 9 6 5 ) . The

s p e e d o f s h o r t e n i n g , t h u s , a p p e a r s t o be an i n h e r e n t p r o p e r t y o f t h e

m y o f i b r i l s , b e i n g c l o s e l y r e l a t e d t o m y o s i n ATPase a c t i v i t y ( B a r a n y and

C l o s e , 1 9 7 1 ) . F a s t - t w i t c h m u s c l e s show h i g h e r m y o s i n ATPase a c t i v i t y

t h a n t h e s l o w - t w i t c h m u s c l e s ( B a r a n y , 1 9 6 7 ) . Th at t h e ATPase a c t i v i t y

may r e f l e c t d i f f e r e n t t y p e s o f c o n t r a c t i l e e l e m e n t i s d e m o n s t r a t e d by

t h e f i n d i n g o f a n t i g e n i c a l l y d i s t i n c t t y p e s o f m y o s i n s i n d i f f e r e n t

t y p e s o f f i b r e s ( A r n d t and P e p e , 1 9 7 5 ; S a r t o r e e t a l . , 1 9 7 8 ) . The

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c u r r e n t c l a s s i f i c a t i o n o f f i b r e t y p e s , w h ic h t a k e s i n t o a c c o u n t t h e

v a r i o u s h i s t o c h e m i c a l and b i o c h e m i c a l e v i d e n c e , d i v i d e s m u s c l e f i b r e s

i n t o 4 b a s i c t y p e s : a) s l o w - t w i t c h - o x i d a t i v e ( t y p e I ) ; b) f a s t - t w i t c h -

o x i d a t i v e - g l y c o l y t i c ( t y p e 1 1 A ) ; c ) f a s t - t w i t c h - g l y c o l y t i c ( t y p e I I B ) ;

d) t o n i c ( o x i d a t i v e - g l y c o l y t i c ; t y p e I I I A and 11 I B ) .

1.2.3 Morphological and ultrastructural characterisation

F a s t - t w i t c h f i b r e s a r e c h a r a c t e r i s e d by a w e l l - d e v e l o p e d

s a r c o t u b u l a r s y s t e m w h i l e t h a t i n s l o w - t w i t c h and t o n i c f i b r e s a r e

r e l a t i v e l y l e s s w e l l d e v e l o p e d ( H e s s , 1 9 6 7 ; P e a c h e y and H u x l e y , 1 9 6 2 ) .

T w i t c h f i b r e s p o s s e s s s t r a i g h t Z d i s k s w h e r e a s t h a t i n s l o w t o n i c f i b r e s

a r e t h i c k and j a g g e d . S l o w - t w i t c h and t o n i c f i b r e s c o n t a i n a l a r g e r

number o f m i t o c h o n d r i a , l i p i d g l o b u l e s and a r e more v a s c u l a r i s e d t h a n

f a s t - t w i t c h f i b r e s .

A n o t h e r d i f f e r e n c e b e t w e e n t h e t w i t c h and t o n i c f i b r e s i s t h e i r

m o t o r e n d p l a t e m o r p h o l o g y . In mammalian m u s c l e s , t h e n e r v e e n d i n g s i n

t h e f a s t - t w i t c h f i b r e s c o v e r a l a r g e r e n d p l a t e r e g i o n and c o n t a i n more

p r e s y n a p t i c v e s i c l e s t h a n t h e s l o w - t w i t c h f i b r e s ( P a d y k u l a and G a u t h i e r ,

1 9 7 0 ; E l l i s m a n _ e t _ a l _ . , 1 9 7 6 ) . Such d i s t i n c t i o n , h o w e v e r , i s n o t a s c l e a r

b e t w e e n t h e t w i t c h and t o n i c f i b r e s i n t h e a v i a n m u s c l e s ( J . C a v a n a g h ,

p e r s o n a l c o m m u n i c a t i o n ) .

1 . 2 . 4 Fibre type composition of muscles

The f o u r t y p e s o f f i b r e s c a n be f o u n d i n v a r i o u s s p e c i e s o f

v e r t e b r a t e s ; h o w e v e r , any g i v e n s p e c i e s o f anim al d o e s n o t n e c e s s a r i l y

p o s s e s s a l l t h e s e t y p e s o f m u s c l e f i b r e s . Q u a n t i t a t i v e d i f f e r e n c e i n t h e

h i s t o c h e m i c a l s t a i n i n g c h a r a c t e r i s t i c s may a l s o o c c u r b e t w e e n f i b r e s o f

d i f f e r e n t s p e c i e s ( G o l d s p i n k and Ward, 1 9 7 9 ; P e t e r e t a l . , 1 9 7 2 ) .

G e n e r a l l y , i n d i v i d u a l m u s c l e c o n t a i n s e i t h e r a p u re p o p u l a t i o n o f o n e

- 1 9 -

t y p e o f f i b r e s o r a m ix e d p o p u l a t i o n o f v a r y i n g p r o p o r t i o n o f s e v e r a l

t y p e s o f f i b r e s ( A r i a n o e t a l . , 1 9 7 3 ) . The f i b r e t y p e c o m p o s i t o n o f a

m u s c l e a p p e a r s t o b e c o n t r o l l e d by t h e p a r t i c u l a r t y p e o f m u s c l e

a c t i v i t y . A good e x a m p l e i s t h e f l i g h t m u s c l e s o f b i r d s . The p e c t o r a l i s

i n n o n - f l y i n g b i r d s s u c h a s t h e c h i c k e n i s w h i t e o r p u r e l y f a s t - t w i t c h -

g l y c o l y t i c , w h i l e t h a t i n t h e f l y i n g b i r d s u c h a s t h e p i g e o n i s r e d o r

f a s t - t w i t c h - o x i d a t i v e (K ha n , 1 9 8 0 ) . The m e t a b o l i c p a t t e r n s a r e r e f l e c t e d

i n t h e number o f m i t o c h o n d r i a and i n t h e c o n t e n t o f g l y c o g e n and l i p i d .

On t h e o t h e r h a n d , t h e c o n t r a c t i l e p r o p e r t i e s a r e c o r r e l a t e d w i t h t h e

s i z e and s h a p e o f t h e m y o f i b r i l s , t h e t h i c k n e s s o f t h e Z d i s k s and t h e

d e v e l o p m e n t o f t h e s a r c o t u b u l a r s y s t e m ( I s h i k a w a , 1 9 8 3 ) .

F i b r e t y p e i s a l s o r e l a t e d t o t h e n a t u r e o f t h e i n n e r v a t i o n . A l l

m u s c l e f i b r e s w i t h i n a m o to r u n i t a r e o f t h e same f i b r e t y p e (B u r k e _et

a l . , 1 9 7 1 ) . C r o s s - i n n e r v a t i o n e x p e r i m e n t s wh er e t h e n e r v e s t o t h e

mammalian f a s t - t w i t c h and s l o w - t w i t c h m u s c l e s a r e i n t e r c h a n g e d , t h e

m u s c l e t y p e s a l s o u n d e r g o an i n t e r c h a n g e ( B u l l e r e t a l . , 1 9 6 0 ; M e l i d i

and S t e f a n i , 1 9 6 9 ; E i s e n b e r g and S a l m o n s , 1 9 8 1 ) . H o w e v e r , i n a v i a n

m u s c l e s , t o n i c and f a s t - t w i t c h m u s c l e s a r e n o t a f f e c t e d by c r o s s

r e i n n e r v a t i o n ( Hni k e t a l . , 1 9 6 7 ) e x c e p t i n n e w ly h a t c h e d c h i c k s ( Z e l e n a

and J i r m a n o v a , 1 9 7 3 ; J i rmanova e t a l . , 1 9 7 1 ) .

The p l a s t i c i t y o f m u s c l e f i b r e s i s a l s o d e m o n s t r a t e d by t h e

e f f e c t o f i n d u c e d a c t i v i t y on m u s c l e f i b r e t y p e c o m p o s i t i o n , w h e t h e r by

c h r o n i c e l e c t r i c a l s t i m u l a t i o n ( S a lm o n s and V r b o v a , 1 9 6 9 ; P e t t e , 1 9 8 0 ;

P e t t e e t a l . , 1 9 7 6 ) o r i n t e n s i v e e x e r c i s e t r a i n i n g (G r e e n e t a l . , 1 9 8 4 ) .

H ence t h e f i b r e t y p e c o m p o s i t i o n o f a m u s c l e i s a d y n a m ic s y s t e m a d a p t e d

t o f u n c t i o n a l d em a n d s . In c o n c l u s i o n , a l t h o u g h t h e t y p i n g o f f i b r e s

p r e s e n t s a somewhat p a r s i m o n i o u s v i e w o f t h e d yn am ic p r o p e r t i e s o f

m u s c l e s , some p r a c t i c a l c l a s s i f i c a t i o n h a v e n e v e r t h e l e s s b e e n p r o v e d

h e l p f u l i n t h e u n d e r s t a n d i n g o f t h e f u n c t i o n a l s i g n i f i c a n c e and t h e

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p a t h o l o g y o f m u s c l e s ( P i z z e y and B a r n a r d , 1 9 8 3 a ; 1 9 8 3 b ; and f o r r e v i e w s

s e e S a lm o n s and H e n r i k s s o n , 1 9 8 1 ; P e t t e , 1 9 8 4 ; B r o o k e and K a i s e r , 1 9 7 4 ) .

1.3 Skeletal muscle AChE in health and disease

1.3.1 Biosynthesis of AChE in skeletal muscles

The m u l t i p l e m o l e c u l a r f o r m s o f AChE i s t h o u g h t t o be

s y n t h e s i z e d i n a s e q u e n t i a l manner by t h e p o s t - t r a n s l a t i o n a l p r o c e s s i n g

and a s s e m b l y o f m u l t i p l e number o f a b a s i c c a t a l y t i c u n i t ( M a s s o u l i e and

B o n , 1 9 8 2 ; B r i m i j o i n , 1 9 8 3 ) . The c o n c l u s i o n t h a t t h e s e c a t a l y t i c

s u b u n i t s a r e s i m i l a r i s b a s e d on k i n e t i c and i m m u n o l o g i c a l o b s e r v a t i o n s .

The k i n e t i c p r o p e r t i e s o f t h e v a r i o u s p u r i f i e d f o r m s o f AChE from

E l e c t r o p h o r u s and T o r p e d o e l e c t r i c o r g a n t i s s u e w e r e f o u n d t o be

s i m i l a r . I m m u n o l o g i c a l i d e n t i f i c a t i o n u s i n g m o n o c l o n a l o r p o l y c l o n a l

a n t i b o d i e s a l s o sh ows t h a t t h e v a r i o u s m o l e c u l a r f o r m s s h a r e

a n t i g e n i c a l l y i d e n t i c a l s u b u n i t s (Marsh e t a l . , 1 9 8 4 ; R o t u n d o , 1 9 8 4 ) .

E l e c t r o p h o r e t i c a n a l y s i s c a r r i e d o u t by W i l s o n and W a lk e r ( 1 9 7 4 )

sh ow ed t h a t t h e s y n t h e s i s o f AChE a f t e r i n h i b i t i o n by o r g a n o p h o s p h a t e

a p p e a r e d f i r s t a s lo w m o l e c u l a r w e i g h t f o r m s , and s u g g e s t e d t h a t t h e s e

l o w m o l e c u l a r w e i g h t fo rm s w e r e p r e c u r s o r s o f t h e h i g h m o l e c u l a r w e i g h t

f o r m s . T h i s was f u r t h e r s u p p o r t e d by t h e r e c o v e r y o f AChE i n

n e u r o b l a s t o m a c e l l c u l t u r e s a f t e r i r r e v e r s i b l e i n h i b i t i o n o f AChE, w h er e

G| ( l_ i ) form a p p e a r s b e f o r e t h e (M) form ( R i e g e r e t a l . , 1 9 7 6 ) . The

r e c o v e r y o f t h e lo w m o l e c u l a r w e i g h t f o r m s p r i o r t o t h e h i g h m o l e c u l a r

w e i g h t f o rm s i s s i m i l a r l y o b s e r v e d i n r a t m u s c l e c u l t u r e ( K o e n i g and

V i g n y , 1 9 7 8 ) , r a t s u p e r i o r c e r v i c a l g a n g l i a ( G i s i g e r and V i g n y , 1 9 7 7 )

and r a t d ia p h r a g m ( S k e l e t j e t a l . , 1 9 8 0 ) .

The m e t a b o l i c r e l a t i o n s h i p b e t w e e n t h e v a r i o u s f o rm s o f AChE h a s

b e e n i n v e s t i g a t e d by m e a s u r i n g t h e t u r n o v e r r a t e o f t h e s e f o r m s . In

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c h i c k m u s c l e c u l t u r e s , i t was f o u n d t h a t t h e AChE m o l e c u l e s w h ic h bound

t o p la s m a membrane ( 7 S and 1 I S ) h a v e h a l f - l i v e s o f a b o u t 50 h o u r s , w h i l e

t h e i n t r a c e l l u l a r po o l o f AChE ( 7 S ) t u r n o v e r more r a p i d l y (R o t u n d o and

Fam broug h, 1 9 8 0 a ) . Such f i n d i n g h a s b e e n c o n f i r m e d by h e a v y i s o t o p e

l a b e l l i n g o f t h e m o l e c u l a r f o r m s i n n e u r a l t i s s u e c e l l l i n e ( L a z a r e t

a l . , 1 9 8 4 ) . The f a c t t h a t t h e membrane bound f o r m s a p p e a r w i t h a 2 - 3

h o u r l a g a f t e r t h e f i r s t a p p e a r a n c e o f t h e i n t r a c e l l u l a r form s u g g e s t s

t h a t t h e membrane bound form i s s y n t h e s i z e d by t h e p r o c e s s i n g o f t h e

i n t r a c e l l u l a r f o rm s ( I n e s t r o s a e t a l . , 1 9 8 5 ) . The s e q u e n c e o f e v e n t s

i n v o l v e d i n t h e p r o c e s s i n g o f t h i s i n t r a c e l l u l a r p o o l o f enzyme h a s b e e n

s t u d i e d i n q u a i l m u s c l e c u l t u r e b a s e d upon t h e c o t r a n s l a t i o n a l

g l y c o s y l a t i o n o f t h e enzym e m o l e c u l e s ( R o t u n d o , 1 9 8 4 a ) . I t was f o u n d

t h a t t h e a s s e m b l y o f t h e m ono m er ic u n i t s i n t o IS and 1 IS fo rm s o c c u r s i n

t h e roug h e n d o p l a s m i c r e t i c u l u m ( RER) , w h i l e t h e c o l l a g e n - t a i l e d f o r m s

a r e p r o b a b l y a s s e m b l e d from t h e 11S f o rm s and t h e c o l l a g e n t a i l i n t h e

G o l g i a p p a r a t u s .

A l t h o u g h t h e p r e c u s o r - p r o d u c t r e l a t i o n s h i p s b e t w e e n t h e g l o b u l a r

and c o l l a g e n - t a i l e d f o rm s o f AChE i s s u p p o r t e d by t h e e v i d e n c e d e s c r i b e d

a b o v e , su c h h y p o t h e s i s d o e s n o t a c c o u n t f o r t h e p o s s i b l e e x i s t e n c e o f

d i f f e r e n t p o o l s o f e n zy m es w h ic h may be r e g u l a t e d s e p a r a t e l y . P r i m a r y

c u l t u r e s o f b o t h m y o tu b e s ( W i l s o n e t a l . , 1 9 7 3 ; B u l g e r e t a l . , 1 9 8 2 ;

R otu n d o and Fam brough, 1 9 7 9 ; W a lk e r and W i l s o n ; C i s s o n and W i l s o n , 1 9 7 7 )

and n e r v e c e l l s ( L a z a r and V i g n y , 1 9 8 0 ; G i s i g e r e t a l . , 1 9 7 7 ) a c t i v e l y

s e c r e t e AChE i n t o t h e medium from a r a p i d l y t u r n o v e r p o o l o f

i n t r a c e l l u l a r enzyme ( R o t u n d o and Fambrough, 1 9 8 0 b ) w h ic h d o e s n o t

i n v o l v e any t r a n s i e n t a s s o c i a t i o n w i t h t h e p la sm a membrane . The s e c r e t e d

and membrance bound AChE, t h e r e f o r e , may r e p r e s e n t tw o d i s t i n c t p o o l s o f

e n z y m e s . F u r t h e r m o r e , l e c t i n b i n d i n g s t u d i e s a l s o s u g g e s t t h a t n e w l y

s y n t h e s i z e d AChE i s n o t n e c e s s a r i l y e n z y m a t i c a l l y a c t i v e ( R o t u n d o ,

1 9 8 4 a ) .

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Some i m m u n o l o g i c a l e v i d e n c e s u g g e s t s t h a t t h e membrane b o u n d ,

d i m e r i c AChE fo rm from T o r p e d o e l e c t r i c o r g a n i s a n t i g e n i c a l l y d i s t i n c t

from t h e s o l u b l e o r a s y m m e t r i c f o rm s ( D o c t o r e t a l . , 1 9 8 3 ) . T o r p e d o AChE

i s a l s o d i f f e r e n t i n t h a t d u r i n g e m b r y o n ic d e v e l o p m e n t , t h e c o l l a g e n

t a i l e d form a p p e a r s e a r l i e r t h a n t h e d i m e r i c form ( B o n , 1 9 8 2 ) .

F u r t h e r m o r e , t h e a s s e m b l y o f t h e c o l l a g e n - t a i l e d form may i n v o l v e non -

c a t a l y t i c s u b u n i t s ( L e e and T a y l o r , 1 9 8 2 ) . T h e s e e v i d e n c e a g a i n s u g g e s t s

t h a t t h e v a r i o u s m o l e c u l a r f o rm s may n o t o r i g i n a t e from a common p o o l o f

' p r e c u r s o r ' u n i t s .

1.3.2 Neurotrophic regulation of AChE

T h e r e i s now a g r e a t d e a l o f e v i d e n c e t o show t h a t AChE o f

s k e l e t a l m u s c l e i s c o n t r o l l e d by t h e m o t o r i n n e r v a t i o n . N e r v e - m u s c l e

c o n t a c t i n v i v o o r i n v i t r o i s a c c o m p a n ie d by a l a r g e i n c r e a s e i n m u s c l e

AChE c o n t e n t i n t h e r a t ( V i g n y e t a l . , 1 9 7 6 ) o r c h i c k ( T o u t a n t e t _ a l _ . ,

1 9 8 3 ) , and s u b s e q u e n t l y f o c a l a c c u m u l a t i o n o f t h e enzyme a p p e a r s

( H a r r i s , 1 9 8 1 ; Z i s k i n d - C o n h a i m e t a l . , 1 9 8 4 ) . A f t e r d e n e r v a t i o n , t h e

e n d p l a t e - a s s o c i a t e d AChE d e c l i n e s i n b o t h mammalian ( H a l l , 1 9 7 3 ; C o l l i n s

and Y o u n k i n , 1 9 8 2 ) and a v i a n m u s c l e s ( S k e t e l j e t a l . , 1 9 7 8 ; S i l m a n e t

a l . , 1 9 7 8 ) . S i m i l a r l y , b l o c k a d e o f n e u r a l t r a n s m i s s i o n by f l a x e d i l

p r e v e n t e d e n d p l a t e AChE a p p e a r i n g i n c h i c k embryo m u s c l e ( B e t z e t a l . ,

1 9 8 0 ) . Such l o s s o f e n d p l a t e AChE c a n b e r e v e r s e d e i t h e r by

r e i n n e r v a t i o n (M al d on ad o e t a l . , 1 9 8 4 ; V i g n y e t a l . , 1 9 7 6 ) o r by d i r e c t

e l e c t r i c a l s t i m u l a t i o n o f t h e m u s c l e c e l l c u l t u r e (L0mo and S l a t e r ,

1 9 8 0 ; W a lk e r and W i l s o n , 1 9 7 5 ; 1 9 7 6 ) . N e r v e - m u s c l e c o - c u l t u r e i n w h ic h

s y n a p t i c t r a n s m i s s i o n i s b l o c k e d by c u r a r e c a n a l s o be r e v e r s e d by

s t i m u l a t i o n ( R u b in e t a l . , 1 9 8 0 ) . M u s c le a c t i v i t y t h u s a p p e a r s t o be

e s s e n t i a l f o r t h e s t a b i l i s a t i o n o f e n d p l a t e AChE. The f o r m a t i o n o f

j u n c t i o n a l ACh r e c e p t o r s and AChE c a n be i n d u c e d by n e u r a l s i g n a l a t t h e

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e a r l i e s t s t a g e s o f j u n c t i o n a l d e v e l o p m e n t , b e f o r e c h a n n e l c o n v e r s i o n

o c c u r s ( B r e n n e r e t a l . , 1 9 8 3 ) . S u b s e q u e n t d e v e l o p m e n t o f t h e j u n c t i o n a l

c h a r a c t e r i s t i c s ca n be m a i n t a i n e d by d i r e c t e l e c t r i c a l s t i m u l a t i o n and

d o e s n o t r e q u i r e t h e p r e s e n c e o f t h e n e r v e . I t i s t h o u g h t t h a t t h e s e

s y n a p t i c s p e c i a l i s a t i o n i s e x e r t e d t h r o u g h t h e m o d i f i c a t i o n o f t h e b a s a l

l a m i n a a t t h e s i t e o f t h e n e r v e - m u s c l e c o n t a c t (W e i n b e r g and H a l l , 1 9 7 9 ;

Lomo and S l a t e r , 1 9 8 0 ; S a n e s e t a l . , 1 9 7 8 ) .

T h e r e i s a l s o e v i d e n c e t o s u g g e s t t h a t some n e u r o t r o p h i c f a c t o r s

may be r e s p o n s i b l e f o r t h e a c t i v i t y - i n d e p e n d e n t r e g u l a t i o n o f t h e n e r v e .

Thus b l o c k a d e o f a x o n a l t r a n s p o r t by c o l c h i c i n e r e s u l t s i n an i n c r e a s e d

r e l e a s e o f e n d p l a t e AChE s i m i l a r t o d e n e r v a t i o n ( I n e s t r o s a e t a l . ,

1 9 7 7 ) , w h e r e a s b o t u l i n u m t o x i n b l o c k a d e o f n e u r o t r a n s m i s s i o n , and t h u s

m u s c l e a c t i v i t y , i s n o t a s e f f e c t i v e a s d e n e r v a t i o n i n d e p l e t i n g

e n d p l a t e AChE (D rachman , 1 9 7 2 ) . F u r t h e r m o r e , n e r v e e x p l a n t s o r t i s s u e

e x t r a c t s h a v e b e e n shown t o i n d u c e ACh r e c e p t o r c l u s t e r i n g i n c l o n e d

m u s c l e c e l l s ( P o d l e s k i e t a l . , 1 9 7 8 ) , i n c r e a s e AChE a c t i v i t y i n newt

o r g a n c u l t u r e ( L e n t z , 1 9 7 4 ) and i n c r e a s e e n d p l a t e AChE i n d e n e r v a t e d r a t

m u s c l e o r g a n c u l t u r e ( D a v e y e t a l . , 1 9 7 9 ; Youn kin e t a l . , 1 9 7 8 ) and

m y o t u b e s ( S a n e s e t a l . , 1 9 8 4 ; L e n t z e t a l . , 1 9 8 1 ) . N e r v e e x t r a c t s a l s o

r e v e r s e t h e d e n e r v a t i o n - i n d u c e d r e d u c t i o n o f AChE i n t h e s u p e r i o r

c e r v i c a l g a n g l i o n o f t h e c a t ( K o e l l e and R u ch , 1 9 8 3 ) . The r e c e n t

• i d e n t i f i c a t i o n o f an ACh r e c e p t o r a g g r e g a t i n g f a c t o r h a s a l s o b e e n shown

t o i n d u c e AChE c l u s t e r i n g ( W a l l a c e e t a l . , 1 9 8 5 ) . T h i s f a c t o r , w h ic h i s

s e c r e t e d by n e u r a l t i s s u e s a s w e l l a s s y n t h e s i z e d by m u s c l e s i s

c o n c e n t r a t e d i n t h e s y n a p t i c c l e f t ( F a l l o n e t a l . , 1 9 8 5 ) and may b e an

i m p o r t a n t f a c t o r i n d i r e c t i n g t h e f o c a l a c c u m u l a t i o n o f AChE d u r i n g

s y n a p t o g e n e s i s and s u b s e q u e n t p r e f e r e n t i a l l o c a l i s a t i o n a t t h e e n d p l a t e .

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1.3.3 Avian muscular dystrophy

M u s c u l a r d y s t r o p h y i n human i s a g e n e t i c a l l y i n h e r i t e d d i s o r d e r .

A number o f ani m al m o d e l s o f m u s c u l a r d y s t r o p h y h a v e b e e n u s e d f o r t h e

s t u d y o f t h i s d i s e a s e . The d y s t r o p h i c c o n d i t i o n i n c h i c k e n i s a

p r o g r e s s i v e a t r o p h y o f t h e f a s t - t w i t c h m u s c l e s , i n p a r t i c u l a r t h e

p e c t o r a l i s , t h e p o s t e r i o r l a t i s s i m u s d o r s i (PLD) and t h e w in g m u s c l e s .

The d i s e a s e i s t h o u g h t t o be o f m y o g e n i c o r i g i n s i n c e no a b n o r m a l i t i e s

c a n be d e t e c t e d i n t h e n e u r a l t r a n s m i s s i o n f u n c t i o n ( H o l l i d a y e t a l . ,

1965; W arnick e t a l . , 1979) a l t h o u g h t h e membrane o f d y s t r o p h i c f i b r e s

e x h i b i t abnormal f i b r i l l a t i o n and h y p e r e x c i t a b i l i t y t o m e c h a n i c a l

s t i m u l i , p o s s i b l y due t o an a l t e r a t i o n i n c a l c i u m c o n d u c t a n c e .

F a s t - t w i t c h f i b r e s s e v e r e l y a f f e c t e d by m u s c u l a r d y s t r o p h y

u n d e r g o r a p i d a t r o p h y . The f i b r e s become l a r g e , r o un de d and i r r e g u l a r .

At t h e same t i m e , s m a l l e r f i b r e s a l s o a p p e a r w h ic h may be a r i s e n from

t h e s p l i t t i n g o f l a r g e f i b r e s . The n u c l e i become more c e n t r a l l y

l o c a l i s e d and more a b u n d a n t , w h ic h i s r e f l e c t e d i n t h e i n c r e a s e o f DNA

c o n t e n t ( Y o s h i k a w a e t a l . , 1984). E x t e n s i v e c o n n e c t i v e and f a t t y t i s s u e

i n f i l t r a t i o n o c c u r s a s t h e d i s e a s e p r o g r e s s e s (Admundson and J u l i a n ,

1956). D e f e c t i v e membrane s t r u c t u r e and p e r m e a b i l i t y i s t h o u g h t t o be

t h e r e s u l t o f i n c r e a s e d l i p i d c o n t e n t i n t h e p la sm a membrane o f

d y s t r o p h i c f i b r e s (Rodan e t a l . , 1974). Such a b n o r m a l i t i e s i n t h e

membrane s t r u c t u r e may l e a d t o t h e l e a k a g e o f m y o f i b r i l l a r p r o t e i n s and

f a u l t y c a l c i u m c o n d u c t a n c e .

A number o f e n z y m e s i n t h e b l o o d h a v e b e e n e m p lo y e d a s m a r k e r s

f o r m o n i t o r i n g t h e p r o g r e s s i o n o f d y s t r o p h y . Abnormal i n c r e a s e i n t h e

l e v e l o f p la sm a a l d o l a s e and g l u t a m i c o x a l i c t r a n s f e r a s e w e r e n o t e d

( C o r n e l i u s e t a l . , 1959). C r e a t i n e k i n a s e and AChE a r e a l s o f o u n d t o be

e l e v a t e d ( Wi l s o n e t a l . , 1973). M u s c le AChE l e v e l was a l s o i n c r e a s e d

w h ic h ca n be s e e n i n m u s c l e a q u e o u s e x t r a c t o f d y s t r o p h i c p e c t o r a l i s a s

-25-

e a r l y a s 5 d a y s e x ov o ( Wi l s o n e t a l . , 1 9 6 9 ) . D e v e l o p m e n t a l s t u d y o f

AChE f o r m s i n normal and d y s t r o p h i c f a s t - t w i t c h m u s c l e s o f c h i c k e n

d y s t r o p h i c l i n e 4 1 3 ( D a v i s , C a l i f o r n i a ) sh ow s t h a t abnormal i n c r e a s e s i n

t h e AChE l e v e l b e g i n a s e a r l y a s 4 d a y s e x ov o and m a i n l y i n v o l v e s t h e

o v e r p r o d u c t i o n o f t h e 7S f o r m . The 20S form i s a l s o i n c r e a s e d m a n y - f o l d

( L y l e s e t a l . , 1 9 7 9 ) when comp ared w i t h normal m u s c l e s o f t h e same a g e

g r o u p . Such d i s t r i b u t i o n r e s e m b l e s t h e e m b r y o n ic d i s t r i b u t i o n o f AChE

f o r m s . In a d d i t i o n , t h e s e d y s t r o p h i c f a s t - t w i t c h f i b r e s r e t a i n a lo w

l e v e l o f l a c t a t e d e h y d r o g e n a s e (LDH) and p h o s p h o r y l a s e , and a r e

c o m p a r a t i v e l y more o x i d a t i v e t h a n t h e normal f i b r e s (A sh m ore and D o e r r ,

1 9 7 1 ) . The l e v e l s o f t h e s e enz ym es a r e s i m i l a r t o t h a t i n im m atu re f a s t -

t w i t c h m u s c l e s , and h e n c e l e a d s t o t h e p r o p o s a l t h a t d y s t r o p h y i s du e t o

a d e r e g u l a t i o n o f f i b r e m a t u r a t i o n ( W i l s o n e t a l . , 1 9 7 9 ; K a p la n and

Ch an , 1 9 6 2 ; Cosmos e t a l . , 1 9 7 9 ) .

I n c r e a s e i n AChE l e v e l s i s a l s o o b s e r v e d i n d e n e r v a t e d c h i c k e n

m u s c l e s . H ow e v e r , i n c r e a s e d AChE l e v e l due t o d y s t r o p h y i s d i f f e r e n t

from d e n e r v a t i o n i n d u c e d i n c r e a s e i n AChE s i n c e t h e H2 C fo rm i s a l s o

e l e v a t e d i n d y s t r o p h i c m u s c l e b u t d i s a p p e a r s i n d e n e r v a t e d m u s c l e .

F u r t h e r m o r e , d y s t r o p h i c m u s c l e s a r e f u n c t i o n a l l y i n n e r v a t e d ; i f a

d y s t r o p h i c m u s c l e i s d e p r i v e d o f i t s n e r v e , t h e AChE l e v e l i n c r e a s e s and

H2 C l e v e l d i s a p p e a r s s i m i l a r t o d e n e r v a t e d normal m u s c l e . H en ce

• d y t r o p h i c m u s c l e s a r e u n d er n e u r a l i n f l u e n c e .

In summary, a number o f s t a b l e , d i s t i n c t i v e m o l e c u l a r f o r m s o f

AChE a r e u b i q u i t o u s i n a v a r i e t y o f v e r t e b r a t e t i s s u e s . The e l u c i d a t i o n

o f t h e s t r u c t u r e s o f t h e s e f o rm s f u r t h e r d e m o n s t r a t e s t h e c o m p l e x

h e t e r o g e n e i t y o f t h e s e m o l e c u l e s . The l o c a l i s a t i o n o f t h e s e m o l e c u l e s ,

i n p a r t i c u l a r t h e d i m e r i c form i n e r t h r o c y t e membranes and t h e c o l l a g e n

t a i l e d f o rm s i n f i s h e l e c t r i c o r g a n , s u g g e s t s t h a t t h e

-26-

c o m p a r t m e n t a l i s a t i o n o f t h e s e f o r m s i s i n h e r e n t i n t h e i r m o l e c u l a r

s t r u c t u r e s . In p u r e s y s t e m s su c h a s t h e two m e n t i o n e d a b o v e , t h i s

r e l a t i o n s h i p se em s i n d i s p u t a b l e . H o w e v e r , t h e m o l e c u l a r form s o f AChE

p r e s e n t i n n e u r a l t i s s u e s and m u s c l e s a r e more c o m p l e x and s u c h

s t r u c t u r e - l o c a l i s a t i o n r e l a t i o n s h i p i s d i f f i c u l t t o d e f i n e .

N e v e r t h e l e s s , t h e l o c a l i s a t i o n o f t h e s e f o rm s h a s g e n e r a l l y b e e n

c o n s i d e r e d t o r e f l e c t , a t l e a s t i n w e l l d e f i n e d s y s t e m s s u c h a s t h e

n e u r o m u s c u l a r j u n c t i o n o f m u s c l e s and n e r v e s y n a p s e s , t h e i r r e s p e c t i v e

f u n c t i o n s . In p a r t i c u l a r , t h e r e l a t i o n s h i p b e t w e e n t h e l o c a l i s a t i o n and

f u n c t i o n o f t h e H2C AChE form i n t h e n e u r o m u s c u l a r j u n c t i o n h as o f t e n

b e e n u s e d a s a m ark er i n d e v e l o p m e n t a l s t u d i e s . H o w e v e r , s i n c e v a r i o u s

e v i d e n c e s u g g e s t s t h a t t h i s form i s n o t e x c l u s i v e t o t h e n e u r o m u s c u l a r

j u n c t i o n no r d o e s i t seem t o be t h e o n l y form i n t h e e n d p l a t e , f u r t h e r

work i s e s s e n t i a l t o c l a r i f y t h e l o c a l i s a t i o n o f t h i s form and i t s r o l e

a s f u n c t i o n a l m a r k e r .

This thesis

I t i s t h e aim o f t h e p r e s e n t work t o e s t a b l i s h more p r e c i s e l y

t h e l o c a l i s a t i o n o f t h e AChE f o rm s and t h e i r r e g u l a t i o n i n s k e l e t a l

m u s c l e s . I t was d e c i d e d t o f o c u s t h e s t u d y on s k e l e t a l m u s c l e s b e c a u s e

o f t h e i r f u n c t i o n a l d i v e r s i t y and t h e i r a m e n a b i l i t y t o f u n c t i o n a l

• m a n i p u l a t i o n by p r o c e d u r e s su c h as d e n e r v a t i o n and a r t i f i c i a l

s t i m u l a t i o n . In a d d i t i o n , a t t e m p t s w e r e made t o s t u d y t h e e f f e c t s o f

m u s c l e a c t i v i t y on t h e abnormal d i s t r i b u t i o n o f AChE f o r m s i n d y s t r o p h i c

a v i a n m u s c l e s i n o r d e r t o g a i n f u r t h e r i n s i g h t i n t o t h e normal

r e g u l a t i o n o f AChE. C h a p t e r 2 d e t a i l s t h e e x p e r i m e n t a l c o n d i t i o n s , n o t

p r e v i o u s l y w e l l - d e f i n e d , w h ich a r e c r i t i c a l f o r t h e r e p r o d u c i b l e i n

v i t r o a n a l y s i s o f AChE m o l e c u l a r f o r m s i n s k e l e t a l m u s c l e s . The

r e l a t i o n s h i p b e t w e e n t h e d i s t r i b u t i o n o f t h e s e m o l e c u l a r f o r m s and

-27-

m u s c l e f u n c t i o n was t h e n d e s c r i b e d . C h a p t e r 3 d e s c r i b e s t h e i n f l u e n c e o f

t h e n e r v e on t h e d i s t r i b u t i o n o f AChE f o rm s i n d i f f e r e n t m u s c l e t y p e s .

C h a p t e r 4 d e s c r i b e s t h e r e s p o n s e o f normal and d y s t r o p h i c m u s c l e s t o

i n d u c e d m u s c l e a c t i v i t y , i n t e r m s o f c h a n g e s i n b o t h t h e m u s c l e

p h y s i o l o g y and t h e d i s t r i b u t i o n o f AChE. The r e l a t i o n s h i p b e t w e e n t h e s e

tw o p a r a m e t e r s was a l s o e x p l o r e d . F i n a l l y , C h a p t e r 5 d e s c i b e d t h e

p u r i f i c a t i o n o f AChE from c h i c k b r a i n and t h e p r o d u c t i o n o f m o n o c l o n a l

a n t i b o d i e s s p e c i f i c a g a i n s t t h i s e n z y m e .

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CHAPTER 2

MULTIPLE MOLECULAR FORMS OF ACHE IN

MMtALIAN AND AVIAN SKELETAL MUSCLES

-29-

2 . 1 INTRODUCTION

In v e r t e b r a t e s k e l e t a l m u s c l e s , t h e m o l e c u l a r form s o f AChE

a r e c o m p r i s e d o f t h e g l o b u l a r L1# l_2 and M fo rm s, and t h e

a s y m m e t r i c c o l l a g e n - t a i l e d H^c and H2c fo rm s ( S e e S e c t i o n

1 . 1 . 3 ) . Some o r a l l o f t h e s e fo rm s c a n o c c u r i n v a r y i n g

p r o p o r t i o n s i n d i f f e r e n t m u s c l e s .

In a v i a n m u s c l e s , i t h a s b e e n p r o p o s e d t h a t t h e d i s t r i b u t i o n

o f t h e m o l e c u l a r for m s o f AChE i s d e p e n d e n t on t h e i r f i b r e t y p e

c o m p o s i t i o n ( B a r n a r d e t a l . , 1 9 8 2 ) . M u s c l e s a r e g e n e r a l l y made

up o f a m i x t u r e o f t y p e I ( s l o w - t w i t c h o x i d a t i v e ) , t y p e IIA

( f a s t - t w i t c h , o x i d a t i v e and g l y c o l y t i c ) , and I IB ( f a s t - t w i t c h

g l y c o l y t i c ) , and t y p e I I I ( t o n i c ) f i b r e s , c l a s s i f i e d by a v a r i e t y

o f h i s t o c h e m i c a l , m o r p h o l o g i c a l , b i o c h e m i c a l and e l e c t r o p h y s i o -

l o g i c a l c h a r a c t e r i s t i c s (V rbova e t a l . , 1 9 7 8 ) . In t h e c h i c k e n

and p i g e o n , t h e H2 c form p r e d o m i n a t e s i n a l l t h e t w i t c h m u s c l e s

s t u d i e d t o - d a t e ( L y l e s e t a l . , 1 9 8 2 ; B a rn a rd e t a l . , 1 9 8 2 ;

B a r n a r d e t a] _ . , 1 9 8 4 ) . S m al l v a r i a t i o n s i n t h e amount o f L and

H^c for m s a p p e a r t o c o r r e l a t e w i t h t h e r e l a t i v e amount o f t y p e

I I and t y p e I f i b r e s . In c o n t r a s t , t o n i c m u s c l e s su c h a s t h e ALD

c o n t a i n l a r g e l y L2 and very l i t t l e H2c fo rm s ( B a r n a r d e t a l . ,

1 9 8 2 ; L y l e s and B a r n a r d , 1 9 8 0 ) . The p r o f i l e o f t h e m o l e c u l a r

f o rm s o f AChE i s t h u s e a s i l y d i s t i n g u i s h a b l e b e t w e e n t h e t w i t c h

and t o n i c m u s c l e s .

I t i s now w e l l e s t a b l i s h e d t h a t H2c i s t h e e n d p l a t e

a s s o c i a t e d form o f AChE i n s k e l e t a l m u s c l e s ( H a l l , 1 9 7 3 ;

J e d r z e j c z y k e t a l _ . , 1 9 8 1 ) . In c h i c k e n s , t h e e n d p l a t e s o f

f a s t - t w i t c h m u s c l e s s u c h a s t h e PLD c o n t a i n l a r g e l y t h e H2c

-30-

form and v e r y l i t t l e g l o b u l a r f o r m s , w h e r e a s t h e form i s

p r e d o m i n a n t i n t h e e n d p l a t e s o f t h e t o n i c ALD ( J e d r z e j c z y k e t

a l . , 1 9 8 4 ) . T h u s , t h e p r e d o m i n a n t m o l e c u l a r f o r m ( s ) o f AChE i n

c r u d e c h i c k e n m u s c l e e x t r a c t s i s l a r g e l y c o n c e n t r a t e d a t t h e

e n d p l a t e .

A l t h o u g h e x t e n s i v e l y s t u d i e d , t h e r e h a s b e e n l i t t l e

c o n s i s t e n c y i n t h e p r o f i l e s o f AChE m o l e c u l a r form s o f mammalian

m u s c l e s c o n t a i n i n g p r e d o m i n a n t l y e i t h e r f a s t o r s l o w f i b r e s .

C o n s i d e r a b l e v a r i a t i o n i n t h e AChE p r o f i l e can o c c u r among

m u s c l e s o f t h e same s p e c i e s , o r b e t w e e n t h e same m u s c l e i n

d i f f e r e n t s p e c i e s ( M a s s o u l i e and B o n , 1 9 8 2 ) . M o r e o v e r , a l t h o u g h

a l l o f t h e s e m u s c l e s ha v e t w i t c h p r o p e r t i e s ( t o n i c m u s c l e s a r e

r a r e i n mammals) , t h e p r o p o r t i o n o f t h e ^ form i s g e n e r a l l y

s m a l l . Thus i n t h e m u s c l e s o f m a tu r e a n i m a l s , t h e amount o f t h e

Hgc f o r m , a s a p r o p o r t i o n o f t h e t o t a l AChE v a r i e s from 5%-7%

i n t h e r a t g a s t r o c n e m i u s o r r e c t u s m u s c l e s ( V i g n y e t a l . . 1 9 7 6 ;

F e r n a n d e z e t a l ., 1 9 7 9 ) , t o 15%-20% i n t h e r a t EDL ( G r o s w a l d and

D e t t b a r n , 1 9 8 3 a ; S e n n i e t a l _ . , 1 9 8 1 ) and t o a b o u t 30% i n t h e

m ous e s o l e u s ( G i s i g e r and S t e p h e n s , 1 9 8 3 ) . As i n a v i a n m u s c l e s ,

t h e H2C form w as a l s o f o u n d t o be a s s o c i a t e d w i t h t h e e n d p l a t e

r e g i o n o f t h e r a t d ia p h r a g m ( H a l l , 1 9 7 3 ; R i e g e r and V i g n y , 1 9 7 6 ;

Y o u n k in e t __ a l . , 1 9 8 2 ) , t h e r a t s t e r n o m a s t o i d (Bon e t a l . , 1 9 7 9 )

and t h e r a b b i t sem im em b ran osu s (B aco u e t a l . , 1 9 8 2 ) . H ow e v e r , a

m a j o r p r o p o r t i o n o f t h e e n d p l a t e - a s s o c i a t e d AChE i n mammalian

m u s c l e s , u n l i k e t h a t i n t h e c h i c k e n , i s p r e s e n t a s g l o b u l a r fo rm s

( M a s s o u l i e and Bon, 1 9 8 2 ) . In o t h e r w o r d s , t h e e n d p l a t e

a s s o c i a t e d H2C AChE c o n s t i t u t e s o n l y a m in o r p a r t o f t h e t o t a l

- 3 1 -

AChE a c t i v i t y , t h e m a j o r i t y b e i n g c o n t r i b u t e d by a v a r y i n g

p r o p o r t i o n o f L and M f o r m s , b o t h i n s i d e and o u t s i d e t h e

e n d p l a t e s .

T h i s s e e m i n g l y d i v e r s e c o m p o s i t i o n o f AChE f o rm s i n v a r i o u s

m u s c l e s may be due t o a number o f r e a s o n s . F i r s t l y , mammalian

m u s c l e s a r e u s u a l l y o f m ix e d f i b r e - t y p e c o m p o s i t i o n ( D u b o w i t z and

B r o o k e , 1 9 7 3 ; V r b o v a , e t a l . , 1 9 7 8 ) ; o n l y a f e w ( e . g . g u i n e a p i g

s o l e u s , r a b b i t sem im em br ano su s a c c e s s o r i u s / p r o p r i u s ) a r e

c o m p o se d p u r e l y o f o n e t y p e o f f i b r e ( P e t e r e t a l . , 1 9 7 2 ) .

F u r t h e r m o r e , t h e f i b r e t y p e c o m p o s i t i o n o f t h e same m u s c l e may

a l s o v a r y b e t w e e n s p e c i e s ( G a u t h i e r and P ac ty ku la , 1 9 6 6 ; s e e a l s o

m ous e and g u i n e a p i g s o l e u s , T a b l e 2 . 1 ) . I f , a s i n t h e c h i c k e n ,

t h e AChE c o n t e n t i n mammalian m u s c l e s i s a l s o d e t e r m i n e d by t h e

f i b r e t y p e c o m p o s i t i o n , t h e n v a r i a t i o n s i n t h e AChE p r o f i l e w o u ld

be e x p e c t e d .

S e c o n d l y , i t i s p o s s i b l e t h a t t h e s e a p p a r e n t v a r i a t i o n s a r e

a r t e f a c t s . As m u s c l e c r u d e e x t r a c t s a r e commonly u s e d f o r t h e

a n a l y s i s o f AChE f o r m s , t h e v a r i a b i l i t y i n t h e i r c o m p o s i t i o n and,

i n p a r t i c u l a r , t h e low l e v e l o f W^c form commonly o b s e r v e d i n

mammalian m u s c l e s c o u l d be due t o t h e p r e s e n c e o f e n d o g e n o u s

p r o t e a s e i n t h e m u s c l e e x t r a c t s . I n d e e d , s e v e r a l g r o u p s ha v e

♦ shown t h a t t h e c o l l a g e n - t a i l e d fo rm s o f AChE a r e s e n s i t i v e t o a

number o f p r o t e a s e s w h ic h c o n v e r t them t o g l o b u l a r fo rm s

( M a s s o u l i e and R i e g e r , 1 9 6 9 ; A n g l i s t e r and S i l m a n , 1 9 7 8 ; V ig n y e t

a l . , 1 9 7 9 ; Mays and R o s e n b e r r y , 1 9 8 1 ) . F u r t h e r m o r e , i t w a s shown

t h a t c o n v e r s i o n o f t h e H form i n t o M and L fo rm s o c c u r r e d d u r i n g

s t o r a g e o f t h e t i s s u e p r e p a r a t i o n e i t h e r a s t i s s u e e x t r a c t s

( R o t u n d o and Fambrough, 1 9 7 9 ) o r a s f r o z e n t i s s u e s ( L y l e s e t a l . ,

1 9 8 2 ) . Mammalian s k e l e t a l m u s c l e s a r e known t o c o n t a i n a w i d e

-32-

TABLE 2.1 The fibre type composition of some manual Ian and chicken skeletal muscles which had been chosen for AChE analysis.

*

Muscle type

Fast-tw1 tch

Slow-twltch

Mixed

Tonic

Species MuscleFibreI

type composition IIA 1 IB

(x)I I I

AChEXL

formsSM XH

Anterior t ib ia lis 4 46 50 - 31 6 62EDL 6 37 57 - 30 - 51

Guinea pig Gastrocnemius 12 32 56 - 18 - 61Semimembranosus accessorius <1 22 77 - 32 - 44Vastus latera lis (red) 4 78 18 - 23 6 69Vastus latera lis (white) 0 29 71 - 26 7 67

Anterior tib ia lis* 6 61 33 . 12 63Hamster EDL* 11 31 58 - 21 13 66

Plantarls 17 82 0 - 31 - 55

Anterior t ib ia lis 2 66 32 38 45 17Rat EDL 5 18 77 62 21 12

Sternomastold N.D. 55 23 13

Rabbit Anterior t ib ia lis 3 40 57 _ 41 45EDLa N.D. 44 - 42Semimembranosus accessorius 2 12 86 - 45 - 35

Mouse Anterior t lb la lls a N.D. 26 44 30

Chicken PLD <3 10 90 - 7 11 82

Plantaris* 100 0 0 20 68Guinea pig Semimembranosus proprlus 100 0 0 - 23 - 63

Soleus 100 0 0 - 17 12 71Hamster Soleus 80 20 0 - 18 - 71Rat Soleus 84 16 0 - 15 - 62Rabbit Semimembranosus proprlus* 100 0 0 - 17 21 61

Soleus 96 4 0 - 33 20 47

Ch1ckenb* Adductor profundus (anterior) 31 69 0 _ 24 12 64Rat Diaphragm 40 27 34 33 34 33Mousec* Soleus 42 58 0 - 31 18 51

Chicken* Adductor profundus (posterior) _ - O O 57 14 19Chicken ALD - - - 100 50 30 20

a These muscles were considered to be typical fast-twitch muscles by their electrophyslologlcal and biochemical characteristics. N.D. * fibre type composition not available,

b For some other chicken limb muscles, see also Chapter 4. c The fibre type composition was determined on three C57 black mice.* Fibre type compositions of these muscles were determined In this laboratory, courtesy of J.A . Plzzey and J . Jarv is.

The fibre type compositions of the other muscles were cited from Arlano et a l . . 1973 ; Peter et a l. , 1972 ; Campbell, 1979; Goldsplnk and Ward, 1979; Barnard et a l .. 1982 and Metzger et al.. 1985

- 3 3 -

v a r i e t y o f p r o t e a s e s ( P e n n i n g t o n , 1 9 7 7 ) , i d e n t i f i a b l e by t h e i r

s u b s t r a t e and i n h i b i t o r s p e c i f i c i t i e s , m a i n l y i n m a s t c e l l s

( B e n d i t t , 1 9 5 6 ; Park e t a l . , 1 9 7 3 ; B r a u n - F a l c o a n d S a l f e l d , 1 9 5 9 ;

Woodbury e t a l 1 9 8 1 ; S c h e c h e t e r e t a l 1 9 8 3 ) a n d c o n n e c t i v e

t i s s u e s ( B o i s , 1 9 6 4 ) . The v a r i e t y and s p e c i f i c i t y o f t h e s e

p r o t e a s e s may d i f f e r b e t w e e n d i f f e r e n t t y p e s o f t i s s u e s ( L a g u n o f f

e t __ a l _ . , 1 9 6 2 ) and a c o m p a r a t i v e stucty o f t h e s e p r o t e a s e s i n

s k e l e t a l m u s c l e s o f d i f f e r e n t s p e c i e s i s i n c o m p l e t e . T h e s e

e n z y m e s a r e r e s p o n s i b l e f o r p r o t e i n c a t a b o l i s m , a n d a r e l i k e l y t o

a t t a c k AChE i n v i t r o d u r i n g t h e t i s s u e h o m o g e n i s a t i o n and

e x t r a c t i o n p r o c e d u r e s when c e l l s t r u c t u r e s a r e d i s r u p t e d . H e n c e ,

p r e c a u t i o n a r y m e a s u r e s s h o u l d be t a k e n t o p r e v e n t t h e i n v i t r o

breakdow n o f t h e H form s so t h a t su ch a n a l y s i s a c c u r a t e l y

r e f l e c t s t h e AChE f o rm s p r e s e n t i n v i v o . P r o t e a s e i n h i b i t o r s a r e

c l e a r l y s u i t e d f o r t h i s p u r p o s e . M o s t o f them a r e l o w m o l e c u l a r

w e i g h t , a c t i v e s i t e - d i r e c t e d o r g a n i c compounds o r p e p t i d e s , w h ic h

a r e e i t h e r s y n t h e t i c o r n a t u r a l l y o c c u r r i n g ( m o s t o f t h e

i n h i b i t o r s u s e d i n t h i s s t u d y b e l o n g t o t h e l a t t e r c l a s s ) ( T a b l e 2.

2 ) . P r o t e a s e i n h i b i t o r s e x h i b i t i n g b r o a d s p e c i f i c i t y , s u c h a s

c ^ - m a c r o g l o b u l i n , a r e a l s o u s e d . A s u i t a b l e c o m b i n a t i o n o f

t h e s e i n h i b i t o r s s h o u l d p r o t e c t t h e H AChE from breakd ow n by

s u p p r e s s i n g a l l t h e p r o t e a s e a c t i v i t y p r e s e n t i n t h e m u s c l e .

The f i n a l r e a s o n f o r t h e v a r i a b i l i t y o f AChE p r o f i l e s may b e

due t o t h e d i f f e r e n t a n a t o m i c a l d i s t r i b u t i o n o f AChE form s i n

mammalian m u s c l e f i b r e s . As m e n t i o n e d e a r l i e r , t h e r e i s , i n a l l

c a s e s r e p o r t e d , a l a r g e p r o p o r t i o n o f g l o b u l a r fo rm s p r e s e n t i n

e n d p l a t e - r i c h r e g i o n s o f mammalian m u s c l e s , u n l i k e t h o s e i n t h e

e n d p l a t e s o f c h i c k e n t w i t c h m u s c l e s w h ic h a r e e n r i c h e d i n t h e

- 3 4 -

TABLE 2.2 Combination of protease inhib itors used during AChE extraction3 from different tissues.

Protease in h ib ito r^ Concen

tra tio n

Spec ific ity Rat and mouse

muscle

Chick muscle

and brain

Hamster, guinea pig

and rabb it muscle

Ref

Bacitracin lmg/ral bacteria l protelnases * * * *

Leupeptln 0.04mg/ml plasmin, tryps in , pepsin * * * Knight, 1981; Kirschke

cathepsln B e t a l. . 1977

N-ethylnalelmide 5nM cysteine proteinases ★ * *

Pepstatln 0.02mg/ml carboxyl protelnases * * * Ba rre tt and Dingle, 1972,

Bestatln O.OlnM ami nopeptidase ★ * * Umezawa and Aoyagl, 1977,

Ovomucoid 0 .2mg/ml chymotrypsin + trypsin * * * Tomimatsu et al,, 1966

Soyabean/Limabean

tryps in In h ib ito r O.lmg/ml tryps in + chymotrypsin * * B1rk, 1976; Haynes and

Feeney, 1967

Apro tin in 0.2mg/ml chymotrypsin + trypsin * Kunitz and Northrop, 1936

EGTA 5nH Ca++ activated protelnases * * * Meyer e t a l .. 1966:

Huston e t a l .. 1968:

Lorand, 1982.

Ylpera ammodytes

Venom inh ib ito r(S V I) O.lmg/ml chymotrypsin + tryps in * R lto r l ja et al.. 1983

c^-macroglobulin O.lmg/ml broad spec if ic ity * Starkey and Ba rre tt, 1977

Rapid processing of the tissue extracts is necessary

Reduction of AChE a c t iv ity , when compared to control sample extracts(no in h ib ito rs added), a fte r addition of these protease in h ib ito rs was <15%.

H£C f o r m . I t i s c o n c e i v a b l e t h a t , i f t h e g l o b u l a r fo rm s a r e

i n d e e d p a r t o f t h e f u n c t i o n a l AChE i n mammalian m u s c l e s

e n d p l a t e s , t h e y w o u ld a c c o u n t f o r t h e h i g h e r l e v e l o f g l o b u l a r

fo rm s i n mammlian m u s c l e s .

The a im o f t h e p r e s e n t s t u d y i s t o d e t e r m i n e i f t h e r e i s a

c o r r e l a t i o n b e t w e e n t h e f i b r e t y p e c o m p o s i t i o n and t h e

d i s t r i b u t i o n o f AChE f o r m s . To do t h i s , m u s c l e s o f d e f i n e d f i b r e

t y p e c o m p o s i t i o n from s e v e r a l mammalian s p e c i e s w e r e a n a l y s e d . A

d e t a i l e d s t u d y was c a r r i e d o u t a t t h e same t i m e t o i d e n t i f y and

r e l a t e t h e a r t e f a c t u a l e f f e c t s o f e n d o g e n o u s p r o t e a s e s on t h e

d i s t r i b u t i o n o f AChE form s i n v i t r o , and p r e c a u t i o n a r y p r o c e d u r e s

w e r e t h e n d e s i g n e d t o e l i m i n a t e o r m i n i m i z e t h e s e a r t e f a c t s .

2 . 2 MATERIALS AND METHODS

2 . 2 . 1 Antals

G u in ea p i g s ( D u n k i n - H a r t l e y , two m onth s o l d ) , r a b b i t s (New

Z e a l a n d w h i t e , %3kg), c h i c k e n s (London l i n e 4 1 2 , 2 - 4 m on th s o l d )

and r a t s ( S p r a g u e - D a w l e y , %200g) w e r e m a i n t a i n e d i n t h e

B i o c h e m i s t r y d e p a r t m e n t , I m p e r i a l C o l l e g e . H a m s t e r s ( G o l d e n , 3

m o n th s o l d ) , w e r e o b t a i n e d from M i d d l e s e x H o s p i t a l , M i d d l e s e x .

• M ic e ( 1 2 9 / R e J , n o r m a l , 2 m on th s o l d ) w e r e o b t a i n e d from C h a r i n g

C r o s s H o s p i t a l , London.

2 . 2 . 2 Muscles

M u s c l e s w h ic h c o n t a i n p r e d o m i n a n t l y t y p e I o r t y p e I I f i b r e s

w e r e s e l e c t e d from t h e a b o v e a n i m a l s a s l i s t e d i n T a b l e 2 . 1 . The

r a t d ia ph rag m w a s c h o s e n t o r e p r e s e n t mammalian m u s c l e s o f m ix e d

f i b r e t y p e c o m p o s i t i o n .

- 3 6 -

2 . 2 . 3 Materials

B a c i t r a c i n , l e u p e p t i n , p e p s t a t i n , c h i c k e n o v o m u c o id t r y p s i n

i n h i b i t o r ( o v o m u c o i c j ) , a p r o t i n i n , b e s t a t i n , N - e t h y l m a l e i m i d e (NEM),

5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB), t e t r a i s o p r o p y l p y r o -

p h o s p h o r a m i d e ( i soOM PA), BW284C51, b o v i n e l i v e r c a t a l a s e , E . c o l i

B - g a l a c t o s i d a s e , a c e t y l c h o l i n e c h l o r i d e , a - c h y m o t r y p s i n from

b o v i n e p a n c r e a s , a z o c o l l and FITC c o n j u g a t e d c a s e i n w e r e o b t a i n e d

from S ig m a ; a c e t y l t h i o c h o l i n e i o d i d e and T r i t o n X - 1 0 0 from

K o c h - L i g h t Ltd ; c ^ - m a c r o g l o b u l i n from B o e h r i n g e r Mannheim;

[ H] a c e t y l c h o l i n e from Arnersham. The V i p e r a am modytes venom

t r y p s i n i n h i b i t o r ( S V I ) was a g i f t from Dr. R. S h i p o l i n i o f t h i s

d e p a r t m e n t .

2 . 2 . 4 Precautions applied in tissue extraction

The f o l l o w i n g p r e c a u t i o n s w e r e t a k e n d u r i n g t i s s u e

e x t r a c t i o n i n o r d e r t o a c h i e v e o p t i m a l p r e s e r v a t i o n o f t h e

m o l e c u l a r fo rm s o f AChE. The e f f e c t i v e n e s s o f t h e s e p r e c a u t i o n s

w as j u d g e d by t h e r e p r o d u c i b i l i t y o f t h e m o l e c u l a r p r o f i l e s

o b t a i n e d and t h e r e l a t i v e amount o f t h e form p r e s e r v e d .

M u s c l e s from t h e h a m s t e r , g u i n e a p i g and r a t w e r e u s e d i n t h e s e

p r e l i m i n a r y e x p e r i m e n t s .

1 . P e r f u s i o n A n im a ls w e r e d e e p l y a n a e s t h e t i s e d w i t h

b a r b i t u r a t e . The t h o r a c i c c a v i t y was o p e n e d by a m i d l i n e

i n c i s i o n and by c a r e f u l l y c u t t i n g t h e d ia p h r a g m from t h e r i b

c a g e . C a r d i o v a s c u l a r p e r f u s i o n was t h e n p e r f o r m e d u s i n g a

p h o s p h a t e b u f f e r e d s a l i n e (PBS) c o n t a i n i n g lOmM EDTA, pH 7 0 .

A p p r o x i m a t e l y 1 m l / g body w e i g h t o f t h e b u f f e r was a p p l i e d a t

1 0 - 2 0 m l / m i n.

- 3 7 -

Protease inhibitors2 . For tissue homogenisation and

extraction, buffers containing the various protease inhibitors,

their concentrations as listed in Table 2.2, were prepared fresh

before use. Selective combinations of the inhibitors were tested

for their ability to preserve the H2C form in tissue extracts.

None of the inhibitors used had any inhibitory activity on AChE

The activities of endogenous proteases in tissue extracts,

and their inhibition by protease inhibitors, were measured using

azocoll or FITC conjugated casein as substrates.

i) Azocoll hydrolysing activity (Saheki and Holzer, 1974)-

batches of azocoll (2.4%) in 1% Triton X-100/1M NaCl phosphate

buffer, pH 7.0,were mixed (5:1) with the muscle crude extract and

incubated, with vigorous shaking, at 25°C. At different time

intervals, up to 24 hours, 0.5ml of the suspension was mixed 1:1

with 10% TCA and centrifuged at 10,000xg for 3 min. The

absorbance of the supernatant was determined at 520nm.

ii) FITC conjugated casein hydrolysing activity — this assay was

modified from the method by Twining, 1983. Batches of

FITC-casein (0.025%), dissolved in 1% Triton X-100/1M NaCl

phosphate buffer, were mixed (4:1) with the muscle crude extract

and incubated at 37°C; at different time intervals, up to 3

hours, the reaction was terminated by mixing 0.05ml of the

mixture with 0.05ml of 10% TCA. The precipitate was centrifuged

at 10,000xg(3 min). 0.08ml of the supernatant was made up to 0.7ml

with 0.1M phosphate buffer, pH 7 0. The supernatant was excited

at 490nm and its fluorescence recorded at 525nm in a Perkin Elmer

(model no. 203) fluorescence spectrophotometer. The combined

protease inhibitors were prepared in the FITC-casein solution,

or included during homogenisation. Dose-response curves were

constructed for SVI, a^-macroglobulin and bacitracin.

-38-

a-chymotrypsin from bovine pancreas was used as a standard

protease for both azocoll and FITC-casein assays. The enzyme

activity of the standard a-chymotrypsin solution was determined

using BTEE as substrate, and;hydrolysis was monitored at 256 nm

in a Cecil spectrophotometer (Hummel, 1959). Enzyme activity was

calculated as:

Uni ts/ml = Afl256/min X 100__________

964 X vol. of reaction mix

3. Rapid processing of tissue extracts

Muscles were excised immediately upon death and rinsed

briefly in PBS. No more than 6 samples were handled at ar\y one

time in order to minimise the processing time. Muscles were

minced briefly on ice, then either the whole muscle, or an

aliquot of the minced muscle, was homogenised in 10 vol of an

ice-cold phosphate buffer containing 1% Triton X-100, 1M NaCl and

an array of protease inhibitors as described in Table 2.2.

Depending on the sample volume, these muscles were either

homogenised with glass/glass hand homogenisers or with a Polytron

(Kinetica) (3x30s) at 4°C. The homogenates were immediately

centrifuged at 43,500xg for 15 min to obtain the crude extracts,

which were then immediately applied onto sucrose gradients for

sedimentation analysis. The whole process of homogenisation and

extraction was accomplished within 60 min.

Long incubations were also carried out in the presence and

absence of protease inhibitors, up to 4 hours before

sedimentation analysis. The efficiency of extraction of AChE

after long or rapid processing was estimated by comparing the

AChE activity present in the extract with that in the muscle

homogenate.

-39-

4. Rapid freezing of muscles The rat soleus muscle was

excised and immediately dropped into liquid nitrogen. The muscle

was then crushed, in liquid nitrogen, into small pieces and

processed as described above.

2.2.5 Hicrodfssection

This technique enables intact whole fibres, endplates and

non-endplate segments to be dissected from muscle. All

manipulations were carried out under a dissecting microscope

fitted with an ice-cooled platform. The muscle was cleaned of

surface connective tissue. A longitudinal slice was then trimmed

from the muscle so that the whole span of the fibre length was

included. Small bundles of fibres were then teased from the edge

of this slice with microforceps, taking care not to stretch or

damage the fibres. Usually a group of 20-30 fibres could be

isolated and, keeping the fibres moist with PBS, further trimmed

to remove any broken fibres. The intact fibres obtained were

then transferred to a fresh cold surface, gently teased and

chopped.

Endplate and non-endplate segments were also dissected from

muscle fibres. The muscle was cleaned of surface connective

• tissues and a suitable slice of the muscle was stained for

endplate AChE (Karnovsky and Roots, 1964). As soon as the

endplates became visible, they were carefully excised from

individual muscle fibre. Non-endplate segments, each about five

times the length of an endplate,were taken from the same fibres

far removed from the endplate region without entering the

tendonous region. Batches of 20-30 endplates or non-endplate

segments were pooled.

-40-

The dissected whole fibres, endplates and non-endplate

segments were immediately extracted by shaking for 60-90 min in

0.25ml of the above medium at room temperature.

2.2.6 Sedimentation analysis

This procedure was carried out as described previously

(Lyles et__aK, 1982). 5% to 20% linear sucrose gradients were

prepared using a Buchler Auto Densi-Flow IIC apparatus (Haake

Buchler), on a 0.5ml 60% sucrose solution in 12ml polyallomer

centrifuge tubes. The sucrose solutions were made up in 1%

Triton X-100/ 1M NaCI phosphate buffer, pH 7.0. Crude muscle

extracts or suspensions of microdissected samples were layered

onto the gradients and centrifuged in a Beckman SW40Ti rotor atip p

4°C, to a preset total of 1.02 x 10 rad for mammaliani i p

tissues, or 9.6 x 10 rad for avian tissues, in a Beckman

L8-70 ultracentrifuge. Catalase and 0-galactosidase were

included as internal protein standards. After sedimentation, the

gradients were fractionated with the Buchler Auto Densi-Flow IIC

apparatus.

2.2.7 Enzyme assay

The AChE activity of crude extracts were assayed by the

colorimetric method of Ellman et al. (1969), essentially

as described previously (Lyles et al.. 1979; Lyles, 1980), in the

presence of isoOMPA, a ^ChE inhibitor. The assay was carried out

at room temperature in a medium containing InM DTNB/ 0.1M

phosphate/ 10"^M isoOMPA. Extracts (10 pi) were preincubated

in 0.5 ml of this medium for 30 min, after which

- 4 1 -

acetylthiocholine iodide was added to a final concentration of

0.75nM to initiate the enzyme reaction. The rate of hydrolysis

of acetylthiocholine iodide was monitored by the increase in

absorbance at 412 nm on a LKB Ultrolab System 2086 Mark II

Kinetic Analyser. The concentration of AChE was calculated using

Beer's law:

C = AOD where 6= 13.6 t l Jt= 1

The sucrose gradient fractions from whole muscle extracts

were assayed by the same method unless otherwise stated. 0.5 ml

of a double strength DTNB/isoOMPA solution was added to each

fraction and preincubated for 30 min. The enzyme reaction was

initiated by adding a further 0.5 ml of 1.5 mM acetylthiocholine

iodide. The length of incubation varied depending on the rate of

colour development. The absorbance of the gradient fractions was

recorded on a Cecil spectrophotometer after suitable incubation.

AChE from gradient fractions of microdissected sample

extracts, or samples containing NEM which interferes with the

colorimetric assay, were analysed radiometrically using

[ H]-acetylcholine (Johnson and Russell, 1973). A typical

assay mixture contained lCf^M isoGMPA, 0.788mM acetylcholineo

chloride with [ H]- acetylcholine and the gradient fraction or

crude extract, in a final volume of 0.3ml and 0.1ml respectively.

The reaction was terminated by adding 0.1ml of stopping mix (1M

chloroacetic acid/ 2M NaCl/ 0.5M NaOH) to crude extract sample

assays, or 0.15ml of a double strength stopping mix to gradient

fraction assays. The whole fraction was mixed in %4.5 ml of 90%

toluene/10% aniyl alcohol/PP0/P0P0P scintillant. AChE activity

was expressed as the proportion of substrate hydrolysed (in cpm)

- 4 2 -

to the total substrate hydrolysed using excess pure AChE from eel

(approximately lO^cpm). AChE activity was calculated as

fol1ows:

Enzyme units _ Sample cpm blank x nmoles substrate added

(nmoles AChE Eel cpm - blank running time x sample vol

hydrolysed/mi n/ml) (min) (ml)q

The non-specific hydrolysis of [ H] acetylcholine, or

acetylthiocholine iodide, was estimated by running a parallel

assay of buffer blanks, or crude extracts/gradient fractions

inhibited with both isoOMPA and 5 x lCf^M BW284C51 (AChE

inhibitor).

Enzyme marker assays

A) Catalase (11.4S)(EC 1.11.1.6) 1 unit/nl sample was loaded

onto a gradient. The position of the marker was subsequently

detected by assaying for the breakdown of HgOg in the

gradient fractions. 1 ml of reaction mix (0.2 ml of 30% HgOg

in 142 ml of 0.05 M sodium phosphate, pH 7.0) was added to 10 ul

of a gradient fraction in a quartz cuvette. The decrease in

absorbance at 240 nm was immediately measured in a

spectrophotometer preset at O.D. = 0.2 with a reagent blank.

B) B-galactosidase (16 S) (EC. 3.2.1.23) 10 units/100 ul

sample was loaded. The position of this enzyme was determined by

the breakdown of its substrate, O-nitrophenylgalactoside (0NPG).

0.3 ml of 0NPG (0.6 g in 1 lit of 0.1 M phosphate, pH 7.2) was

mixed with 10 ill of a gradient fraction and incubated until a

strong yellow colour developed. The reaction was then stopped by

adding 0.6 ml of 1 M I^COg and the absorbance of the fraction

recorded at 416 nm.

- 4 3 -

2.3 RESULTS

SECTION I. Effects of extraction conditions on the

distribution of ACHE forms.

2.3.1 Protease activities in muscle extracts and the effects of

protease inhibitors on AChE extraction

The possibility that the variable amount of L forms observed

in tissue extracts may be a result of proteolytic degradation of

the higher molecular weight forms was assessed by monitoring the

azocoll or casein hydrolysing activity of the extracts. Both of

these proteins are substrates for a wide spectrum of proteases

which may be present in muscle extracts (Cabib and Ulane, 1973;

Reimerdeo and Klostermeyer, 1976). These non-specific substrates

were employed, instead of the many synthetic peptides used for

quantitative determination of specific proteases, for three

reasons. Firstly, assay systems employing synthetic peptides

could not be used because Triton X-100, which was present in the

medium, interfered with absorbance at 280nm. Secondly, the

non-specific substrates provided an estimate of total proteolytic

activity present in crude extracts so that the combined

inhibitory effect of the protease inhibitors could be assessed.

Finally, an accurate quantitative measurement of individual

protease was not essential.

The measurement of muscle protease activity using azocoll as

substrate was negative. This was probably because the level of

enzyme activity in the extracts was below the sensitivity range

of this assay, since the hydrolysing activity of a standard

- 4 4 -

a-chymotrypsin solution could be measured by this method.

However, the possibility that azocoll was an unsuitable substrate

for muscle proteases could not be ruled out.

The hydrolysis of FITC conjugated casein was a more

sensitive assay system for measuring endogenous protease activity

in muscle extracts. With purified bovine a-chymotrypsin, the

rate of hydrolysis was linear with enzyme concentrations as

previously reported (Twining, 1983). The present results showed

that rat muscles contained a large amount of protease activity

(Fig. 2.1), which was low or negligible in brain tissues and

guinea pig muscles.

The protease activities of three rat muscles, v.t. soleus.

sternomastoid and gastrocnemius were studied. They were found to

have a similar activity, roughly equivalent to 1.2 mil of bovine

pancreatic a-chymotrypsin per ml of extract. The protease

inhibitors, used in combination (Table 2.2), completely inhibited

the protease activity observed in these muscles (Fig. 2.1).

Interestingly, a2-macroglobulin produced an apparent total inhibition at concentrations of O.lmg/ml or above for a period of

60-90 min (Fig. 2.2C). This indicates that ag-macroglobulin

may be one of the most potent protease inhibitors within the

• processing time period. Bacitracin and SVI, on the other hand,

only produced a partial inhibition (Fig. 2.2A,B). The amount of

SVI required to produce 50% inhibition of bovine a-chymotrypsin

was 30 ug/unit of enzyme activity (Fig. 2.3).

2.3.2 Extraction of AChE by rapid processing in the presence of

protease inhibitors

Immediate analysis of freshly prepared crude extracts of

mammalian muscles by sucrose density gradients showed that the

-45-

FIG- 2-1 Casein hyrolysing activity of tissue crude extracts

from various species. 0.02% final concentration of

FITC-conjugated casein in 1M NaCl/1% Triton/O.IM phosphate, pH

7.0, incubated with crude extracts from: O , rat sternomastoid/

rat soleus (16.5 units/hr); # , rat brain/chick brain (1.4

units/hr); ^ , guinea pig hind limb muscles (undetectable);

■ , muscle and brain extracts in the presence of a full array

of protease inhibitors (Table 2.2.). All incubations were

carried out at 37°C. Activity is expressed in arbitrary

fluorescence units per hour. See text for calibration with

bovine a-chymotrypsin. Results were pooled from at least 2

animals.

- 4 6 -

FIG. 2.2 Dose dependent inhibition of casein hydrolysing

activity of rat muscle extracts by bacitracin, SVI and

c^-macroglobulin. (A) 0 , rat sternomastoid; Q , rat

soleus; (B) rat hind limb muscles; (C) rate of hydrolysis of

casein by rat soleus extract in the presence of different

concentrations of c^-macroglobulin; # , O.Olmg/ml; O ,

0 . 0 5 mg/ml; ■ , 0 . 1 mg/ml; □ , 0 . 5 mg/ml. Results were from

a typical experiment; repeat experiments gave similar inhibition

character!' sties.- 4 7 -

i

FIG- 2-3 Dose dependent inhibition of bovine a-chymotrypsin

activity by SVI. 0.5 itM final concentration of BTEE in 0.05 M

CaCl2/0.025 M Tris HC1, pH 7.8 was used as substrate. Reactions

were carried out at room temperature.

-48-

Hg form predominated in most type I and type II muscles, (e.g.

Fig. 2.4A,C). In contrast, if muscle homogenates or crude

extracts were stored (on ice) for a period of time, up to 4 hours

before sedimentation analysis, the H forms were greatly

diminished. At the same time, a proportional increase in the L

forms became apparent. Such changes occurred when the extracts

contained few (e.g. bacitracin, ovomucoid and NEM only) or no

protease inhibitors (Fig. 2.4B,D). The difference observed was

not due to limited extraction of L forms during rapid processing,

as tissue homogenisation and extraction in 1% Triton X-100/1M

NaCl solubilised at least 90% of total AChE (Appendix 1). There

was no difference between the AChE activities extracted slowly or

rapidly from samples of the same muscle.

If protease inhibitors were present in the muscle extracts,

the profile of AChE forms remained stable even after prolonged

incubation at 4°C (Fig. 2.5B). The H2c form in guinea pig

muscles predominated even in the absence of protease inhibitors,

provided that the extracts were processed rapidly (Fig. 2.4 A,C).

In contrast, rat muscles always require protease inhibitors to

suppress the breakdown of the H forms if reproducible results

were to be obtained. Such difference between guinea pig and rat

• muscle extracts correlated with the levels of protease activity

observed in the muscles of the two species. Rapid processing in

the presence of protease inhibitors, therefore, is an essential

procedure for the extraction and analysis of AChE forms.

The particular combination of protease inhibitors used

during AChE extraction differed between species, depending on the

level of protease activities in the particular tissues. Some

effective inhibitor combinations tested in various species are

listed in Table 2.2.

- 4 9 -

IS A 15 C

AA

FR A CTIO N S

FIG- 2-4 Effect of prolonged storage of guinea pig muscle crude

extracts on the distribution of the molecular forms of AChE.

A,C, sucrose gradient profiles of AChE from freshly prepared

extracts of soleus and EDL, respectively; B,D, sucrose gradient

profiles of AChE from extracts of soleus and EDL, respectively,

after storing at 4°C for up to 4 hours before analysis. No

protease inhibitors were included in any of the extracts.

Results were from a typical experiment. Similar effect of

short-term storage was observed in other mammalian muscles

tested. Arrows on these and other sucrose gradient profiles

indicate the position of catalase, unless otherwise stated.

- 5 0 -

*

-5 1 -

FIG- 2-5 P r o t e a s e i n h i b i t o r s s t a b i l i s e t h e d i s t r i b u t i o n o f AChE

fo rm s in m u s c le c r u d e e x t r a c t s . (A) s u c r o s e g r a d i e n t p r o f i l e o f

AChE i n r a t s o l e u s , p r e p a r e d 1n t h e a b s e n c e o f p r o t e a s e

i n h i b i t o r s and t h e e x t r a c t s t o r e d a t 4°C f o r 1 h o u r b e f o r e

g r a d i e n t a n a l y s i s . D o t t e d l i n e r e p r e s e n t s b a ck g ro u n d a b s o r b a n c e

o f s u c r o s e g r a d i e n t f r a c t i o n s w h ich w e r e i n c u b a t e d i n t h e

p r e s e n c e o f b o th isoOMPA (1 x 1 0 “^M)and BW 284C51 ( 5 x

10-*). (B) s u c r o s e g r a d i e n t p r o f i l e o f AChE i n r a t s o l e u s

p r e p a r e d i n t h e p r e s e n c e o f a f u l l a r r a y o f p r o t e a s e i n h i b i t o r s

( T a b l e 2 . 2 ) , and t h e e x t r a c t a n a l y s e d by g r a d i e n t e i t h e r

im m e d ia t e ly o r a f t e r s t o r i n g a t 4°C f o r 1 h o u r . Same am ount o f

e x t r a c t s was a n a l y s e d i n (A) and ( B ) . U n l e s s i n d i c a t e d ( a s in A ) ,

g r a d i e n t p r o f i l e s shown have b e e n c o r r e c t e d f o r b a c k g r o u n d

a b s o r b a n c e . R e p e a t e x p e r i m e n t s g a v e s i m i l a r r e s u l t s .

FRACTIONS

- 5 2 -

2 . 3 . 3 The effect of perfusion

B lo o d c o n t a i n s L and M fo rm s o f AChE ( s e e g e n e r a l

i n t r o d u c t i o n ) and serum p r o t e a s e s ( f o r r e v i e w , s e e L o r a n d ,1 9 7 6 ) .

P e r f u s i o n w as i n c l u d e d a s a p r e c a u t i o n i n t h e e x t r a c t i o n o f AChE

p r i m a r i l y t o rem ove b l o o d from m u s c le c a p i l l a r i e s . T h is

p r o c e d u r e h a s b e e n em p lo y e d p r e v i o u s l y i n t h e s t u d y o f c h i c k e n

m u s c l e s ( L y l e s e t a l_ . , 1 9 8 2 ) . H ow ever, when u s e d h e r e no

d i f f e r e n c e i n t h e AChE p r o f i l e s w as o b s e r v e d . B e c a u s e o f t h i s ,

and i n v i e w o f t h e f a c t t h a t p e r f u s i o n may in d u c e o s m o t i c damage

t o c a p i l l a r i e s and t h e s u r r o u n d in g t i s s u e , and t h a t f o r m o st

m u s c l e s t h e am ount o f c a p i l l a r y b l o o d * i s s m a l l , p e r f u s i o n was

s u b s e q u e n t l y o m i t t e d from t h e e x t r a c t i o n p r o c e d u r e .

* The re d c o l o u r o f m u s c l e s su ch a s t h e d iap h ragm i s l a r g e l y due

t o m y o g lo b in and m i t o c h o n d r i a l c y t o c h r o m e s i n t h e s lo w f i b r e

( G a u t h i e r , 1 9 6 9 ) and n o t t o b l o o d .

2 . 3 . 4 The effect of freezing nuscle tissues

T h is p r o c e d u r e h a s b een em p lo y e d in s e v e r a l l a b o r a t o r i e s

( G i s i g e r and S t e p h e n s , 1 9 8 3 ; Bacou e t _ a l . , 1 9 8 2 ) f o r t h e s t o r a g e

o f t i s s u e s b e f o r e e x t r a c t i o n . P r e s u m a b ly , a t v e r y lo w

t e m p e r a t u r e , t h e p r o t e a s e a c t i v i t y w o u ld b e i n s t a n t a n e o u s l y

• a r r e s t e d upon f r e e z i n g . T h is was t h e r e f o r e i n c l u d e d a s a

p r e c a u t i o n a r y p r o c e d u r e f o r e x t r a c t i n g AChE from r a t m u s c l e s ,

a l o n g w i t h r a p i d p r o c e s s i n g . H o w ev er , i t was fo u n d t h a t f r e e z i n g

d i d n o t a p p e a r t o make any d i f f e r e n c e in p r e s e r v i n g t h e H£C

AChE, p o s s i b l y b e c a u s e p r o t e o l y s i s was n o t e x t e n s i v e b e f o r e

h o m o g e n i s a t i o n .

In c o n c l u s i o n , i t w as e s t a b l i s h e d t h a t r a p i d p r o c e s s i n g o f

-53-

m u s c le c r u d e e x t r a c t s from f r e s h l y e x c i s e d m u s c l e s , a n d t h e u s e o f

an a p p r o p r i a t e c o m b in a t io n o f p r o t e a s e I n h i b i t o r s , w e r e t h e

s i m p l e s t w ays o f e n s u r i n g r e p r o d u c i b i l i t y In t h e vn v l t r o

a n a l y s i s o f AChE f o r m s .

SECTION II. Distribution of AChE torus in u u n l i a n and

avian skeletal uuscles

2 . 3 . 5 Molecular profiles of AChE in uauualian skeletal uuscles

The r e l a t i v e p r o p o r t i o n s o f AChE fo rm s In t h e v a r i o u s

m u s c l e s a n a l y s e d a r e l i s t e d In T a b le 2 . 1 .

A. F a s t - t w i t c h m u s c l e s

M u s c le s w h ich a r e com p osed p r e d o m in a n t ly o f t y p e I I f i b r e s

w ere a n a l y s e d f o r t h e i r AChE c o n t e n t ( T a b le 2 . 1 ) . T h e s e t y p e I I

m u s c l e s from h a m s t e r , g u i n e a p i g and r a b b i t show ed t h a t t h e r e

w ere two p r i n c i p a l t y p e s o f m o l e c u l a r p r o f i l e s . The f i r s t t y p e

c o n s i s t e d o f a b o u t e q u a l a m o u n ts o f t h e L an d H f o r m s , t h e

s e c o n d c o n s i s t e d p r e d o m in a n t ly o f t h e ^ fo r m . The p r o f i l e s

o f AChE fo rm s i n a l l t h e r a b b i t f a s t - t w i t c h m u s c l e s , a s w e l l a s

i n t h e EDL ( F i g . 2 .4 C ) an d t h e sem im em branosus a c c e s s o r i u s o f

t h e g u in e a p i g , a r e o f t h e fo r m e r t y p e . F i g u r e 2 . 6 i l l u s t r a t e s

• some r e p r e s e n t a t i v e p r o f i l e s o f t h i s t y p e o f d i s t r i b u t i o n .

The s e c o n d t y p e o f d i s t r i b u t i o n o f AChE fo rm s was e x h i b i t e d

by a l l t h e h a m s te r t y p e I I m u s c l e s and some o f t h e g u i n e a p i g

m u s c l e s ( F i g . 2 . 7 ) , w h ere AChE w as d o m in a n t . T h er e w a s no

d i f f e r e n c e i n t h e c o m p o s i t i o n o f AChE form s i n t y p e IIA a n d I IB

f i b r e s , a s shown by t h e r e d and w h i t e r e g i o n s o f t h e g u in e a p i g

v a s t u s m u s c l e . ( T a b le 2 . 1 ) .

- 5 4 -

FIG. 2-6 T y p ic a l s u c r o s e g r a d i e n t p r o f i l e s o f AChE fo rm s in

some mammalian t y p e I I m u s c l e s , A, r a b b i t EDL; B, r a b b i t AT; C,

g u in e a p i g sem i-m em b ra n o su s a c c e s s o r i u s . A l l e x t r a c t s w e r e

p r e p a r e d in t h e p r e s e n c e o f p r o t e a s e i n h i b i t o r s .

-55-

aa

FRACTIONS

FIG- 2.7 T y p ic a l s u c r o s e g r a d i e n t p r o f i l e s o f AChE fo rm s in

some mammalian t y p e I I m u s c l e s . A, h a m s te r AT; B, h a m s te r EDL;

C, g u i n e a p i g v a s t u s l a t e r a l i s ; D, g u i n e a p ig g a s t r o c n e m i u s . A l l

e x t r a c t s w ere p r e p a r e d i n t h e p r e s e n c e o f p r o t e a s e i n h i b i t o r s .

-56-

In c o n t r a s t , t h e t y p e I I m u s c l e s i n r a t and m ouse c o n t a i n e d

a s u b s t a n t i a l am ount o f L and M fo rm s ( F i g . 2 . 8 ) , t h e p r o p o r t i o n s

o f w h ic h r e m a in e d l a r g e e v e n when p r o t e a s e i n h i b i t o r s w e r e u se d

d u r in g e x t r a c t i o n .

B . S l o w - t w i t c h m u s c l e s

Type I m u s c l e s from t h e v a r i o u s mammalian s p e c i e s c l e a r l y

sh ow ed t h e p r e d o m in a n c e o f t h e H2 c form ( F i g . 2 . 9 ) . In

a d d i t i o n , t h e r e w as a l s o an i m p o r t a n t com p onent o f H^c f o r m ,

m o st p r o m in e n t i n t h e r a t s o l e u s ( F i g . 2 . 5 B ) , t o g e t h e r w i t h a

m in o r am ount o f t h e l o w e r m o l e c u l a r w e i g h t fo r m s . The r e l a t i v e

am ount o f t h e L fo rm s v a r i e d b e tw e e n m u s c l e s , r a n g i n g from 15%

( r a t s o l e u s ) t o 33% ( r a b b i t s o l e u s ) o f t o t a l a c t i v i t y . The M

form on t h e o t h e r hand a c c o u n t e d f o r a r e l a t i v e l y m in o r

p r o p o r t i o n o f t o t a l AChE a c t i v i t y . T h e s e AChE p r o f i l e s o f t y p e I

m u s c l e s a r e t h e r e f o r e v e r y s i m i l a r t o some p r o f i l e s o f t y p e I I

m u s c l e s d e s c r i b e d a b o v e ( F i g . 2 . 7 ) .

C. A n a l y s i s o f AChE fo rm s by m i c r o d i s s e c t i o n

S i n c e t h e a b u n d a n c e o f L fo rm s i n r a t m u s c l e s c o i n c i d e d w i t h

t h e e x c e p t i o n a l l y h ig h p r o t e o l y t i c a c t i v i t y fo u n d i n th em , i t was

e s s e n t i a l t o a s c e r t a i n w h e t h e r o r n o t t h e l a r g e p r o p o r t i o n o f L

form s was an a r t e f a c t o f p r o t e o l y s i s . A l t h o u g h p r o t e a s e

i n h i b i t o r s had b e e n shown t o r e d u c e t h e p r o t e o l y t i c a c t i v i t y in

r a t m u s c l e s t o v e r y lo w l e v e l s , i t d id n o t p r e c l u d e t h e

p o s s i b i l i t y o f some r e s i d u a l p r o t e a s e a c t i v i t y . AChE a n a l y s i s

was t h e r e f o r e c a r r i e d o u t u s i n g m i c r o d i s s e c t e d s a m p le s t o

m in i m is e c o n t a m i n a t i o n by e n d o g e n o u s p r o t e a s e s . The AChE c o n t e n t

-57-

FIG. 2.8 T y p ic a l s u c r o s e g r a d i e n t p r o f i l e s o f AChE fo rm s in

some mammalian t y p e I I m u s c l e s . A, r a t s t e r n o m a s t o i d ; B, r a t

EDL; C, m ouse AT. A l l e x t r a c t s w ere p r e p a r e d in t h e p r e s e n c e o f

p r o t e a s e i n h i b i t o r s .

-58-

FIG- 2-9 T y p ic a l s u c r o s e g r a d i e n t p r o f i l e s o f AChE form s in

some mammalian t y p e I m u s c l e s . A, h a m s te r s o l e u s ; B, r a b b i t

sem i-m em b ra n o su s p r o p r i u s ; C, g u in e a p i g sem i-m em b ra n o su s

p r o p r i u s . A l l e x t r a c t s w ere p r e p a r e d in t h e p r e s e n c e o f p r o t e a s e

i n h i b i t o r s .

-59-

o f w h o le m u s c le f i b r e s a s w e l l a s o f e n d p l a t e and n o n - e n d p l a t e

r e g i o n s w e r e a n a l y s e d .

U s in g t h e s t e r n o m a s t o i d and s o l e u s o f t h e r a t a s e x a m p le s o f

t y p e I I and t y p e I m u s c l e s r e s p e c t i v e l y , t h e p r o f i l e s o f AChE

fo rm s o b t a i n e d from w h o le m u s c l e f i b r e s w e r e v e r y s i m i l a r t o

t h o s e from w h o le m u s c le e x t r a c t s ( c f . F i g . 2 .1 0 A ,B and F i g . 2 .

8A , F i g . 2 . 5 B ) . The AChE p r o f i l e from t h e s t e r n o m a s t o i d

c o n s i s t e d o f a s u b s t a n t i a l am ount o f t h e L f o r m s , w h i l e t h e

s o l e u s c o n t a i n e d a much s m a l l e r L c o m p o n e n t , w i t h a b o u t eq u a l

a m o u n ts o f t h e H^c an d H2 c f o r m s . D i s s e c t i o n o f t h e e n d p l a t e

and n o n - e n d p l a t e r e g i o n s o f t h e s e m u s c l e s c o n f i r m e d t h e

e n d p l a t e - s p e c i f i c l o c a l i z a t i o n o f t h e H2c form a s o b s e r v e d

p r e v i o u s l y i n t y p e I I m u s c l e s ( J e d r z e j c z y k e t a l . , 1 9 8 1 ; F i g .

2 . 1 1 A , B ) . The l a r g e a m o u n ts o f L and M fo rm s in t h e

s t e r n o m a s t o i d w ere m a in ly l o c a t e d o u t s i d e t h e e n d p l a t e s ( F i g .

2 . 1 1 C ) . The l o w e r m o l e c u l a r w e i g h t fo rm s a t t h e e n d p l a t e s c o u l d

be c o n t a m i n a n t s from t h e n o n - e n d p l a t e r e g i o n s i n c l u d e d in t h e

s a m p le , e s p e c i a l l y s i n c e t h e AChE a c t i v i t y o u t s i d e t h e e n d p l a t e s

i n t h i s c a s e was h i g h . F o r t h e s l o w - t w i t c h s o l e u s ( F i g . 2 . 1 1

B , D , E ) , t h e form w a s fo u n d i n s i d e a s w e l l a s o u t s i d e t h e

e n d p l a t e s , a s i n t h e c a s e o f t h e g u in e a p i g s o l e u s ( s e e C h a p te r

• 3 , F i g . 3 .5 B and 3 .6 B ) and t h e r a b b i t sem im em b ran osu s p r o p r i u s

( J . J e d r z e j c z y k , p e r s o n a l c o m m u n i c a t i o n ) . T h i s e x t r a j u n c t i o n a l

H2C i n t h e r a t s o l e u s a p p e a r e d t o l o c a l i s e c l o s e t o t h e

e n d p l a t e r e g i o n , s i n c e r e g i o n s away from t h e e n d p l a t e s to w a r d t h e

e n d s o f t h e f i b r e s c o n t a i n v e r y much l e s s H2 c form ( F i g . 2 .

1 1 E ) . In t h e g u in e a p i g s o l e u s , h o w e v e r , t h e e x t r a j u n c t i o n a l

H2 c form w as fo u n d a l o n g t h e e n t i r e l e n g t h o f t h e f i b r e . The

- 6 0 -

4 0 A

FRACTIONSFIG- 2-10 M o l e c u l a r form s o f AChE from t y p e I an d t y p e I I m u s c le

f i b r e s . A, r a t s t e r n o m a s t o i d ( t y p e I I ) ; B, r a t s o l e u s ( t y p e I ) .

D o t t e d l i n e in (A) r e p r e s e n t s b a c k g r o u n d a b s o r b a n c e

o f s u c r o s e g r a d i e n t f r a c t i o n s i n c u b a t e d in t h e p r e s e n c e o f b o th

isoOMPA (1 x 1 0 _ 4M) an d BW284C51 ( 5 x 1 0 ’ ^M). A rrow s d e n o t e

t h e c o r r e s p o n d i n g p o s i t i o n s o f c a t a l a s e ( c a t ) and 8- g a l a c t o s i d a s e

( 8- g a l ) . B oth e x t r a c t s w ere p r e p a r e d i n t h e p r e s e n c e o f p r o t e a s e

i n h i b i t o r s . R e p e a t e x p e r im e n t s g a v e s i m i l a r p r o f i l e s .

- 6 1 -

FIG. 2.11 The d i s t r i b u t i o n o f m o l e c u l a r fo rm s o f AChE i n t h e

e n d p l a t e and n o n - e n d p l a t e r e g i o n s from t y p e I and t y p e I I

m u s c l e s . A,B, e n d - p l a t e r e g i o n o f r a t s t e r n o m a s t o i d ( t y p e I I )

and s o l e u s ( t y p e I ) , r e s p e c t i v e l y ; C, n o n - e n d p l a t e r e g i o n o f r a t

s t e r n o m a s t o i d ; D, p e r i j u n c t i o n a l r e g i o n o f r a t s o l e u s ; E,

n o n - e n d p l a t e r e g i o n o f r a t s o l e u s . D o t t e d l i n e d e n o t e s t h e

b a c k g r o u n d a b s o r b a n c e o f s u c r o s e g r a d i e n t f r a c t i o n s . The am ount

o f t i s s u e sa m p le u s e d i n e a c h a n a l y s i s was a r b i t r a r y , . A l l

e x t r a c t s w e r e p r e p a r e d i n t h e p r e s e n c e o f p r o t e a s e i n h i b i t o r s .

R e p e a t e x p e r i m e n t s g a v e s i m i l a r p r o f i l e s .

- 6 2 -

H^c fo r m , a m a jo r form o f t h e AChE i n t h e s o l e u s , w as a l s o

l o c a l i s e d o u t s i d e t h e e n d p l a t e s . T h e s e r e s u l t s s u g g e s t ,

t h e r e f o r e , t h a t t h e a n a l y s i s o f AChE form s by m i c r o d i s s e c t i o n

c o r r e l a t e d w e l l w i t h w h o le m u s c le e x t r a c t s . I t i s a l s o e v i d e n t

from t h e a n a l y s i s o f e n d p l a t e and n o n - e n d p l a t e AChE t h a t t h e h ig h

l e v e l o f L form s i n t h e s t e r n o m a s t o i d m u s c le i s n o t a d e g r a d a t o r y

p r o d u c t o f t h e 1 ^ form s i n c e t h e two a r e s p a t i a l l y s e p a r a t e .

H ow ever , t h e p o s s i b i l i t y t h a t some o f t h e L fo rm s w e r e breakdow n

p r o d u c t s o f t h e M form c o u l d n o t be r u l e d o u t .

A f e a t u r e o f t h e t y p e I m u s c l e s w h ich d i s t i n g u i s h e s them

from t y p e I I m u s c l e s i s t h e r e f o r e t h e p r e s e n c e o f e x t r a s y n a p t i c

p o p u l a t i o n o f and H£C f o r m s . F o r t y p e I a n d t y p e I I

m u s c l e s w h ic h have s i m i l a r HgQ and AChE c o n t e n t p e r u n i t w e i g h t

( T a b l e 2 . 3 ) , t h e am ount o f H£C form i n t h e e n d p l a t e s o f t y p e I

m u s c l e s w o u ld be l e s s th a n t h a t in t h e t y p e I I m u s c l e s s i n c e p a r t

o f t h e H£C AChE i n t h e f o r m e r i s d i s t r i b u t e d o v e r a l a r g e r

vo lu m e o f t h e m u s c l e .

2 . 3 . 6 Molecular profiles of AChE in avian ouseles

P r e v i o u s s t u d i e s show ed t h a t m u s c l e s o f m ix ed t y p e I and

t y p e IIA f i b r e s ( e . g . t h e s a r t o r i u s re d s e c t o r , 40% t y p e I , 60%

• t y p e I I ) c o n t a i n a h i g h e r l e v e l o f t h e L and Hl c AChE th a n a

p u r e t y p e I IB m u s c le su c h a s t h e PLD (B a rn a rd e t a l . . 1 9 8 2 ) . In

t h i s s t u d y t h e a n t e r i o r r e g i o n o f t h e a d d u c t o r p r o f u n d u s (31%

t y p e I , 69% t y p e I IA ) was a l s o fo u n d t o c o n t a i n a s i g n i f i c a n t

am ount o f t h e L form and a d i s t i n c t H^c co m p o n en t ( F i g . 2 . 1 2 ) ,

T h e s e fo rm s w e r e p r o b a b ly c h a r a c t e r i s t i c s o f t h e a v i a n t y p e I an d

IIA f i b r e s , s i n c e a p u r e f a s t - t w i t c h m u s c l e , su c h a s t h e PLD,

- 6 3 -

TABLE 2.3 AChE a ctiv ity in some mammalian muscles

Species Muscle Fibre type [AChE] (U/g) n

guinea pig EDL I I 0.79 ± 0.03 2

SAa 0.37 ± 0.04 7

Vastus la te ra lis (white) 0.45 1

Vastus la te ra lis (red) 0.71 1

Rabbit SA 0.49 ± 0.03 7

Rat EDL 1.03 ± 0.0 2

Sternomastold (red) 1.00 ± 0.02 2

Sternomastoid (white) 1.17 ± 0.12 2

Guinea pig Soleus I 0.49 ± 0.06 5

SPb 0.56 ± 0.04 6

Plantarls 0.41 1

Rabbi t Soleus 0.6 ± 0.07 6

SP 0.55 ± 0.07 7

Rat Soleus 0.31 ± 0.02 2

Rat Diaphragm I + I I 0.69 ± 0.02 2

a Semimembranosus accessorius (see also chapter 3),

b Semimembranosus proprlus (see also chapter 3),

The a c t iv ity of AChE 1s expressed as units per gram tissue wet weight, and

the standard error 1s indicated for n number of determinations.

- 6 4 -

FIG- 2-12 M o l e c u l a r form s o f AChE i n t h e a n t e r i o r and p o s t e r i o r

r e g i o n s o f t h e c h i c k e n a d d u c t o r p r o f u n d u s . Dotted line: s u c r o s e

g r a d i e n t p r o f i l e o f AChE in t h e a n t e r i o r r e g i o n , Solid line:

s u c r o s e g r a d i e n t p r o f i l e o f AChE in t h e p o s t e r i o r r e g i o n .

E x t r a c t s w e r e p r e p a r e d i n t h e p r e s e n c e o f p r o t e a s e i n h i b i t o r s

( T a b l e 2 . 2 ) . R e s u l t s w e r e from a t y p i c a l e x p e r i m e n t .

-65-

d o e s n o t c o n t a i n s i g n i f i c a n t am ou n ts o f t h e s e fo rm s ( F i g . 2 .1 3 ) .

T h i s f i n d i n g i n c h i c k e n m u s c l e s i s c l e a r l y d i f f e r e n t from t h a t in

mammalian m u s c l e s . The t y p e I I I o r t o n i c m u s c l e , su ch a s t h e ALD

( L y l e s e t __ a l . . , 1 9 8 1 ) and t h e p o s t e r i o r r e g i o n o f t h e a d d u c t o r

p r o f u n d u s ( F i g . 2 . 1 2 ) c o n s i s t e d o f L^ and M fo r m s , w i t h v e r y

l i t t l e H£C fo r m .

2 . 3 . 7 Molecular profiles of AChE in anscles of Mixed fibre type

M u s c le s o f m ix ed f i b r e t y p e c o m p o s i t i o n , su ch a s t h e r a t

d ia p h r a g m , w e r e s t u d i e d . In t h i s mammalian m u s c l e , a b o u t eq u a l

am ou n ts o f L, M and H fo rm s w e r e fo u n d ( F i g . 2 . 1 4 ) . The l a r g e L

and M c o m p o n e n ts w e r e p r o b a b ly c o n t r i b u t e d by t h e t y p e I I f i b r e s ,

i f t h e y w ere a ssu m ed t o h a v e a s i m i l a r d i s t r i b u t i o n o f form s a s

t h e s t e r n o m a s t o i d o r t h e EDL. A n a l y s i s o f e n d p l a t e and

n o n - e n d p l a t e AChE a g a i n r e v e a l e d t h e form a s t h e j u n c t i o n a l

co m p o n en t o f AChE, w h i l e t h e L an d M fo rm s w ere r e s t r i c t e d t o t h e

e x t r a j u n c t i o n a l r e g i o n s ( F i g . 2 . 1 5 ) .

2 . 4 DISCUSSION

2 . 4 . 1 Autolysis of AChE in tissue crude extracts

The t e c h n i q u e o f s e d i m e n t a t i o n a n a l y s i s h a s b e e n u s e d

e x t e n s i v e l y f o r t h e s tu d y o f m u l t i p l e m o l e c u l a r form s o f AChE i n

t i s s u e e x t r a c t s . The p r e s e n t r e s u l t s show t h a t t h e r e l a t i v e

p r o p o r t i o n s o f t h e s e form s c o u l d i n a d v e r t e n t l y be g r e a t l y

a l t e r e d i n v i t r o by t h e a c t i v i t i e s o f e n d o g e n o u s p r o t e a s e s , b u t

t h e p ro b lem c o u l d l a r g e l y be a v o i d e d by p r o c e s s i n g c r u d e e x t r a c t s

r a p i d l y in t h e p r e s e n c e o f a s u i t a b l e a r r a y o f p r o t e a s e

i n h i b i t o r s .

-66-

1.0

F IG - 2 - 1 3 S u c r o s e g r a d i e n t p r o f i l e o f AChE in t h e c h i c k e n PLD

m u s c l e .

- 6 7 -

10 3 0 5 0

Fractions

FIG. 2.14 S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n some m u s c l e s o f

m ix e d f i b r e t y p e c o m p o s i t i o n . A, r a t d ia p h ra g m ; B, m ou se s o l e u s .

E x t r a c t s w ere p r e p a r e d i n t h e p r e s e n c e o f p r o t e a s e i n h i b i t o r s .

R e p e a t e x p e r i m e n t s showed s i m i l a r d i s t r i b u t i o n o f m o l e c u l a r

f o r m s .

-68-

FIG- 2-15 D i s t r i b u t i o n o f m o l e c u l a r forms o f AChE i n t h e

e n d p l a t e and n o n - e n d p l a t e r e g i o n s o f t h e r a t d i a p h r a g m . A,

s u c r o s e g r a d i e n t p r o f i l e o f AChE i n t h e e n d p l a t e ; B, s u c r o s e

g r a d i e n t p r o f i l e s o f AChE o u t s i d e t h e e n d p l a t e s . R e s u l t s o f J .

• J e d r z e j c z y k .

-69-

The breakdown o f t h e m u l t i - s u b u n i t AChE fo rm s i n e l e c t r i c

o r g a n t i s s u e e x t r a c t was f i r s t r e p o r t e d by M a s s o u l i e a n d R i e g e r

( 1 9 6 9 ) . L a t e r , R i e g e r a nd V i g n y ( 1 9 7 6 ) n o t e d a t i m e - d e p e n d e n t

r e d u c t i o n o f t h e a n d M fo rm s and a p r o p o r t i o n a l i n c r e a s e

o f L f o r m s i n r a t d ia p h r a g m e x t r a c t s o v e r a p e r i o d o f s e v e r a l

d a y s , a t room t e m p e r a t u r e , and r e g a r d e d t h e s e c h a n g e s t o o s l o w t o

h a v e a n y s i g n i f i c a n t e f f e c t on t h e a n a l y s i s o f AChE f o r m s ,

n o r m a l l y c o m p l e t e d w i t h i n 1 d a y . Rotun do and Fambrough ( 1 9 7 9 )

o b s e r v e d t h a t s t o r i n g c h i c k m u s c l e c r u d e e x t r a c t s l e d t o a r a p i d

l o s s o f H f o rm s i n t h e s e e x t r a c t s , w h i l e i s o l a t e d m o l e c u l a r fo rm s

r e t a i n e d t h e i r s e d i m e n t a t i o n c h a r a c t e r i s t i c s f o r a l o n g t i m e ,

w h e t h e r s t o r e d f r o z e n a t - 2 0 ° C o r i n s o l u t i o n a t 5 ° C . S i l m a n e t

a l . ( 1 9 7 8 ) showed t h a t r e p e a t e d f r e e z i n g and t h a w i n g o f m u s c l e

e x t r a c t s , o r f r e e z e - s t o r i n g t i s s u e s a l w a y s g i v e r i s e t o some

d e g r e e o f breakdown o f t h e f o rm ; t h e y f u r t h e r showed t h a t a

m i x t u r e o f p r o t e a s e i n h i b i t o r s c o u l d p r e v e n t t h e breakdow n o f t h e

h i g h e r m o l e c u l a r w e i g h t f o rm s d u r i n g e x t r a c t i o n (1 h o u r a t 3 8 °C )

from f r e s h c h i c k e n m u s c l e . T h e s e r e s u l t s c l e a r l y show t h a t t h e

c o n v e r s i o n o f t h e AChE f o rm s i n v i t r o i s n o t due t o a s p o n t a n e o u s

i n t e r c o n v e r s i o n o f t h e s e fo rm s, b u t a r e s u l t o f t h e br eakdo w n o f

t h e m u l t i - s u b u n i t c o m p le x i n p a r t by e n d o g e n o u s p r o t e o l y t i c

• d e g r a d a t i o n .

2 . 4 . 2 Correlation of the degradation of the H2c fora with the

proteolytic activity in tissue crude extracts

The t i m e c o u r s e o f t h e i n v i t r o breakdown o f t h e H f o r m s i s

n o t c l e a r b e c a u s e t h e e x p e r i m e n t a l c o n d i t i o n s and t h e t i s s u e

e x t r a c t s u s e d i n v a r i o u s s t u d i e s d i f f e r e d w i d e l y . H o w e v e r , a s

- 7 0 -

t h i s s t u d y h a s shown , t h e r a t e a t w h ic h su ch breakdown t a k e s

p l a c e d u r i n g e x t r a c t i o n h a s b een g r e a t l y u n d e r e s t i m a t e d ,

e s p e c i a l l y i n t h e c a s e o f AChE i n r a t m u s c l e e x t r a c t s .

S u b s t a n t i a l breakdo wn o f H fo rm s o c c u r r e d w i t h i n 1 ho ur a t 4 ° C ,

w h e t h e r i t was k e p t a s c r u d e e x t r a c t ( F i g . 2 . 5 A ) o r a s h o m o g e n a te

( r a t d i a p h r a g m , n o t s h o w n ) . For g u i n e a p i g m u s c l e s , r a p i d

p r o c e s s i n g a l o n e may i n c r e a s e t h e p r o p o r t i o n o f H fo rm s o b s e r v e d ,

b u t t h e m o l e c u l a r p r o f i l e i s h i g h l y v a r i a b l e . The c o n v e r s i o n

from H fo rm s t o L fo rm s may v a r y w i t h t h e p r o t e o l y t i c a c t i v i t y

p r e s e n t i n d i f f e r e n t t i s s u e e x t r a c t s a n d , d e p e n d i n g on s u c h

a c t i v i t y and t h e t i m e t h e H fo rm s a r e e x p o s e d t o t h e p r o t e a s e s ,

w o u ld g i v e r i s e t o a g r e a t v a r i e t y o f m o l e c u l a r p r o f i l e s w h ic h do

n o t r e p r e s e n t t h e d i s t r i b u t i o n i n v i v o ,

2 . 4 . 3 Inhibition of proteolysis stabilised the Molecular

distribution of AChE forus

A v a r i e t y o f p r o t e a s e i n h i b i t o r s w i t h w e l l - d e f i n e d

s p e c i f i c i t y w e r e u s e d i n c o m b i n a t i o n t o i n h i b i t a l l known

p r o t e a s e s w h ic h c o u l d be p r e s e n t i n m u s c l e e x t r a c t s . S e r i n e

p r o t e a s e s a c c o u n t f o r a s i g n i f i c a n t p r o p o r t i o n o f p r o t e a s e

a c t i v i t y i n r a t m u s c l e s (Katnuma and Kominami , 1 9 7 7 ) , p r o b a b l y

• o f m a s t c e l l o r i g i n ( L a g u n o f f and B e n d i t t , 1 9 6 3 ) . A number o f

s e r i n e p r o t e a s e i n h i b i t o r s w h ich do n o t e x h i b i t i n h i b i t o r y

a c t i v i t y on AChE w e r e u s e d i n a d d i t i o n t o e n s u r e t h e i n h i b i t i o n

o f t h e s e p r o t e a s e s , w h ic h may e x h i b i t d i f f e r e n t i n h i b i t o r

s p e c i f i c i t y i n d i f f e r e n t s p e c i e s ( G l e n n e r a nd C oh en , 1 9 6 0 ) . The

c o n c e n t r a t i o n o f t h e s e i n h i b i t o r s r e q u i r e d f o r maximum i n h i b i t i o n

was h i g h , a s e x e m p l i f i e d by t h e d i f f e r e n c e i n t h e amount o f SVI

- 7 1 -

r e q u i r e d f o r 50% i n h i b i t i o n o f b o v i n e a - c h y m o t r y p s i n and o f r a t

m u s c l e p r o t e a s e s ( c . f . F i g . 2 .2 C and F i g . 2 . 3 ) , H o w ev er ,

O g - m a c r o g l o b u l i n a p p e a r e d t o be an e x c e p t i o n : a t a

c o n c e n t r a t i o n o f O . lm g/m l ( 0 . 1 4 p M ) , i t r e d u c e d t h e r a t m u s c l e

p r o t e a s e a c t i v i t y t o m inim al l e v e l . The a p p a r e n t r e c o v e r y o f

p r o t e a s e a c t i v i t y i s p r o b a b l y due t o r e s i d u a l p r o t e a s e and n o t t o

r e v e r s i b l e i n h i b i t i o n , a s p r e v i o u s e x p e r i m e n t s ha ve d e m o n s t r a t e d

t h e i r r e v e r s i b l e n a t u r e o f t h e a g - m a c r o g l o b u l i n - e n z y m e c o m p le x

( B a r r e t t and S t a r k e y , 1 9 7 3 ; S a u n d e r s e t a l . , 1 9 7 1 ) . R e s i d u a l

p r o t e a s e a c t i v i t y was o f t e n a c c o m p a n ie d by a d i s c r e p a n c y i n t h e

o b s e r v e d AChE p r o f i l e s , s u g g e s t i n g t h a t e v e n l o w p r o t e a s e

a c t i v i t y p r e s e n t i n m u s c l e h o m o g e n a t e s c o u l d be s u f f i c i e n t t o

a l t e r t h e r e l a t i v e p r o p o r t i o n o f AChE fo rm s i n v i t r o , and t h a t

t h e d e g r a d a t i o n o f t h e s e fo rm s p r o b a b l y i n v o l v e d a v a r i e t y o f

p r o t e a s e s i n t h e e x t r a c t s . T h i s w o u ld a l s o e x p l a i n t h e h i g h

l e v e l o f L fo rm s o b s e r v e d i n p r e v i o u s i n v e s t i g a t i o n s i n w h ic h

some p r o t e a s e i n h i b i t o r s w ere e m p lo y e d ( G i s i g e r a n d S t e p h e n s ,

1 9 8 3 ; Bacou e t __ a L , 1 9 8 2 ) , s i n c e u n s u i t a b l e o r i n s u f f i c i e n t

i n h i b i t o r s w ou ld be i n e f f e c t i v e . Hence c o m p l e t e i n h i b i t i o n o f

p r o t e a s e a c t i v i t y i s i m p o r t a n t f o r t h e a n a l y s i s o f AChE f o r m s .

# 2 . 4 . 4 . Tiae course and nechanisa of ACHE degradation

The p r o c e s s o f h o m o g e n i s a t i o n f a c i l i t a t e s p r o t e o l y s i s by

e x p o s i n g AChE t o t h e p r o t e a s e s ; t h u s t h e breakdown o f H fo rm s i n

c r u d e e x t r a c t s w o u ld t a k e p l a c e more r e a d i l y t h a n w o u ld o c c u r i n ,

s a y , f r o z e n i n t a c t t i s s u e s . I n i t i a l p e r f u s i o n o r r a p i d f r e e z i n g

b e f o r e h o m o g e n i s a t i o n , t h e r e f o r e , h a d l i t t l e e f f e c t i n p r e v e n t i n g

s u c h br ea k d o w n . The r a p i d i t y w i t h w h ic h t h e t r a n s f o r m a t i o n o f H

- 7 2 -

t o L fo rm s o c c u r r e d d u r i n g t h e f i r s t few h o u r s o f e x t r a c t i o n

c o n t r a s t s w i t h t h e much s l o w e r r a t e o f c o n v e r s i o n o f t h e M f o rm s

t o L f o r m s o b s e r v e d by R i e g e r and V i g n y ( 1 9 7 6 ) . Such d i f f e r e n c e s

c o u l d be due t o t h e i n s t a b i l i t y o r d e g r a d a t i o n o f t h e p r o t e a s e s

t h e m s e l v e s w i t h l o n g i n c u b a t i o n . M o r e o v e r , t h e d i f f e r e n c e i n

t h i s i n i t i a l p r o t e o l y t i c a c t i v i t y o b s e r v e d i n d i f f e r e n t s p e c i e s ,

a p a r t from t h e a c t u a l amount and v a r i e t y o f p r o t e a s e s p r e s e n t ,

may a l s o be i n f l u e n c e d by t h e i r e n z y m i c s t a t e when t h e y a r e

m e c h a n i c a l l y r e l e a s e d : e v i d e n c e s u g g e s t s t h a t r a t m a s t c e l l

p r o t e a s e s a r e s t o r e d i n t h e i r a c t i v e fo rm s i n t h e g r a n u l e s ( C h iu

and L a g u n o f f , 1 9 7 2 ) w h i l e g u i n e a p i g m a s t c e l l p r o t e a s e s a r e

s t o r e d a s z y m o g e n s (U n gar a n d Damgaard, 1 9 5 5 ) .

The q u e s t i o n o f how t h e c o l l a g e n - t a i l e d o r g l o b u l a r fo rm s

a r e b r o k e n down i n v i t r o r e q u i r e s t h e s t r u c t u r a l e l u c i d a t i o n o f

t h e c a t a l y t i c s u b u n i t s and t h e i r a s s o c i a t i o n t o form s t a b l e

q u a t e r n a r y p o l y m e r s . The g l o b u l a r s u b u n i t s a p p e a r t o a s s o c i a t e

w i t h e a c h o t h e r a s w e l l a s w i t h t h e c o l l a g e n - t a i l v i a d i s u l p h i d e

b r i d g e s ( s e e G e n e r a l I n t r o d u c t i o n ) . In a d d i t i o n , e v i d e n c e

s u g g e s t s t h a t s u c h q u a t e r n a r y a s s o c i a t i o n a l s o i n v o l v e s

n o n - c o v a l e n t i n t e r - s u b u n i t i n t e r a c t i o n , s i n c e s u b u n i t

d i s s o c i a t i o n w o u ld n o t o c c u r e v e n a f t e r d i s u l p h i d e r e d u c t i o n

• u n l e s s t h e enzym e i s s u b j e c t e d t o d e n a t u r a t i o n o r l i m i t e d

p r o t e o l y t i c d i g e s t (Bon and M a s s o u l i e , 1 9 7 6 ; Mays and R o s e n b e r r y ,

1 9 8 1 ; G r a s s i e t __ a l . , 1 9 8 2 ; Lee e t __ a l _ . , 1 9 8 2 ) . I t may b e

e n i v s a g e d , t h e r e f o r e , t h a t t h e d i s s o c i a t i o n o f t h e s u b u n i t s from

e a c h o t h e r r e q u i r e s some e n z y m i c c l e a v a g e on t h e p o l y p e p t i d e

b a c k b o n e a t a s i t e w h ic h w o u ld s e p a r a t e t h e b u l k o f t h e c a t a l y t i c

s u b u n i t from t h e r e g i o n w h ic h c o n t a i n s t h e h a l f c y s t e i n e a nd

- 7 3 -

w h i c h i s i n v o l v e d i n t h e i n t e r s u b u n i t i n t e r a c t i o n . Such e n z y m i c

c l e a v a g e i s l i k e l y t o r e l e a s e t h e L fo rm s from t h e M a n d H f o r m s ,

w h ic h i s m o s t commonly a s s o c i a t e d w i t h t h e breakdow n o f

Hg AChE. F u r t h e r m o r e , su ch e n z y m i c c l e a v a g e c a n a l s o o p e r a t e

b e t w e e n t h e g l o b u l a r s u b u n i t s and t h e c o l l a g e n t a i l , g i v i n g r i s e

t o t h e g l o b u l a r M f o r m . L i m i t e d t r y p s i n d i g e s t , f o r e x a m p l e , h a s

b e e n shown t o d e g r a d e t h e ^ form from c h i c k m u s c l e ( R o tu n d o

and Fambrough, 1 9 7 9 ) and e l e c t r i c o r g a n ( M a s s o u l i § a n d R i e g e r ,

1 9 6 9 ) i n a s e q u e n t i a l m an n er , y i e l d i n g L and M f o r m s . The

r e s u l t s p r e s e n t e d i n t h i s c h a p t e r show t h a t i n mammalian m u s c l e

t i s s u e s , t h e d i s s o c i a t i o n o f t h e g l o b u l a r s u b u n i t s from H AChE

o c c u r s r a p i d l y i n v i t r o , s u g g e s t i n g t h a t c e r t a i n r e g i o n s on t h e

p o l y p e p t i d e i n v o l v e d i n q u a t e r n a r y i n t e r a c t i o n s may be

s u s c e p t i b l e t o a t t a c k s by e n d o g e n o u s e n d o p e p t i d a s e . T h a t t h e

s u b u n i t s w h ic h a r i s e from d e g r a d a t i o n a r e i n d i s t i n g u i s h a b l e from

t h e n a t i v e L and M f o rm s by t h e i r s e d i m e n t a t i o n c o e f f i c i e n t s

s u g g e s t s t h a t d e g r a d a t i o n d o e s n o t r e s u l t i n any e x t e n s i v e l o s s

o f t h e s u b u n i t m o l e c u l a r s i z e o r c a t a l y t i c a c t i v i t y . I n d e e d ,

t r y p s i n t r e a t m e n t o f t h e t e t r a m e r i c human serum AChE g e n e r a t e s

a c t i v e monomers w i t h a v e r y s m a l l c h a n g e i n t h e a p p a r e n t

m o l e c u l a r w e i g h t o f t h e f u l l y r e d u c e d c a t a l y t i c s u b u n i t

( L o c k r i d g e and La Du, 1 9 8 2 ) . C t e r m i n a l s e q u e n c i n g o f p u r i f i e d

human serum AChE by L o c k r i d g e ( 1 9 8 4 ) sh ow s t h a t

a c y s t e i n e r e s i d u e p r e s e n t c l o s e t o t h e C t e r m i n u s may b e

i n v o l v e d i n t h e i n t e r s u b u n i t d i s u l p h i d e b o n d i n g . I t i s

t h e r e f o r e , r e a s o n a b l e t o s u g g e s t t h a t t h e p r o x i m i t y o f t h e

d i s u l p h i d e bond t o t h e C t e r m i n u s , and i t s a c c e s s i b i l i t y t o

p r o t e o l y t i c d e g r a d a t i o n c o u l d d e t e r m i n e t h e s u s c e p t i b i l i t y o f t h e

- 7 4 -

m o l e c u l a r fo rm s t o breakdown i n v i t r o . Such s p e c u l a t i o n c a n o n l y

b e v e r i f i e d by t h e e l u c i d a t i o n o f t h e s u b u n i t p e p t i d e s e q u e n c e o f

AChE i n v a r i o u s s p e c i e s .

2 . 4 . 5 is a dominant component of M i l fan skeletal ouscle

AChE

S k e l e t a l m u s c l e s from a number o f mammalian s p e c i e s ha v e

r e v e a l e d t h a t t h e H2 c form c o n s t i t u t e s a s i g n i f i c a n t p r o p o r t i o n

(30%-71%) o f t o t a l AChE a c t i v i t y i n b o t h f a s t - and s l o w - t w i t c h

m u s c l e s ( T a b l e 2 . 1 ) e x c e p t i n r a t f a s t - t w i t c h m u s c l e s w h e r e t h e

am ou nt o f Hg d o e s n o t e x c e e d 20% o f t o t a l AChE. A g r o s s

u n d e r e s t i m a t i o n o f t h e H2 c p o p u l a t i o n i n mammalian m u s c l e s h as

b e e n a r e s u l t o f e x p e r i m e n t s p e f o r m e d i n t h e a b s e n c e o f p r o t e a s e

i n h i b i t o r s l e a d i n g t o a r a p i d breakdown o f H2 c , e s p e c i a l l y i n

r a t m u s c l e s . I t i s a l s o due t o t h e l a c k o f a c o m p r e h e n s i v e s t u d y

o f t h e d i s t r i b u t i o n o f AChE f o rm s i n m u s c l e s from d i f f e r e n t

s p e c i e s , and b a s i n g o b s e r v a t i o n s m o s t l y on r a t f a s t - t w i t c h

m u s c l e s w h o se AChE p r o f i l e s a r e a t y p i c a l i n c o n t a i n i n g a l a r g e

p r o p o r t i o n o f L f o r m s .

2 . 4 . 6 AChE profiles in fast- and slow-twitch wuscles

S l o w - t w i t c h m u s c l e s w h ic h a r e co m p o se d p r e d o m i n a n t l y o f t y p e

I f i b r e s a l l e x h i b i t a d o m in a n t p r o p o r t i o n o f H^AChE and a

l a r g e , d i s t i n c t H^c c o m p o n e n t . No s i g n i f i c a n t d i f f e r e n c e

b e t w e e n t h e s e p r o f i l e s , e x c e p t t h e somewhat v a r i a b l e H^c

c o m p o n e n t , i s a p p a r e n t . On t h e o t h e r h a n d , t h e f a s t - t w i t c h

m u s c l e s w h ic h c o n s i s t l a r g e l y o f t y p e I I f i b r e s r e v e a l a more

v a r i e d and a p p a r e n t l y s p e c i e s - s p e c i f i c d i s t r i b u t i o n o f t h e AChE

- 7 5 -

f o r m s . A g e n e r a l p a t t e r n o f d i s t r i b u t i o n i s p r e v a l e n t i n f a s t

m u s c l e s from t h e same s p e c i e s b u t may d i f f e r b e t w e e n t h e same

m u s c l e from d i f f e r e n t s p e c i e s . Thus a l l r a b b i t f a s t m u s c l e s ha v e

e q u a l a m o u n ts o f L and H f o r m s ; h a m s t e r f a s t m u s c l e s a l w a y s

h a v e a p r e d o m i n a n t H^ c o m p o n e n t w h i l e t h a t i n r a t f a s t m u s c l e s

i s s m a l l . G uin ea p i g f a s t m u s c l e s h a v e a s i m i l a r AChE

d i s t r i b u t i o n a s t h e h a m s t e r f a s t m u s c l e s e x c e p t t h e EDL and t h e

s e m i- m e m b r a n o su s a c c e s s o r i u s w h ic h r e s e m b l e more c l o s e l y t h a t o f

t h e r a b b i t f a s t m u s c l e s . I n t e r - s p e c i e s d i f f e r e n c e s a r e m o s t

s t r i k i n g l y i l l u s t r a t e d by t h e d i f f e r e n t d i s t r i b u t i o n o f AChE

f o rm s i n t h e EDL o f t h e r a t ( F i g . 2 . 8B) a n d g u i n e a p i g ( F i g .

2 . 4 C ) d e s p i t e t h e s i m i l a r i t y b e t w e e n t h e i r f i b r e t y p e

c o m p o s i t i o n . F o r a p a r t i c u l a r s p e c i e s , m u s c l e s w i t h d i f f e r e n t

p r o p o r t i o n s o f t y p e IIA and I IB f i b r e s e x h i b i t s i m i l a r

d i s t r i b u t i o n o f AChE f o r m s , a s shown by t h e two r e g i o n s o f t h e

g u i n e a p i g v a s t u s l a t e r a l i s . T h i s s u g g e s t s t h a t t h e d i s t r i b u t i o n

o f AChE fo rm s i s n o t s i m p l y r e l a t e d t o t h e m e t a b o l i c p r o p e r t i e s

o f m u s c l e f i b r e s . F o r g u i n e a p i g and h a m s t e r , s i m i l a r p r o f i l e s

o f AChE fo rm s a r e f o u n d i n t h e f a s t - and s l o w - t w i t c h m u s c l e s ,

w h e r e a s d i s t i n c t p a t t e r n s o f d i s t r i b u t i o n o f t h e s e fo rm s a r e

a s s o c i a t e d w i t h t h e two f i b r e t y p e s i n r a b b i t ,

mouse and r a t . In m u s c l e s o f m ixe d f i b r e t y p e s s u c h a s t h e r a t

d i a p h r a g m , t h e AChE p r o f i l e i s c h a r a c t e r i s t i c o f a m i x t u r e o f t h e

c o n s t i t u e n t t y p e s , i . e . t y p e I a nd I I f i b r e s . Taken t o g e t h e r ,

t h e s e r e s u l t s s u g g e s t t h a t t h e d i s t r i b u t i o n o f AChE fo rm s

c o r r e l a t e s w i t h t h e f i b r e t y p e c o m p o s i t i o n o f t h e m u s c l e s ;

h o w e v e r , t h e two f i b r e t y p e s do n o t n e c e s s a r i l y h a v e d i f f e r e n t

AChE p r o f i l e s , a s s e e n i n g u i n e a p i g and h a m s t e r s . Where a

- 7 6 -

d i f f e r e n c e i s o b s e r v e d , i t i s m o s t l y due t o a l a r g e r p r o p o r t i o n

o f L f o rm s i n f a s t m u s c l e s r e l a t i v e t o t h a t i n s l o w m u s c l e s . The

p h y s i o l o g i c a l s i g n i f i c a n c e o f t h i s v a r y i n g am ount o f L fo rm s i n

d i f f e r e n t m u s c l e s i s n o t c l e a r ; h o w e v e r , some s p e c u l a t i o n f o r

t h e i r v a r i a b i l i t y i s s u g g e s t e d b e l o w i n s e c t i o n 2 . 4 . 8 . In t h e

c a s e o f r a t f a s t m u s c l e s , a l t h o u g h t h e H2 c form i s a m in o r AChE

c o m p o n e n t , t h e a c t u a l a c t i v i t y i t c o n s t i t u t e s i s s i m i l a r t o t h a t

i n t h e r a t s o l e u s , w h er e H2 c i s m a j o r . T h i s i s due t o t h e

e x c e p t i o n a l l y h i g h l e v e l o f AChE i n t h e f a s t m u s c l e s o f t h e r a t

( T a b l e 2 . 3 ) . Thus t h e H2 c a c t i v i t i e s a r e c o m p a r a b l e i n a l l t h e

m u s c l e s t h a t h a v e b e e n s t u d i e d d e s p i t e t h e p r e s e n c e o f d i f f e r e n t

p r o p o r t i o n s o f t h e l o w e r m o l e c u l a r w e i g h t f o rm s i n d i f f e r e n t

m u s c l e s

2 . 4 . 7 AChE profiles in chicken twitch and tonic auscles

C h ic k e n s k e l e t a l m u s c l e s p r o v i d e f u r t h e r e v i d e n c e f o r t h e

c o r r e l a t i o n b e t w e e n t h e d i s t r i b u t i o n o f AChE fo rm s and t h e f i b r e

t y p e c o m p o s i t i o n o f m u s c l e s . As ha s b e e n m e n t i o n e d a b o v e ,

t y p i c a l f a s t - t w i t c h m u s c l e s suc h a s t h e PLD c o n t a i n l a r g e l y H2c

( F i g . 2 . 1 3 ) , w h i l e t h e t o n i c ALD m u s c l e c o n t a i n s l a r g e l y L and M

f o r m s , w i t h a m in o r H2 c c o m p o n e n t t h a t d i m i n i i i s h e s w i t h

m a t u r a t i o n ( L y l e s and B a r n a r d , 1 9 8 0 ) . T h i s d i f f e r e n t i a l

d i s t r i b u t i o n o f AChE i s a l s o s e e n i n t h e a d d u c t o r p r o f u n d u s

m u s c l e , w h ic h i s d i v i d e d i n t o an a n t e r i o r r e g i o n o f t w i t c h f i b r e s

and a p o s t e r i o r r e g i o n o f t o n i c f i b r e s ( T a b l e 2 . 1 ) . The AChE

fo rm s e x h i b i t e d by t h e two r e g i o n s o f t h i s m u s c l e c o r r e l a t e w e l l

w i t h t h e i r r e s p e c t i v e f i b r e t y p e c h a r a c t e r i s t i c s ( F i g . 2 . 1 2 ) .

- 7 7 -

2 . 4 8 Non-endplate ACHE in type I and type II fibres

The o t h e r f e a t u r e w h ic h d i s t i n g u i s h e s t y p e I f i b r e s from

t y p e I I f i b r e s i s t h e p r e d o m in a n c e o f e x t r a j u n c t i o n a l H^ f o rm s

i n t y p e I f i b r e s , w h e r e a s L and M fo rm s a r e m o s t common o u t s i d e

t h e e n d p l a t e s o f t y p e I I f i b r e s ( F i g . 2 . 1 1 , F i g . 2 . 1 5 and C h a p t e r

3 , F i g s . 3 . 5 a n d 3 . 6 ) . T h e s e d i f f e r e n c e s i n t h e e x t r a j u n c t i o n a l

AChE b e t w e e n t h e two f i b r e t y p e s a r e t h u s r e s p o n s i b l e f o r t h e

v a r i a t i o n s i n t h e o v e r a l l AChE p r o f i l e s . The p h y s i o l o g i c a l and

f u n c t i o n a l s i g n i f i c a n c e f o r s u c h d i f f e r e n c e s i n t h e

e x t r a j u n c t i o n a l AChE p oo l b e t w e e n t h e two f i b r e t y p e s h a s y e t t o

b e d e t e r m i n e d . H ow e v e r , a number o f f a c t o r s may be i m p o r t a n t i n

d e t e r m i n i n g su c h d i s t r i b u t i o n . As h a s b e e n o b s e r v e d i n

s t i m u l a t i o n e x p e r i m e n t s , n o n - e n d p l a t e H fo rm s w e r e i n d u c e d i n

f a s t - t w i t c h m u s c l e s upon c o n t i n u o u s 1 o w - f r e q u e n c y s t i m u l a t i o n

( s e e C h a p t e r 4 ) ; t h e s e e x t r a s y n a p t i c H fo rm s t h u s a p p e a r t o be

a s s o c i a t e d w i t h t h e f u n c t i o n a l o u t p u t o f t h e m u s c l e s . T h i s may

a l s o be t r u e o f t h e mammalian s l o w - t w i t c h m u s c l e s w h ic h r e c e i v e

c o n t i n u o u s t r a i n s o f i m p u l s e s ( V r b o v a , Gordon a n d J o n e s , 1 9 7 8 ) .

The f a s t m u s c l e s , on t h e o t h e r h a n d , a r e r e l a t i v e l y i n a c t i v e i n

a n i m a l s i n c a p t i v i t y . Such i n a c t i v i t y may g i v e r i s e t o a l a r g e

e x t r a j u n c t i o n a l po o l o f g l o b u l a r f o r m s . T h e r e i s no o b v i o u s

• e x p l a n a t i o n f o r t h e s p e c i e s d i f f e r e n c e s o b s e r v e d i n t h e

d i s t r i b u t i o n o f AChE fo rm s i n t h e f a s t t w i t c h m u s c l e s . P r e v i o u s

o b s e r v a t i o n s show t h a t h i g h l e v e l s o f L and M f o rm s w e r e

m a i n t a i n e d i n f a s t m u s c l e s o f r a t t h r o u g h o u t d e v e l o p m e n t and

m a t u r a t i o n ( V i g n y e t a K , 1 9 7 6 ) , w h e r e a s t h e H form

p r o g r e s s i v e l y d i m i n i s h e s d u r i n g t h e same t i m e c o u r s e . The L and

M f o r m s , t h e r e f o r e , a p p e a r t o be r e g u l a t e d d i f f e r e n t l y from t h e

- 7 8 -

H20 AChE. S p e c i e s d i f f e r e n c e s i n t h e l e v e l o f L a n d M form s i n

f a s t m u s c l e s may p o s s i b l y be due t o d i f f e r e n c e s i n t h e

b i o s y n t h e s i s o r p o s t t r a n s l a t i o n a l p r o c e s s i n g o f t h e s e fo rm s.

The p r e s e n c e o f e x t r a j u n c t i o n a l H2 c form i n t y p e I f i b r e s

a l s o a p p e a r s t o c o m p le m e n t t h e l o w c o n c e n t r a t i o n o f

e x t r a j u n c t i o n a l ACh r e c e p t o r s a l o n g t h e e n t i r e l e n g t h o f t h e s e

f i b r e s (McA rdle a nd A l b u q u e r q u e , 1 9 7 3 , M i l e d i a n d Z e l e n a , 1 9 6 6 ) .

The p o s s i b l e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h i s form w i l l be

d i s c u s s e d i n C h a p t e r 3 ( s e e S e c t i o n 3 . 4 . 3 ) .

2 . 4 . 9 Endplate AChE in twitch and tonic fibres

The e n d p l a t e - s p e c i f i c n a t u r e o f t h e H2 c form i s c o n f i r m e d

i n b o t h t y p e I and t y p e I I m u s c l e s . In a d d i t i o n , t h e r e i s a

s i g n i f i c a n t H^c co m p o n e n t a t t h e e n d p l a t e s . T h i s e n d p l a t e

c o m p o n e n t a c c o u n t s f o r t h e common o c c u r r e n c e o f an H^c m o i e t y

i n b o t h t y p e I and t y p e I I f i b r e s o f mammalian m u s c l e s , and

s u g g e s t s t h a t i t may be a f u n c t i o n a l m o l e c u l a r form a t t h e

e n d p l a t e , a l t h o u g h t h e p o s s i b i l i t y t h a t i t i s a d e g r a d a t o r y

p r o d u c t o f t h e HgQAChE c a n n o t be e n t i r e l y r u l e d o u t . The

g l o b u l a r fo rm s o c c u r l a r g e l y a s c o n t a m i n a n t s a t t h e e n d p l a t e s o f

t h e s e t w i t c h f i b r e s , b u t become p r e d o m i n a n t i n t o n i c f i b r e

e n d p l a t e s , s u g g e s t i n g a d i f f e r e n t o r g a n i s a t i o n o f e n d p l a t e AChE

i n t h e t w i t c h and t o n i c f i b r e s ( J e d r z e j c z y k e t a l . . 1 9 8 4 ) .

W h e r e a s ^ i s p r o b a b l y l o c a l i s e d a t t h e b a s a l l a m i n a by

a n c h o r i n g v i a i t s c o l l a g e n - t a i l ( A n g l i s t e r a n d S i l m a n , 1 9 7 8 ;

McMahan e t a l . . 1 9 7 8 ) , t h e L a nd M fo rm s a t t h e e n d p l a t e s may be

a s s o c i a t e d w i t h t h e p la sm a membrane ( L y l e s and B a r n a r d , 1 9 8 0 ) a s

o b s e r v e d f o r t h e b r a i n AChE ( R i e g e r and V i g n y , 1 9 7 6 ; R o t u n d o ,

1 9 8 4 ) .

- 7 9 -

2 . 5 CONCLUSION

In c o n c l u s i o n , b o t h p a s t and p r e s e n t e v i d e n c e s u g g e s t s t h a t

t h e m u l t i p l e m o l e c u l a r f o rm s o f AChE a r e s u s c e p t i b l e t o

p r o t e o l y t i c d e g r a d a t i o n jm v i t r o . Such m o l e c u l a r c o n v e r s i o n

o c c u r s r e a d i l y d u r i n g h o m o g e n i s a t i o n and e x t r a c t i o n i n

c o r r e l a t i o n w i t h t h e l e v e l o f p r o t e o l y t i c a c t i v i t y p r e s e n t i n t h e

t i s s u e s . The d i f f e r e n t l e v e l o f p r o t e o l y t i c a c t i v i t y i n

d i f f e r e n t t i s s u e s , a s w e l l a s t h e d u r a t i o n o f s a m p le p r o c e s s i n g

a r e some o f t h e f a c t o r s w h ic h g i v e r i s e t o v a r i a b i l i t y and l a r g e

r a t i o s o f L form: H form o b s e r v e d f o r d i f f e r e n t m u s c l e s . T h a t

s u c h v a r i a b i l i t y i s a r e s u l t o f p r o t e o l y s i s i s s u p p o r t e d by t h e

f a c t t h a t when p r o t e o l y s i s i s a b o l i s h e d by an e f f e c t i v e

c o m b i n a t i o n o f p r o t e a s e i n h i b i t o r s i n t h e e x t r a c t i o n medium, t h e

m o l e c u l a r p r o f i l e s e x h i b i t a s t a b l e a n d , i n m o s t c a s e s , a

p r e d o m i n a n t form i n a d u l t mammalian m u s c l e s . The breakdown

o f t h e AChE fo rm s p r o b a b l y i n v o l v e s a v a r i e t y o f p r o t e a s e s , a s

i n c o m p l e t e i n h i b i t i o n c o u l d n o t p r e v e n t su c h a r t e f a c t s .

F u r t h e r m o r e , t h e e n d o g e n o u s p r o t e a s e a c t i v i t y p r o b a b l y d e c r e a s e s

w i t h l o n g i n c u b a t i o n , and t h u s i t s i n i t i a l , s h o r t - t e r m e f f e c t h a s

l a r g e l y b e e n o v e r l o o k e d i n t h e p a s t .

The d i s t r i b u t i o n o f AChE form s and t h e f i b r e t y p e

c o m p o s i t i o n o f a m u s c l e a r e i n t e r - r e l a t e d . T h e r e a r e c e r t a i n

c o r r e l a t i o n s b e t w e e n t h e f i b r e t y p e c o m p o s i t i o n and t h e AChE

p r o f i l e s w h i c h , i n t h e c a s e o f f a s t - t w i t c h m u s c l e s a l s o a p p e a r t o

b e s p e c i e s - s p e c i f i c . A t t h e c e l l u l a r l e v e l , t h e d i s t r i b u t i o n o f

AChE a l o n g a m u s c l e f i b r e i s d i c t a t e d by t h e c h a r a c t e r i s t i c s o f

t h e f i b r e t y p e w h e t h e r a t t h e s i t e o f t h e n e u r o m u s c u l a r c o n t a c t ,

- 8 0 -

o r i n t h e r e s t o f t h e f i b r e . E n d p l a t e AChE a p p e a r s t o be

r e g u l a t e d by t h e t y p e o f m o t o n e u r o n e w h ic h s y n a p s e s w i t h t h e

m u s c l e , w h i l e t h e e x t r a j u n c t i o n a l AChE i s p o s s i b l y d e t e r m i n e d by

m u s c l e a c t i v i t y ( W a lk e r and W i l s o n , 1 9 7 5 ; Drachman and W i t z k e ,

1 9 7 2 ) . The f u n c t i o n a l s i g n i f i c a n c e o f t h e e x t r a j u n c t i o n a l AChE

a n d i t s r e l a t i o n s h i p t o t h e e n d p l a t e AChE n e e d s t o be c l a r i f i e d .

I t i s a l s o i m p o r t a n t t o e s t a b l i s h t h e m ec h a n is m s w h ic h d e t e r m i n e

t h e d i f f e r e n t s p a t i a l o r g a n i s a t i o n o f t h e e n d p l a t e AChE i n

d i f f e r e n t f i b r e t y p e s and t h e r o l e t h e s e o r g a n i s a t i o n s p l a y i n

m a i n t a i n i n g t h e c h a r a c t e r i s t i c i n n e r v a t i o n d i f f e r e n t f i b r e t y p e s

r e c e i v e (McArdle and A l b u q u e r q u e , 1 9 7 3 ; Gordon a n d Y r b o v a , 1 9 7 5 ;

C l o s e , 1 9 7 2 ) .

♦

- 8 1 -

CHAPTER 3

THE EFFECT OF CHRONIC DENERVATION ON THE ENDPLATE AND NON-ENDPLATE ACHE

IN MAMMALIAN FAST AND SLOW-TWITCH MUSCLES

♦

- 8 2 -

3 . 1 INTRODUCTION

I t h a s b e e n shown i n C h a p t e r 2 t h a t t h e d i s t r i b u t i o n o f t h e

m o l e c u l a r fo rm s o f AChE i s o n e o f t h e many d i s t i n c t i v e f e a t u r e s o f f a s t

and s l o w m u s c l e s . Much e f f o r t s h a v e b een made i n t h e l a s t t h r e e d e c a d e s

t o i d e n t i f y f a c t o r s w h ic h c o n t r o l t h e e x p r e s s i o n o f m u s c l e t y p e

c h a r a c t e r i s t i c s ( f o r r e v i e w s s e e G u th , 1 9 6 8 ; J o l e s z and S r e t e r , 1 9 8 1 ) .

The d i f f e r e n t p h y s i o l o g i c a l and m e t a b o l i c c h a r a c t e r i s t i c s e x h i b i t e d by

t h e v a r i o u s m u s c l e f i b r e t y p e s may be r e l a t e d t o d i f f e r e n t p r o p e r t i e s o f

t h e i r r e s p e c t i v e m o t o n e u r o n e s ( C l o s e , 1 9 7 2 ; P a d y k u l a and G a u t h i e r ,

1 9 7 0 ) . One way o f e l u c i d a t i n g t h e r e g u l a t o r y r o l e o f t h e n e r v e i s t o

s t u d y c h a n g e s w h ic h t a k e p l a c e i n a m u s c l e o r m u s c l e f i b r e when t h e

i n n e r v a t i o n h a s b e e n r e m o v e d . The h i s t o c h e m i c a l and m o r p h o l o g i c a l

c h a n g e s w h ic h o c c u r i n m u s c l e s upon d e n e r v a t i o n h a v e b e e n s t u d i e d i n

d e t a i l ( T o w e r s , 1 9 3 5 ; T e n n y so n e t a l . , 1 9 7 7 ) . I t was f o u n d t h a t t h e

rem ova l o f m o t o n e u r o n e s was t h e m a jo r f a c t o r w h ich i n d u c e d d e g e n e r a t i o n

o f t h e m u s c l e . I t was shown t h a t d e v e r v a t i o n i n d u c e d s e v e r e a t r o p h i c

symptoms p a r t l y due t o an i n c r e a s e i n t h e p r o t e o l y t i c c a p a c i t y o f t h e

m u s c l e and t h e s u r r o u n d i n g t i s s u e s ( M a s k r e y e t a l . , 1 9 7 7 ; F e r n a n d e z and

D u e l l , 1 9 8 0 ) . F u r t h e r , t h e r e was a r a p i d r e d u c t i o n o f m y o f i b r i l l a r

p r o t e i n s and a c o n c o m i t a n t i n c r e a s e i n c y t o p l a s m i c p r o t e i n s i n

d e n e r v a t e d r a b b i t m u s c l e s ( S y r o v y e t a l . , 1 9 7 1 ) .

+ D e n e r v a t i o n i n d u c e s s p e c i e s - s p e c i f i c c h a n g e s i n t h e s y n t h e s i s

and d i s t r i b u t i o n o f AChE. D e n e r v a t e d c h i c k e n ( L i n k h a r t and W i l s o n , 1 9 7 5 )

and r a b b i t ( T e n n y s o n e t a l . , 1 9 7 7 ; Bacou e t a l , , 1 9 8 2 ) m u s c l e s e x h i b i t

i n c r e a s e d AChE a c t i v i t y w h e r e a s r e d u c e d AChE a c t i v i t y was o b s e r v e d i n

d e n e r v a t e d m u s c l e s o f r a t ( S e n n i e t a l . , 1 9 8 1 ) and m i c e (McCaman, 1 9 6 6 ) .

D e n e r v a t e d r a t and r a b b i t m u s c l e s b o t h e x h i b i t a r e d u c t i o n o f AChE

c o n c e n t r a t i o n a t t h e e n d p l a t e s i t e s , a s t h e s i z e and i n t e n s i t y o f AChE

s t a i n i n g d i m i n i s h e s w i t h t h e p r o g r e s s i o n o f a t r o p h y . The s t a i n i n g

- 8 3 -

i n t e n s i t y i n c r e a s e s c o n c o m i t a n t l y i n t h e e x t r a - j u n c t i o n a l r e g i o n o f t h e

d e n e r v a t e d r a b b i t g a s t r o c n e m i u s b u t s u c h s t a i n i n g was a b s e n t i n t h e r a t

m u s c l e u n d er t h e same e x p e r i m e n t a l c o n d i t i o n s ( B r z i n and M a j c e n - T r a c e v ,

1 9 8 3 ; T en n y so n e t a l . , 1 9 7 7 ) . Such h i s t o c h e m i c a l o b s e r v a t i o n a g r e e s w i t h

t h e f i n d i n g s o f t h e q u a n t i t a t i v e a n a l y s i s o f j u n c t i o n a l and

e x t r a j u n c t i o n a l AChE a c t i v i t i e s . Thus t h e i n c r e a s e i n t h e AChE a c t i v i t y

i n t h e e x t r a - j u n c t i o n a l r e g i o n i n r a b b i t s e em s t o be r e s p o n s i b l e f o r t h e

o v e r a l l i n c r e a s e i n t o t a l a c t i v i t y ; l a c k o f su c h d e n e r v a t i o n - i n d u c e d

i n c r e a s e s i n t h e e x t r a - j u n c t i o n a l AChE i n t h e r a t m u s c l e t h e r e f o r e

r e s u l t s i n an o v e r a l l d e c r e a s e i n a c t i v i t y . D e n e r v a t i o n a l s o g i v e s r i s e

t o an i n c r e a s e i n t h e r e l e a s e o f e n d p l a t e AChE ( I n e s t r o s a e t a l . , 1 9 7 7 )

and t h e t u r n o v e r o f AChE (Newman e t a l . , 1 9 8 4 ) . F u r t h e r m o r e , C a r t e r and

B r i m i j o i n ( 1 9 8 1 ) f o u n d t h a t t h e 10S fo rm a c c o u n t e d f o r a b o u t 80% o f

AChE r e l e a s e d from t h e d e n e r v a t e d r a t d i a p h r a g m . In c o n t r a s t , e l e c t r i c a l

s t i m u l a t i o n i n t h e a b s e n c e o f t h e n e r v e r e s t o r e d t h e AChE a c t i v i t y t o

normal ( W a lk e r and W i l s o n , 1 9 7 5 ) .

The m ost n o t i c e a b l e c h a n g e i n t h e m o l e c u l a r d i s t r i b u t i o n o f AChE

upon d e n e r v a t i o n i s t h e d e p l e t i o n o f t h e e n d - p l a t e s p e c i f i c , c o l l a g e n

t a i l e d form ( H a l l , 1 9 7 3 ; S e n n i e t a l . , 1 9 8 1 ; C o l l i n s and Y o u n k i n , 1 9 8 2 ;

S k e t e l j e t a l . , 1 9 7 8 ) . F u r t h e r m o r e , AChE c l u s t e r i n g a t t h e o r i g i n a l

e n d p l a t e s i t e o r e c t o p i c n e u r a l c o n t a c t c a n be m a i n t a i n e d by

♦ r e i n n e r v a t i o n ( S e n n i e t a l . , 1 9 8 1 ; M aldo nad o e t a l . , 1 9 8 4 ; V i g n y e t a l . ,

1 9 7 6 ; Lptoo and S l a t e r , 1 9 8 0 ) . M u s c le a c t i v i t y i s t h o u g h t t o b e o n e o f

t h e m o st i m p o r t a n t f a c t o r s i n d e t e r m i n i n g t h e a p p e a r a n c e and t h e

s t a b i l i t y o f e n d p l a t e AChE (R u b in e t a l . , 1 9 8 0 ; K o e n ig e t a l . , 1 9 8 2 ) .

E v i d e n c e a l s o s u g g e s t s t h e r o l e o f m u s c l e a c t i v i t y i n t h e r e g u l a t i o n o f

e x t r a - j u n c t i o n a l AChE ( L i n k h a r t and W i l s o n , 1 9 7 5 ) . The m a i n t e n a n c e o f

AChE by some n e u r o t r o p h i c f a c t o r h a s a l s o been p r o p o s e d ( K o e l l e and

R u ch , 1 9 8 3 ; L e n t z e t a l . , 1 9 8 1 ) .

- 8 4 -

S y r o v y e t a l . ( 1 9 7 1 ) r e p o r t e d t h a t t h e p a t t e r n o f c h a n g e ,

e s p e c i a l l y t h e r a t e o f d e v e l o p m e n t o f a t r o p h y , t h e c o n t e n t o f m y o s i n and

lo w m o l e c u l a r w e i g h t p r o t e i n s , t h e ATPase a c t i v i t y and t h e c o n t r a c t i l e

p r o p e r t i e s o f d e n e r v a t e d r a b b i t f a s t and s l o w m u s c l e s w e r e d i s t i n c t l y

d i f f e r e n t . The m u s c l e t y p e s r e m a i n e d d i s t i n c t e v e n a f t e r d e n e r v a t i o n

( L e w i s e t a l . , 1 9 7 4 ) . T h e s e m u s c l e t y p e - s p e c i f i c c h a n g e s may r e f l e c t

d i f f e r e n t modes o f r e g u l a t i o n by w h ic h t h e f a s t and s l o w n e r v e s n o r m a l l y

e x e r t on t h e i r m u s c l e s (M a g a z a n ik e t a l . , 1 9 7 8 ; McArdle and A l b u q u e r q u e ,

1 9 7 3 ) . R e c e n t l y , Bacou e t a l . ( 1 9 8 2 ) r e p o r t e d an a p p a r e n t i n c r e a s e i n

t h e 16S (H2C) a c t i v i t y i n t h e d e n e r v a t e d se m i- m e m b r a n o su s p r o p r i u s , a

p u r e s l o w - t w i t c h m u s c l e i n t h e r a b b i t , s u g g e s t i n g t h a t t h i s fo rm i s

n o r m a l l y s u p p r e s s e d by t h e n e r v e . Such o b s e r v a t i o n c o n t r a s t s w i t h t h e

n e u r a l i n d u c t i o n o f ^ a c t i v i t y d u r i n g s y n a p t o g e n e s i s i n e m b r o y o n ic r a t

( K o e n i g and V i g n y , 1 9 7 8 ) and c h i c k m u s c l e s ( T o u t a n t e t a l . , 1 9 8 3 ) . The

e x p e r i m e n t s d e s c r i b e d i n t h i s s e c t i o n aim t o c l a r i f y t h e e f f e c t o f

d e n e r v a t i o n on AChE a c t i v i t y and t h e d i s t r i b u t i o n o f t h e m o l e c u l a r f o rm s

i n f a s t and s l o w m u s c l e s i n a number o f mammalian s p e c i e s , and how t h e

v a r i o u s m o l e c u l a r f o rm s c h a n g e i n r e l a t i o n t o t h e o v e r a l l i n c r e a s e o r

d e c r e a s e i n t o t a l AChE a c t i v i t y i n d i f f e r e n t m u s c l e s . The d i f f e r e n t i a l

e f f e c t s o f d e n e r v a t i o n on e n d p l a t e and n o n - e n d p l a t e AChE f o r m s w e r e a l s o

i n v e s t i g a t e d .

3.2 MATERIALS AND METHODS

3.2.1 Animals

New Z e a l a n d W h it e and Old E n g l i s h B l a c k r a b b i t s (^3 k g ) , D u n k in -

H a r t l e y g u i n e a p i g s ( t w o m onth s o l d ) and G olde n h a m s t e r s ( 3 m o n th s o l d )

w e r e u s e d f o r t h i s s t u d y .

- 8 5 -

3.2.2 Muscles

The f o l l o w i n g p e l v i c l im b m u s c l e s o f s i n g l e f i b r e - t y p e

c o m p o s i t i o n w e r e s t u d i e d : t h e f a s t - t w i t c h EDL ( e x t e n s o r d i g i t o r u m

l o n g u s ) ( A r i a n o e t a l . , 1 9 7 3 ) , t h e s l o w - t w i t c h s o l e u s ( P e t e r e t a ! . ,

1 9 7 2 ; C a m p b e l l , 1 9 7 9 ) and t h e se m i- m e m b r a n o su s w h ic h i s made up o f tw o

d i s t i n c t r e g i o n s — t h e sem im em b ran osu s a c c e s s o r i u s (SA) ( f a s t - t w i t c h ) ,

o r t h e o u t e r r e g i o n , and t h e se m im em br ano su s p r o p r i u s (S P ) ( s l o w -

t w i t c h ) , o r t h e i n n e r r e g i o n . The f i b r e - t y p e c o m p o s i t i o n o f t h e s e

m u s c l e s a r e l i s t e d i n T a b l e 1 . 1 . The p u re f i b r e t y p e c o m p o s t i o n o f t h e s e

m u s c l e s f a c i l i t a t e s s e p a r a t e a n a l y s e s o f t h e e f f e c t o f d e n e r v a t i o n on

t h e t y p e I I ( f a s t - t w i t c h ) and t y p e I ( s l o w - t w i t c h ) f i b r e s . The f a c t t h a t

t h e s e m u s c l e s a r e a l l p a r t o f t h e p e l v i c l i m b m u s c u l a t u r e s i m p l i f i e s t h e

s u r g e r y r e q u i r e d f o r d e n e r v a t i o n .

3.2.3 Surgical procedures

A n i m a l s w e r e o p e r a t e d on u n d e r f l u o t h a n e a n a e s t h e s i a . One o f t h e

h i n d l i m b s w e r e d e n e r v a t e d ; t h e m u s c l e s i n t h e u n d e n e r v a t e d l i m b w e r e

u s e d a s c o n t r a l a t e r a l c o n t r o l s . *

The r a b b i t sem im em br ano su s i s i n n e r v a t e d by t h e n . c u t a n e u s

f e m o r i s c a u d a l i s , R r . m u s c u l a r e s n . i s c h i a d i c i and b r a n c h e s o f t h e N.

o b t u r a t o r i u s ( B o r o n e e t a l . , 1 9 7 3 ) . C o m p l e te d e n e r v a t i o n o f t h e s e

m u s c l e s r e q u i r e d s e c t i o n i n g a l l t h e n e r v e b r a n c h e s d i s t a l l y c l o s e t o t h e

m u s c l e . The g u i n e a p i g sem im em br ano su s i s i n n e r v a t e d by t h e N.

i s c h i a d i e u s , t h e n . c u t a n e u s f e m o r i s c a u d a l i s , t h e n . f e m o r i s and t h e n .

s a p h e n u s ( C o o p e r and S c h i l l e r , 1 9 7 5 ) . The EDL and s o l e u s o f r a b b i t ,

g u i n e a p i g and h a m s t e r w e r e r o u t i n e l y d e n e r v a t e d by s e c t i o n i n g t h e N.

i s c h i a d i e u s i n t h e m i d - t h i g h r e g i o n and r e m o v i n g a p p r o x i m a t e l y 6mm o f

t h e d i s t a l n e r v e t r u n k .

* I w i s h t o t h a n k Mr. D a v i d Green f o r k i n d l y p e r f o r m i n g t h e s u r g i c a l

d e n e r v a t i o n p r o c e d u r e s .

-86-

3.2.4 Partial denervation

To s t u d y t h e e f f e c t o f p a r t i a l d e n e r v a t i o n , i t was d e c i d e d t o

u s e t h e r a b b i t se m im em br ano su s w h ic h h a s a c o m p le x n e u r a l i n p u t . P a r t i a l

d e n e r v a t i o n was a c h i e v e d e i t h e r by s e c t i o n i n g t h e n . c u t a n e u s f e m o r i s

c a u d a l i s a l o n e o r o n l y t h e R r . m u s c u l a r i s n . i s c h i a d i c i .

The g u i n e a p i g s w e r e s a c r i f i c e d 2 t o 4 w e e k s p o s t - d e n e r v a t i o n ;

t h e r a b b i t s and h a m s t e r s w e r e a l w a y s s a c r i f i c e d 4 w e e k s p o s t

d e n e r v a t i o n .

3 . 2 . 5 Muscle extract preparation

A n i m a l s w e r e k i l l e d by an o v e r d o s e o f p e n t o b a r b i t o n e . M u s c l e s

w e r e d i s s e c t e d from t h e d e n e r v a t e d and t h e c o n t r a l a t e r a l l i m b s ,

i m m e d i a t e l y w e i g h e d , m in c e d b r i e f l y and h o m o g e n iz e d i n 10 v o l o f an i c e -

c o l d p h o s p h a t e b u f f e r , pH 7 . 0 , c o n t a i n i n g 1M N a C l , 1% T r i t o n , 5 mM EGTA

and t h e f o l l o w i n g p r o t e a s e i n h i b i t o r s : b a c i t r a c i n ( l m g / m l ) , b e n z e m i d i n e

HC1 (2mM), c h i c k e n o v o m u c o id t r y p s i n i n h i b i t o r ( 0 . 2 m g / m l ) , s o y - b e a n

t r y p s i n i n h i b i t o r ( 0 . 1 m g / m l ) , l e u p e p t i n ( 0 . 0 4 m g / m l ) , p e p s t a t i n ( 0 . 2

m g/m l) and NEM (5mM) ( s e e S e c t i o n 2 . 2 . 3 ) . Smal l m u s c l e s w e r e h o m o g e n i s e d

w h o l e , a f t e r m i n c i n g b r i e f l y , w i t h g l a s s / g l a s s hand h o m o g e n i s e r s . L a r g e

m u s c l e s w e r e m in c e d and a b o u t l g o f t h e m i n c e s w e r e h o m o g e n i s e d w i t h a

P o l y t r o n ( K i n e t i c a ) ( 3 x 3 0 s ) a t 4 ° C . The h o m o g e n a t e s w e r e i m m e d i a t e l y

* c e n t r i f u g e d a t 4 3 , 5 0 0 x g f o r 15 min t o y i e l d t h e m u s c l e c r u d e e x t r a c t s .

3.2.6 Measurement of endplate and non-endplate AChE

E n d p l a t e and n o n - e n d p l a t e r e g i o n s w e r e d i s s e c t e d from m u s c l e s

( w h e r e i n d i c a t e d ) and e x t r a c t e d a s d e s c r i b e d p r e v i o u s l y ( s e e S e c t i o n

2 . 2 . 5 ) . T h e s e s a m p le c r u d e e x t r a c t s w e r e f o l l o w e d i m m e d i a t e l y by

s e d i m e n t a t i o n and enzyme a n a l y s i s a s d e s c r i b e d i n S e c t i o n 2 . 2 . 6 and

2 . 2 . 7 .

- 8 7 -

3 . 3 Results

3.3.1 Denervation-induced changes in AChE activities in different

species

C h r o n i c a l l y d e n e r v a t e d m u s c l e s e x h i b i t s e v e r e a t r o p h y and

e x t e n s i v e f a t t y i n f i l t r a t i o n ( F i g . 3 . 1 ) . T h e r e was s u b s t a n t i a l v a r i a t i o n

i n t h e w e i g h t l o s s o f t h e d e n e r v a t e d m u s c l e s b e t w e e n d i f f e r e n t a n i m a l s

and t r e a t m e n t ; on a v e r a g e , t h e t o t a l l y d e n e r v a t e d SA and SP s u f f e r e d

s i m i l a r w e i g h t l o s s , p a r t i a l l y d e n e r v a t e d SA was t h e m o st a f f e c t e d w h i l e

t h e p a r t i a l l y d e n e r v a t e d SP e x h i b i t e d r e l a t i v e l y m in o r w e i g h t c h a n g e s .

In t h e c o n t r a l a t e r a l i n n e r v a t e d f a s t - t w i t c h m u s c l e s , i . e . t h e

EDL and t h e SA, t h e AChE a c t i v i t y r a n g e d b e t w e e n 0 . 3 and 1 . 0 U / g ( w e t

w e i g h t ) ; i n t h e i n n e r v a t e d s l o w - t w i t c h m u s c l e s , t h e a c t i v i t y was b e t w e e n

0 . 5 and 0 . 7 U / g . T h e r e was no a p p a r e n t c o r r e l a t i o n b e t w e e n t h e l e v e l o f

AChE a c t i v i t y and t h e f i b r e t y p e c o m p o s i t i o n o f t h e s e m u s c l e s ( s e e a l s o

T a b l e 2 . 3 ) , c o n t r a r y t o t h a t o b s e r v e d i n c h i c k e n w h er e t y p e I m u s c l e s

o f t e n d i s p l a y e d much h i g h e r a c t i v i t y t h a n t y p e I I m u s c l e s ( L y l e s e t a l . ,

1 9 8 0 ; L y l e s e t a l . , 1 9 8 2 ) .

The AChE a c t i v i t y i n t h e h a m s t e r m u s c l e s d e c r e a s e d upon

d e n e r v a t i o n ( T a b l e 3 . 1 ) , s i m i l a r t o t h a t o b s e r v e d i n r a t m u s c l e s ( H a l l ,

1 9 7 3 ; S e n n i e t a l . , 1 9 8 1 ) . H ow ever , i n c r e a s e s i n AChE a c t i v i t y w e r e

• o b s e r v e d i n t h e d e n e r v a t e d m u s c l e s o f b o t h r a b b i t and g u i n e a p i g when

comp ared t o t h e i r c o n t r a l a t e r a l c o n t r o l m u s c l e s ( T a b l e 3 . 1 ) ( T e n n y s o n e t

a l . , 1 9 7 7 ) . T h e s e c h a n g e s a r e e x p r e s s e d a s t h e r a t i o o f AChE a c t i v i t i e s

o r ' r e l a t i v e l y a c t i v i t y 1 i n t h e d e n e r v a t e d and c o n t r a l a t e r a l c o n t r o l

m u s c l e s . Such r a t i o s v a r i e d b e t w e e n t h e m u s c l e s t h a t w e r e s t u d i e d , b u t

on t h e w h o l e t h e y w e r e somewhat l o w e r i n t h e f a s t m u s c l e s ; f u r t h e r m o r e ,

t h e d e n e r v a t e d sem im em br an osu s from b o t h r a b b i t and g u i n e a p i g d i s p l a y e d

h i g h e r r e l a t i v e a c t i v i t i e s t h a n o t h e r m u s c l e s o f s i m i l a r f i b r e t y p e

-88-

Fig.3.1 Changes in the structure and myosin ATPase staining of the denervated SA (B) and SP (0) rabbit muscles compared to the contralateral control innervated (A, SA; C, SP) muscles. Staining was after preincubation at pH 4.5. Three types of fibres can be distinguished: type 11A stains weakly, type 11B stains intermediately and type I stain intensely. The SP has only type I fibres and the intense ATPase reaction is retained on denervation. The type II fibres atrophy more marked than that seen in the type I fibres. Bar represents 30ym. Results of J. Pizzey.

- 9 0 -

TABLE 3.1. Relative activities of AChE in denervated rabbit,

guinea pig and hamster muscles.

species muscle

fibre

type

relative

activity n

hamster EDL 0.5 ± 0.0 2

guinea pig EDL 1.3 ± 0.2 2

guinea pig SA type II 2.7 ± 0.4 5

rabbit SA(partial)* 4.5 ± 0.6 2

(total) 3.7 ± 0.8 5

hamster soleus 0.8 1

guinea pig soleus 2.5 ± 0.4 2

guinea pig SP 3.5 ± 0.3 5

rabbit soleus type I 2.5 ± 0.4 3

rabbit SP (partial)* 3.3 ± 0.2 2

(total) 4.4 ± 0.7 5

1 Denervation by sectioning the Rr. muscularis N. ischiadici.

composition. The relative activity of the denervated rabbit SP was

similar to that of the denervated guinea pig.

3.3.2 Effects of denervation on the AChE profiles of fast and slow

muscles

In all the type II muscles used in this study, the L and are

equally prominant (Fig. 3.3), except the hamster EDL where the form

predominates (Fig.3.2). Type I muscles contain a large ^ component and

also significant amounts of M and H^c forms (Fig.3.4), contrary to that

previously reported where the H2 C form accounted for a minor proportion

of total activity (Bacou et al., 1982). This discrepancy is likely to be

due to the use of stored, frozen tissue samples, and an inadequate

protection of the AChE from proteolytic degradation, as discussed in

Chapter 2.

It is clear from Fig 3.2, 3.3 and 3.4 that the proportion of

form in these muscles invariably diminished upon denervation. Such

reduction was more pronounced in the fast muscles (averaged 34%) than

that in the slow muscles (averaged 17%). A concomitant increase in the L

forms was observed in denervated guinea pig and rabbit muscles (Fig.3.3

and 3.4), but not in the hamster muscles (Fig.3.2). The fraction of the

area under each peak corresponding to a molecular form was taken to

• represent the proportion of total AChE activity of that molecular form.

The relative change in terms of activity of the different molecular

forms upon denervation is illustrated in Table 3.3.

These figures show that the L forms were increased by many folds

in all denervated muscles except those of the hamster. The activity of

the H2C form, on the other hand, was only marginally reduced in the

denervated fast muscles (Table 3.2), except in the denervated guinea pig

EDL where activity dropped significantly. Almost all the slow

-92-

20 40 60FRACTIONS

FIG- 3-2 Sucrose gradient profile of contralateral control and

chronically denervated hamster EDL. # , contralateral control;

O , denervated (4-week). All extracts in this and subsequent

figures in this chapter were prepared in the presence of protease

inhibitors. Arrows indicate the position of the catalase

marker.- 9 3 -

FIG. 3.3 Sucrose gradient profiles of AChE in control and denervated type II muscles.

Top row: AChE in control muscles. A, guinea pig EDL; B, guinea pig SA; C, rabbit SA. Bottom row:

AChE in denervated muscles. D, guinea pig EDL; E, guinea pig SA; F, rabbit SA (partially

denervated)*, G, rabbit SA (totally denervated). The amount of samples analysed in each case in

this and Figs. 4, 5 and 6 was arbitrary.

*

-95-

1.5 B

#

t FIG. 3.4 Sucrose gradient profiles of AChE in contralateral

control and denervated type I muscles. Top row: AChE in control

muscles. A, guinea pig soleus; B, guinea pig SP; C, rabbit

soleus; D, rabbit SP. Second row: AChE in denervated muscles.

E, guinea pig soleus; F, guinea pig SP; G, rabbit soleus; H,

rabbit SP (partially denervated); I, rabbit SP (totally

denervated).

- 9 5 a -

muscles exhibited an increase in the H^ activity upon denervation, the

increase being most pronounced in the SP muscles. Therefore, although

the relative proportion of W^c form in these slow muscles was reduced

upon denervation, the total activity contributed by this molecular form

actually became greater in most cases (Table 3.2). Hence increases in

the total AChE activity in these denervated muscles were largely due to

the presence of a great excess of L and to a lesser extent the M forms.

The H~ form also contributed to the overall increase in AChE in 2cdenervated slow muscles.

Partial denervation of the rabbit semimembranosus by sectioning

the n. cutaneus femoris caudal is alone had little effect on the muscle.

The muscle underwent only minor weight loss (^5%); neither the AChE

content nor its molecular distribution was altered (Table 3.3). In

contrast, when partial denervation of the muscle was achieved by

sectioning the branches from the sciatic nerve (Rr. muscularis N.

ischiadici), the effect on the semimembranosus was profound (Table 3.1

and 3.2). The AChE activity increased in both the SA and the SP.

Increased activity of the molecular forms was similar in magnitude to

the totally denervated semimembranosus. The differences in the relative

activities, whether of total AChE or of molecular forms, observed

between partial and total denervation were not significant at a 99%

confident limits. There was also no significant difference between the

changes in the proportion of the molecular forms in partially and

totally denervated semimembranosus, as shown in Fig 3.3 and 3.4. If the

relative activities were corrected for the weight differences between

the denervated and control muscles (Table 3.4), the data shows that the

AChE activity per muscle increased slightly; the L forms increased by

similar amount in both type I and type II muscles, while the form

was clearly reduced in the type II but not the type I muscles.

-96-

TABLE 3.2 Relative activities of L and forms in denervated

rabbit, guinea pig and hamster muscle.

species muscle

fibre

type

relative activity

L H2C

hamster EDL 0.4 0.3

guinea pig EDL 2.2 0.4

guinea pig SA II 6.2 1.1

rabbit SA (partial)* 12.1 0.7

(total) 10.3 0.8

hamster soleus 1.0 0.4

guinea pig soleus 4.0 1.4

guinea pig SP I 6.2 2.0

rabbit soleus 5.8 0.7

rabbit SP (partial)* 5.3 2.3

(total) 7.0 3.0

1 Denervation by sectioning the Rr. muscularis N. ischiadici.

- 9 7 -

TABLE 3.3. Changes in total AChE and its molecular forms in the 2

partially denervated rabbit semimembranosus.

relative activity

species muscle total AChE L H2C n

rabbi t SA 1.1 ± 0.2 1.0 1.0 3

rabbit SP 0.9 ± 0.1 0.9 0.95 3

2The semimembranosus was denervated by sectioning the n.

cutaneus femoris caudal is only.

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TABLE 3 . 4 The r e l a t i v e a c t i v i t i e s ( p e r m u s c l e ) o f AChE and i t s

m o l e c u l a r f o rm s i n t h e d e n e r v a t e d g u i n e a p i g and r a b b i t

se m im e m b r a n o s u s .

s p e c i e s m u s c l e

f i b r e

t y p e

r e l a t i v e a c t i v i t y ( p e r m u s c l e )

t o t a l AChE L n

r a b b i t SA ( p a r t i a l ) 1 . 6 ± 0 . 1 4 . 3 0 . 2 2

( t o t a l ) II 1 . 6 ± 0 . 3 4 . 4 0 . 3 5

g u i n e a p i g SA 1 . 2 ± 0 . 1 2 . 8 0 . 5 5

r a b b i t SP ( p a r t i a l ) 2 . 6 ± 0 . 4 4 . 1 2 . 0 2

( t o t a l ) I 1 . 7 ± 0 . 3 2 . 7 1 . 2 5

g u i n e a p i g SP 1 . 7 ± 0 . 2 3 . 0 1 . 0 5

1 D e n e r v a t i o n by s e c t i o n i n g t h e R r . m u s c u l a r i s N . i s c h i a d i c i

*

-99-

3.3.3 Differential analysis of endplate and non-endplate AChE in

normal and denervated muscles

D i f f e r e n t i a l a n a l y s i s o f t h e j u n c t i o n a l and e x t r a - j u n c t i o n a l

AChE by m i c r o d i s s e c t i o n showed t h a t t h e H^c and f o r m s p r e d o m i n a t e a t

t h e e n d p l a t e s o f b o t h t y p e I and t y p e I I f i b r e s ( F i g 3 . 5 A and 3 . 5 B ) .

C h r o n i c d e n e r v a t i o n up t o 4 w e e k s r e d u c e d b u t d i d n o t e l i m i n a t e t h e s e

f o r m s from t h e e n d p l a t e s ( F i g 3 . 5 C and 3 . 5 D ) . The p e r s i s t e n c e o f t h e s e

f o r m s i s c o n s i s t e n t w i t h t h e w ea k en ed b u t v i s i b l e e n d p l a t e AChE s t a i n i n

b o t h t y p e s o f m u s c l e f i b r e s . T h i s i s i l l u s t r a t e d i n F i g 3 . 7 w h i c h s h o w s

t h e s t a i n e d t e a s e d f i b r e s from d e n e r v a t e d and c o n t r a l a t e r a l c o n t r o l

s o l e u s m u s c l e s o f t h e g u i n e a p i g . F u r t h e r m o r e , L f o r m s w h i c h a r e

n o r m a l l y f o u n d o n l y i n t h e n o n - e n d p l a t e r e g i o n s a p p e a r i n t h e e n d p l a t e s

o f t h e d e n e r v a t e d t y p e I f i b r e s ( F i g 3 . 6 A and 3 . 6 B ) . E x t r a j u n c t i o n a l H

f o r m s a r e p r e s e n t i n t h e s o l e u s o f t h e g u i n e a p i g ( F i g 3 . 6 B ) and t h e SP

o f t h e r a b b i t ( J e d r z e j c z y k J . , p e r s o n a l c o m m u n i c a t i o n ) , b u t a r e

n e g l i g i b l e i n t h e t y p e II f i b r e s ( F i g 3 . 6 A ) . T h e s e e x t r a j u n c t i o n a l H

f o r m s r e m a in s i g n i f i c a n t i n t h e d e n e r v a t e d t y p e I f i b r e s ( F i g 3 . 6 C )

w i t h i n t h e 4 week p o s t - d e n e r v a t i o n p e r i o d .

3.4 DISCUSSION

3.4.1 Changes in AChE activities induced by chronic denervation

D e n e r v a t i o n - i n d u c e d c h a n g e s i n AChE a c t i v i t i e s o f s k e l e t a l

m u s c l e s a r e s p e c i e s s p e c i f i c ( B r z i n and M a jc e n - T r a f c e v , 1 9 6 3 ) . In m u s c l e s

w h i c h e x h i b i t an o v e r a l l r e d u c t i o n i n AChE a c t i v i t y upon c h r o n i c

d e n e r v a t i o n , e . g . h a m s t e r EDL, t h e r e i s an e q u a l r e d u c t i o n i n t h e L and

H f o r m s . In c o n t r a s t , d e n e r v a t e d r a b b i t and g u i n e a p i g m u s c l e s e x h i b i t

a l a r g e i n c r e a s e i n AChE a c t i v i t y , w h ic h i s a t t r i b u t e d t o a marked

e l e v a t i o n o f t h e L and M f o r m s . T h e r e i s a l s o an a p p a r e n t d e c r e a s e i n

t h e p r o p o r t i o n o f H fo r m s i n b o t h f a s t and s l o w m u s c l e s , c o n t r a r y t o

-100-

FIG- 3-5 S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n t h e e n d p l a t e

r e g i o n o f c o n t r o l and d e n e r v a t e d g u i n e a p i g m u s c l e f i b r e s . EDL

( t y p e I I ) : A, c o n t r a l a t e r a l c o n t r o l ; C, d e n e r v a t e d . S o l e u s ( t y p e

I): B, c o n t r a l a t e r a l c o n t r o l ; D, d e n e r v a t e d . D o t t e d l i n e

r e p r e s e n t s b a c k g r o u n d a b s o r b a n c e o f g r a d i e n t f r a c t i o n s i n c u b a t e d

i n t h e p r e s e n c e o f b o t h isoOMPA (1 x 1 0 - 4 M) a n d BW284C51 ( 5 x

10"^M). Where b a c k g r o u n d i s n o t i n d i c a t e d , i t h a s b een

s u b t r a c t e d from t h e s a m p le p r o f i l e .

-101-

FRACTIONS FRACTIONS

FIG. 3-6 S u c r o s e g r a d i e n t p r o f i l e s o f e x t r a j u n c t i o n a l AChE i n

c o n t r o l and d e n e r v a t e d g u i n e a p i g m u s c l e f i b r e s . EDL ( t y p e I I ) :

A, c o n t r a l a t e r a l c o n t r o l ; C, d e n e r v a t e d . S o l e u s ( t y p e I): B,

c o n t r a l a t e r a l c o n t r o l ; D, d e n e r v a t e d .

-102-

F i g . 3 . 7 Changes in the intensity of endplate AChE staining in

denervated guinea pig soleus. Teased fibres from control (A) and 4-

weeks-denervated (B) guinea pig soleus were stained for AChE

(Karnovsky and Roots, 1964) for the same period of incubation in the

presence of 10"^ M isoOMPA. Much weaker staining was observed in

denervated endplates (arrows). Extrajunctional staining (small

arrows) were also seen in the denervated fibres. Bar represents 25y

m.

- 1 0 3 -

t h a t r e p o r t e d by Ba cou e t a l . ( 1 9 8 2 ) . T h e s e w o r k e r s sh o w ed t h a t w h i l e

t h e p r o p o r t i o n o f ^ f o r m s was r e d u c e d i n t h e d e n e r v a t e d f a s t - t w i t c h SA

( t y p e I I ) , i t was m a r k e d l y i n c r e a s e d i n t h e d e n e r v a t e d s l o w - t w i t c h SP

( t y p e I ) . I t i s c l e a r t h a t t h e d i s c r e p a n c y b e t w e e n t h e p r e s e n t r e s u l t

and t h a t r e p o r t e d p r e v i o u s l y l i e s i n t h e d i f f e r e n t e s t i m a t i o n s o f t h e H

AChE co m p o n e n t i n t h e c o n t r o l S P . A n a l y s i s o f t h e AChE c o m p o s i t i o n i n a

v a r i e t y o f p u r e t y p e I m u s c l e s d e m o n s t r a t e s u n e q u i v o c a l l y t h a t t h e H

AChE i s a m a j o r c o m p o n e n t i n t y p e I m u s c l e s ( s e e C h a p t e r 2 ) , t h u s t h e

a p p a r e n t i n c r e a s e i n t h e p r o p o r t i o n o f t h e H AChE c o m p o n e n t r e p o r t e d by

Bacou e t a l . ( 1 9 8 2 ) i s an a r t e f a c t du e t o t h e u n d e r e s t i m a t i o n o f t h e

amount o f H f o r m s i n t h e c o n t r o l S P . I t may be c o n c l u d e d , t h e r e f o r e ,

t h a t i n b o t h f a s t and s l o w m u s c l e s o f t h e r a b b i t and t h e g u i n e a p i g , t h e

s u b s t a n t i a l i n c r e a s e i n t o t a l AChE a c t i v i t i e s upon d e n e r v a t i o n i s

l a r g e l y due t o an i n c r e a s e i n t h e a c t i v i t y o f t h e L and M form i n t h e

m u s c l e s . The a c c u m u l a t i o n o f L and M f o r m s p r o b a b l y a r i s e s from an

e x c e s s s y n t h e s i s o f t h e s e f o rm s i n t h e d e n e r v a t e d m u s c l e s , s i n c e

d e t a i l e d s t u d i e s by T e n n y s o n e t a l . ( 1 9 7 7 ) showed t h a t l a r g e amount o f

c y t o p l a s m i c AChE a p p e a r e d upon c h r o n i c d e n e r v a t i o n , c l e a r l y

d i s t i n g u i s h a b l e from t h e p a r t i c u l a t e AChE w h ic h a c c o u n t s f o r m o st o f t h e

AChE a c t i v i t y i n normal m u s c l e s . Some r e g e n e r a t i n g f i b r e s , a s w e l l a s

t h e l a r g e number o f AChE c o n t a i n i n g m o n o n u c l e a r c e l l s m u st a l s o

c o n t r i b u t e t o t h e i n c r e a s e i n AChE a c t i v i t y i n t h e a t r o p h i c m u s c l e .

The d e n e r v a t e d t y p e I m u s c l e s o f b o t h r a b b i t and g u i n e a p i g a l s o

e x h i b i t an e l e v a t e d a c t i v i t y o f t h e H2 c f o r m , e v e n t h o u g h s u c h i n c r e a s e

i s m i n o r when comp ared t o t h e i n c r e a s e i n t h e L and M f o r m s ( T a b l e 3 . 2 ) .

T h i s i n c r e a s e d H AChE a c t i v i t y i s p e c u l i a r t o t h e t y p e I m u s c l e s , s i n c e

i n t y p e II m u s c l e s b o t h t h e p r o p o r t i o n and t h e t o t a l a c t i v i t y o f t h e H2 c

a r e r e d u c e d . T h i s i n c r e a s e i n t h e t o t a l H2 c AChE i n t h e t y p e I m u s c l e s

a g r e e s w i t h t h e f i n d i n g s o f Bacou e t a l . ( 1 9 8 2 ) .

-104-

3.4.2 AChE in the endplates of normal and denervated muscle fibres

Bo th t h e anc* a s i g n i f i c a n t amount o f t h e H^c AChE a r e

c o n f i n e d t o t h e e n d p l a t e s o f t y p e I I f i b r e s . T h e s e fo r m s r e m a in

d e t e c t a b l e i n d e n e r v a t e d m u s c l e s 4 w e e k s a f t e r d e n e r v a t i o n and c o n f i n e d

t o t h e e n d p l a t e s i t e . The o v e r a l l r e d u c t i o n i n t h e H f o r m s i n t h e

d e n e r v a t e d t y p e I I f i b r e s t h e r e f o r e r e p r e s e n t s t h e l o s s o f AChE from t h e

e n d p l a t e . A f t e r 4 w e e k s , t h e s e f o rm s a r e r e d u c e d by 40-60%, w h ic h means

t h a t a s i g n i f i c a n t p r o p o r t i o n o f H2 c and Hl c AChE r e m a in a t t h e e n d p l a t e

e v e n when t h e n e r v e i s a b s e n t . T h i s a g r e e s w i t h t h e w e a k e n e d b u t s t i l l

c l e a r l y d i s c e r n a b l e e n d p l a t e AChE s t a i n i n g t y p i c a l o f d e n e r v a t e d m u s c l e s

( B r z i n and M a j c e n - T r a c e v , 1963). AChE s t a i n i n g a l s o sh ow s t h a t t h e

e n d p l a t e s i n t h e d e n e r v a t e d t y p e I f i b r e s a r e now s m a l l e r and o n l y

w e a k l y s t a i n e d ( F i g . 3.7). Hence a l t h o u g h t h e s e f i b r e s a r e c a p a b l e o f

s y n t h e s i z i n g e x c e s s H f o r m s , t h e e n d p l a t e p o p u l a t i o n o f t h e s e form s m us t

h a v e d i m i n i s h e d , a s o b s e r v e d i n t h e d e n e r v a t e d t y p e I I f i b r e s . An

abnormal p o p u l a t i o n o f L AChE i s a l s o a p p a r e n t i n t h e e n d p l a t e s o f

d e n e r v a t e d t y p e I f i b r e s . They may a r i s e from t h e p r o t e o l y t i c breakdown

o f H AChE a t t h e e n d p l a t e , a l t h o u g h i t se em s u n l i k e l y s i n c e v e r y l i t t l e

L f o r m s i s o b s e r v e d i n t h e e n d p l a t e o f d e n e r v a t e d t y p e II f i b r e s .

A l t e r n a t i v e l y , t h e s e fo rm s may r e p r e s e n t n e w l y s y n t h e s i z e d L AChE w h ic h

e i t h e r h a s a s p e c i f i c l o c a l i s a t i o n a t t h e e n d p l a t e s i t e o r a r i s e s from

• t h e g e n e r a l i n c r e a s e i n t h e s y n t h e s i s o f l o w e r m o l e c u l a r w e i g h t f o r m s

a l o n g t h e l e n g t h o f t h e f i b r e s .

The r e d u c t i o n i n e n d p l a t e H AChE s u g g e s t s t h a t t h e c o n c e n t r a t i o n

o f AChE a t t h e n e u r o m u s c u l a r j u n c t i o n i s i n d e p e n d e n t o f t h e s y n t h e t i c

c a p a c i t y o f t h e m u s c l e . I n c r e a s e d s y n t h e s i s o f t h e L, M and i n t h e c a s e

o f t h e d e n e r v a t e d t y p e I f i b r e s , t h e H f o rm s d o e s n o t p r e v e n t t h e

r e d u c t i o n o f AChE a t t h e e n d p l a t e , w h ic h i n v a r i a b l y o c c u r s upon c h r o n i c

d e n e r v a t i o n . I t s u g g e s t s , t h e r e f o r e , t h a t t h e c o n c e n t r a t i o n o f AChE a t

- 105 -

t h e e n d p l a t e may be c o n t r o l l e d by some o t h e r n e u r a l l y r e g u l a t e d f a c t o r s ,

e . g . t h e number o f b i n d i n g s i t e s f o r t h e a t t a c h m e n t o f t h e H f o r m s .

I n d e e d much i n d i r e c t e v i d e n c e ha s s u g g e s t e d t h e a s s o c i a t i o n o f t h e

a s y m m e t r i c fo rm s w i t h t h e b a s a l l a m i n a w h ic h a p p e a r s t o c o n t a i n u n i q u e

e l e m e n t s a t t h e n e u r o m u s c u l a r j u n c t i o n ( S a n e s and H a l l , 1979; S a n e s and

C h e n e y , 1982; S a n e s , 1983). T h e s e e n d p l a t e - s p e c i f i c c h a r a c t e r i s t i c s a r e

a s s o c i a t e d w i t h t h e o r g a n i s a t i o n o f r e c e p t o r s and AChE ( F a l l o n e t a l . ,

1985; W a l l a c e e t a l . , 1985). Cha nge s i n t h e a r c h i t e c t u r e o f t h e b a s a l

l a m i n a upon d e n e r v a t i o n c o u l d t h e r e f o r e be o n e o f t h e c r u c i a l f a c t o r s i n

d e t e r m i n i n g t h e c o n c e n t r a t i o n o f e n d p l a t e AChE. H o w e v e r , t h e f a c t t h a t

t h e H f o r m s re m ai n s p e c i f i c t o t h e e n d p l a t e r e g i o n i n t h e t y p e I I f i b r e s

a l s o s u g g e s t s t h a t e v e n t h o u g h t h e number o f b i n d i n g s i t e s may h a v e b een

r e d u c e d , t h e a t t a c h m e n t o f AChE r e m a i n s s e l e c t i v e . I t h a s b e e n shown

t h a t n e u r a l i n d u c t i o n l e a v e s a p erm a n en t s i g n a l f o r t h e f o c a l

a c c u m u l a t i o n o f t h e H fo r m s (B u rd en e t a l . , 1979). I n c r e a s e d t u r n o v e r o f

t h e en zy m e i n d e n e r v a t e d m u s c l e s (Newman e t a l . , 1984) i s n o t a

s u f f i c i e n t e x p l a n a t i o n f o r t h e r e d u c t i o n o f e n d p l a t e AChE i n c a s e s wh er e

t h e r e a r e c o m p e n s a t o r y i n c r e a s e s i n AChE s y n t h e s i s . H ow ever ,

n e u r o m u s c u l a r i n t e r a c t i o n i s e s s e n t i a l f o r t h e s t a b i l i s a t i o n o f t h e

normal c l u s t e r i n g o f t h e s y n a p t i c AChE. The d e n e r v a t e d t y p e I f i b r e s , on

t h e o t h e r h a n d , c o n t a i n L a s w e l l a s H f o r m s a t t h e e n d p l a t e s i t e s . The

* n a t u r e and l o c a l i s a t i o n o f t h i s L co m p o n en t r e m a i n s t o be c o n f i r m e d ;

h o w e v e r , s i n c e t h e amount o f H fo rm s a t t h e s e e n d p l a t e s i s a l s o r e d u c e d ,

t h e e f f e c t o f d e n e r v a t i o n on t h e e n d p l a t e H fo rm s i s t h e r e f o r e s i m i l a r .

3.4.3 AChE in the non-endplate regions of normal and denervated muscle

fibres

The e x c e s s s y n t h e s i s o f t h e L and M fo rm s i n d e n e r v a t e d t y p e II

m u s c l e i s e n t i r e l y e x t r a s y n a p t i c . The c y t o p l a s m i c AChE s t a i n i n g o b s e r v e d

- 106 -

by T e n n y so n e t a l . ( 1 9 7 7 ) i s l i k e l y t o be c o n t r i b u t e d by t h e s o l u b l e

f r a c t i o n o f t h e s e f o r m s , n o r m a l l y w e l l l o c a l i s e d w i t h i n t h e s a r c o p l a s m i c

r e t i c u l u m . T h e s e l a r g e i n c r e a s e s i n AChE c o u l d a l s o a c c o u n t f o r t h e

i n c r e a s e i n c y t o p l a s m i c p r o t e i n s s e e n i n d e n e r v a t e d r a b b i t m u s c l e s

( S y r o v y e t a l . , 1 9 7 1 ) . In t y p e I m u s c l e s , a p a r t from t h e L and M f o r m s ,

an e x t r a - j u n c t i o n a l p o p u l a t i o n o f F ^ i s n o t e n d p l a t e - s p e c i f i c .

F u r t h e r m o r e , t h i s e x t r a - j u n c t i o n a l H^c AChE m us t b e o v e r - p r o d u c e d

b e c a u s e o f t h e n e t i n c r e a s e i n t h e a c t i v i t y i n d e n e r v a t e d t y p e I

m u s c l e s , d e s p i t e t h e r e d u c t i o n o f t h e e n d p l a t e H AChE c o m p o n e n t . T h i s

s u g g e s t s t h a t t h e e x t r a - j u n c t i o n a l F ^ AChE u n d e r g o e s s i m i l a r c h a n g e s

w h ic h a f f e c t t h e L and M f o rm s o u t s i d e t h e e n d p l a t e , and i s q u i t e

i n d e p e n d e n t o f t h e e n d p l a t e F ^ . In t y p e I I m u s c l e s , d e n e r v a t i o n d o e s

n o t i n d u c e t h e p r o d u c t i o n o f e x t r a - j u n c t i o n a l H d e s p i t e s i m i l a r

e x c e s s i v e s y n t h e s i s o f t h e L and M f o r m s . I t i s c o n c e i v a b l e , t h e r e f o r e ,

t h a t i n c r e a s e d am ounts o f t h e n o n - e n d p l a t e F ^ AChE i s n o t m e r e l y du e t o

an o v e r p r o d u c t i o n o f t h e l o w e r m o l e c u l a r w e i g h t AChE f o r m s . T h i s

a p p a r e n t l a c k o f c o r r e l a t i o n b e t w e e n t h e s y n t h e s i s o f AChE

e x t r a j u n c t i o n a l l y and t h e p r e s e n c e o f j u n c t i o n a l f o rm s i n d i c a t e s t h a t

t h e e x t r a j u n c t i o n a l AChE i s n o t n e c e s s a r i l y t h e p r e c u r s o r s o f t h e

j u n c t i o n a l AChE. D en e jv a t io n h a s b een shown t o i n c r e a s e t h e s e c r e t i o n o f

AChE from m u s c l e t i s s u e ( C a r t e r and B r i m i j o i n , 1 9 8 1 ) , w h ic h r e s e m b l e s

• t h e i n t r a c e l l u l a r s e c r e t o r y po ol o f AChE i n a n e u r a l m u s c l e c u l t u r e

( R o t u n d o and Fam brough, 1 9 8 0 ) .

The p r e s e n c e o f e x t r a j u n c t i o n a l H2C i n t y p e I f i b r e s and i t s

i n c r e a s e d s y n t h e s i s upon d e n e r v a t i o n o f t h e s e f i b r e s may be r e l a t e d t o

some s p e c i f i c l o c a l i s a t i o n o f t h i s form o u t s i d e t h e e n d p l a t e . T h e r e i s

y e t no a v a i l a b l e e v i d e n c e c o n c e r n i n g t h e p o s s i b l e u l t r a - s t r u c t u r a l

l o c a l i s a t i o n o f t h i s e x t r a - j u n c t i o n a l p o p u l a t i o n o f AChE, h o w e v e r ,

t h e p r e s e n c e o f t h i s form n o r m a l l y i n t y p e I f i b r e s and n o t t y p e II

- 107 -

f i b r e s s u g g e s t s t h a t i t may be r e l a t e d t o t h e many p h y s i o l o g i c a l

d i s t i n c t i o n s b e t w e e n t h e two f i b r e t y p e s . One s u c h p e r t i n e n t d i s t i n c t i o n

i s t h e e x t r a j u n c t i o n a l ACh s e n s i t i v i t y due t o t h e p r e s e n c e o f

e x t r a s y n a p t i c ACh r e c e p t o r s i n normal t y p e I f i b r e s . The p r e s e n c e o f

e x t r a - j u n c t i o n a l H AChE may b e r e l a t e d t o t h e f u n c t i o n o f t h i s

p o p u l a t i o n o f r e c e p t o r s . E x t r a j u n c t i o n a l ^ AChE h a s a l s o b e e n o b s e r v e d

i n n e o n a t a l r a t m u s c l e s ( S k e t e l j and B r z i n , 1 9 8 0 ) and human i n t e r c o s t a l

m u s c l e s ( C a r s o n e t a l . , 1 9 7 9 ) . F u r t h e r m o r e , imm ature f i b r e s e x h i b i t t y p e

I c h a r a c t e r s i t i c s ( E c c l e s e t a l . , 1 9 6 0 a ) and c o n t a i n e x t r a j u n c t i o n a l ACh

r e c e p t o r s ( H a r r i s , 1 9 8 1 ) . B a se d on t h e s e o b s e r v a t i o n s , t h e r e f o r e , t y p e I

f i b r e s may r e p r e s e n t a c l a s s o f p a r t i a l l y d i f f e r e n t i a t e d f i b r e s w h ic h

r e t a i n c e r t a i n e m b r y o n ic c h a r a c t e r i s t i c s .

3.4.4 Effect of partial and total denervation on the two regions of

semi membranosus

The e f f e c t o f d e n e r v a t i n g t h e s c i a t i c b r a n c h t o t h e

se m im em b ra n o su s was a s e f f e c t i v e a s when a l l 3 n e r v e s w e r e r e m o v e d ,

s u g g e s t i n g t h a t t h e s c i a t i c t r u n k i s t h e main m o t o n e u r o n e i n p u t t o t h e

tw o r e g i o n s o f t h e m u s c l e . The o b t u r a t o r n e r v e c a r r i e s a m ix e d s e n s o r y

and m o t o r i n p u t w h ic h i s i m p o r t a n t i n t h e r e f l e x m ec h a n ism f o r

c o o r d i n a t e d l o c o m o t i o n . The o b s e r v a t i o n t h a t t h i s n e r v e and t h e c a u d a l

f e m o r a l c u t a n e o u s n e r v e i n p u t do n o t r e d u c e t h e e x t e n t o f d e n e r v a t i o n -

i n d u c e d e l e v a t i o n o f AChE s u g g e s t s t h a t t h e s e n e r v e s p r o b a b l y r e g u l a t e a

r e l a t i v e l y s m a l l number o f m o t o r u n i t s , and may n o t r e i n n e r v a t e t h e

d e n e r v a t e d f i b r e s n o r m a l l y i n n e r v a t e d by t h e s c i a t i c n e r v e .

The AChE c o n t e n t p e r m u s c l e i n d e n e r v a t e d r a b b i t and g u i n e a p i g

m u s c l e s i s f r a c t i o n a l l y h i g h e r t h a n t h a t i n t h e c o n t r o l a n i m a l s d e s p i t e

t h e s e v e r e a t r o p h y o f t h e s e m u s c l e s . T h i s i n d i c a t e s t h a t t h e i n c r e a s e i s

a r e s u l t o f i n c r e a s e d s y n t h e s i s and n o t an a p p a r e n t i n c r e a s e due t o

- 108 -

w e i g h t l o s s . The n o t i c e a b l y h i g h e r AChE a c t i v i t y i n t h e p a r t i a l l y

d e n e r v a t e d SP m u s c l e when compared t o i t s t o t a l l y d e n e r v a t e d c o u n t e r

p a r t i s a r e s u l t o f i t s r e l a t i v e l y m in o r a v e r a g e w e i g h t l o s s . H o w e v e r ,

t h e s e m u s c l e s e x h i b i t e d s i m i l a r d e n e r v a t i o n - i n d u c e d c h a n g e s i n t h e AChE

a c t i v i t i e s a s t h e t o t a l l y d e n e r v a t e d m u s c l e s . Type I f i b r e s a r e known t o

a t r o p h y more s l o w l y t h a n t h e t y p e I I f i b r e s ( N i e d e r l e and Mayr, 1 9 7 8 ) ,

and p e r h a p s some r e s i d u a l a c t i v i t y i n t h e m u s c l e due t o t h e o b t u r a t o r

i n p u t may s l o w down t h e d e g r a d a t i o n o f t h e m y o f i b r i l l a r p r o t e i n s i n t h e

t y p e I m u s c l e . T h i s o b s e r v a t i o n , h o w e v e r , r e q u i r e s c o n f i r m a t i o n s i n c e

t h e number o f s a m p l e s i n v e s t i g a t e d i s s m a l l and e x h i b i t l a r g e

d i f f e r e n c e s . The p a r t i a l l y d e n e r v a t e d SA was s i m i l a r t o t h e t o t a l l y

d e n e r v a t e d SA i n t e r m s o f t h e c h a n g e i n AChE a c t i v i t y a s w e l l a s t h e

d e g r e e o f a t r o p h y , s u g g e s t i n g t h i s r e s i d u a l i n n e r v a t i o n i s i n e f f e c t i v e

i n m a i n t a i n i n g normal r e g u l a t i o n o f t h e t y p e I I f i b r e s . The e f f e c t o f

p a r t i a l d e n e r v a t i o n t h e r e f o r e d i f f e r s l i t t l e from t h a t o f t o t a l

d e n e r v a t i o n on t h e s y n t h e s i s o f AChE i n t y p e I and t y p e I I f i b r e s , b u t

may h a v e d i f f e r e n t i n f l u e n c e s on t h e s t r u c t u r a l e l e m e n t s b e t w e e n t h e two

f i b r e t y p e s . T h e r e a r e c e r t a i n p e c u l i a r i t i e s a s s o c i a t e d w i t h t h e r a b b i t

SP: t h e m u s c l e i s t o t a l l y e n v e l o p e d by t h e SA; h i s t o c h e m i c a l l y , t h e t y p e

I f i b r e s i n t h i s m u s c l e , l i k e t h o s e i n t h e s o l e u s , d i s p l a y much h i g h e r

r e s i s t a n c e t o a l k a l i n e p r e t r e a t m e n t t h a n t h e t y p e I f i b r e s f o u n d i n

• m ix e d m u s c l e s ( J . P i z z e y , p e r s o n a l c o m m u n i c a t i o n ) . T h e r e f o r e t h e s e

f i b r e s i n a p u r e s l o w - t w i t c h m u s c l e may be c o n s i d e r e d t e n t a t i v e l y ,

a c c o r d i n g t o t h e i r h i s t o c h e m i c a l c h a r a c t e r i s t i c s , a s a s u b - t y p e o f

mammalian t y p e I f i b r e s . I t i s n o t w i t h i n t h e s c o p e o f t h i s s t u d y t o

c l a r i f y t h e p r o p e r t i e s o f t h e s e t y p e I f i b r e s . The q u e s t i o n s a s t o

w h e t h e r t h e r e s p o n s e o f t h e SP t o p a r t i a l d e n e r v a t i o n i s p e c u l i a r t o t h e

m u s c l e i t s e l f , o r t y p i c a l o f t h i s s u b - t y p e o f t y p e I f i b r e s , o r w h e t h e r

i t d e m o n s t r a t e s t h e d i f f e r e n c e s b e t w e e n t y p e I and t y p e II f i b r e s a r e

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s t i l l t o be s e t t l e d .

3.5 Conclusion

In c o n c l u s i o n , i t was f o u n d t h a t b o t h t h e t y p e I and t y p e I I

f i b r e s e x p r e s s s i m i l a r AChE a c t i v i t i e s and c o n t a i n a s i g n i f i c a n t

p r o p o r t i o n o f H fo r m s w h ic h i s e n d p l a t e s p e c i f i c . In a d d i t i o n , t y p e I

f i b r e s c o n t a i n a s i g n i f i c a n t amount o f e x t r a - j u n c t i o n a l H f o r m s . Bo th

p a r t i a l and t o t a l d e n e r v a t i o n o f m u s c l e s i n g u i n e a p i g and r a b b i t

i n d u c e d an i n c r e a s e i n t o t a l a c t i v i t y , a t t r i b u t e d t o a l a r g e i n c r e a s e i n

t h e s y n t h e s i s o f e x t r a - j u n c t i o n a l AChE f o r m s , i n p a r t i c u l a r t h e L f o r m s .

The i n c r e a s e i n e x t r a - j u n c t i o n a l H2 c form i n t h e t y p e I f i b r e s was

c o n c o m i t a n t w i t h t h e i n c r e a s e i n t h e l o w e r m o l e c u l a r w e i g h t f o r m s , and

a c c o u n t e d f o r t h e i n c r e a s e i n t h e t o t a l H2 c a c t i v t i y . The e n d p l a t e AChE

f o r m s w e r e r e d u c e d upon d e n e r v a t i o n , b u t a s i g n i f i c a n t f r a c t i o n r e m a i n e d

a t t h e e n d p l a t e s i t e . D e n e r v a t e d t y p e I and t y p e I I f i b r e e x h i b i t

s i m i l a r i n c r e a s e i n e x t r a - j u n c t i o n a l AChE and s i m i l a r r e d u c t i o n o f

e n d p l a t e AChE; i n t h e c a s e o f t y p e I f i b r e s , t h e e n d p l a t e and n o n -

e n d p l a t e H2 c AChE w e r e t h u s r e g u l a t e d s e p a r a t e l y . The p r e s e n c e o f no n -

e n d p l a t e H2 c form i n t y p e I m u s c l e s and i t s i n c r e a s e upon d e n e r v a t i o n i s

i n marked d i s t i n c t i o n from t y p e I I m u s c l e s . T h e s e d i f f e r e n c e s may be

r e l a t e d t o t h e e x t r a - j u n c t i o n a l s e n s i t i v i t y o f t y p e I f i b r e s t o ACh.

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CHAPTER 4

CHANGES IN MOLECULAR FORMS OF AChE IN CHRONICALLY STIMULATED

CHICKEN MUSCLE.

- 1 1 1 -

4 . 1 INTRODUCTION

The n e u r o n a l c o n t r o l o f a m u s c l e c o u l d be e x e r t e d by t h e

a c t i o n o f ( a ) some c h e m i c a l m o d u l a t o r ( s ) o r i g i n a t i n g i n t h e m o to r

n e r v e ; ( b ) t h e r e p e t i t i v e d e p o l a r i s a t i o n o f t h e m u s c l e membrane

by t h e c o n d u c t e d a c t i o n p o t e n t i a l s - ( c ) c o m b in e d e f f e c t s o f ( a )

and ( b ) . I t h a s b e e n s u g g e s t e d t h a t t h e s e a c t i o n s may have

d i f f e r e n t i a l e f f e c t s i n d e t e r m i n i n g t h e v a r i o u s p r o p e r t i e s o f t h e

m u s c l e ( W a lk e r and W i l s o n , 1 9 7 5 ) .

M u s c l e a c t i v i t y i s e s s e n t i a l f o r AChE c l u s t e r i n g a t t h e

n e u r o m u s c u l a r c o n t a c t . The b l o c k a d e o f c o n d u c t i o n i n t h e s c i a t i c

n e r v e t r u n k by t h e s u b - p e r i n e u r a l a p p l i c a t i o n o f t e t r o d o t o x i n

( B u t l e r e t a K , 1 9 7 8 ) o r by c o m p r e s s i o n ( C a n g i a n o e t a K , 1 9 8 0 )

i n d u c e d a 50% d e c r e a s e i n r a t h i n d - l i m b m u s c l e AChE and i t s l o s s

from t h e e n d p l a t e . L i k e w i s e , t h e b l o c k a d e o f m yo tub e c o n t r a c t i o n

i n m u s c l e c e l l c u l t u r e by t e t r o d o t o x i n ( W a lk e r and S t r o h m a n ,

1 9 7 6 ; W a lk e r and W i l s o n , 1 9 7 6 ; R i e g e r e t a l . . 1 9 8 0 ) , and i n

i n n e r v a t e d c u l t u r e s by d - t u b o c u r a r i n e ( R u b in e t a l .. 1 9 8 0 ) p r e v e n t

t h e a p p e a r a n c e o f j u n c t i o n a l AChE. C h r o n ic s t i m u l a t i o n ( a t 0 . 5

Hz) t h r o u g h e l e c t r o d e s i n s e r t e d a t t h e s p i n a l c o r d o f t h e c h i c k

embryo i n c r e a s e d t h e number o f n e u r a l c o n t a c t s on t h e PLD m u s c l e

f i b r e w i t h a p l a q u e o f AChE a c t i v i t y p r o d u c e d a t e a c h c o n t a c t

(Renaud e t a ^ . , 1 9 7 8 ) . D i r e c t e l e c t r i c a l s t i m u l a t i o n r e s t o r e s

t h e normal AChE l e v e l i n d e n e r v a t e d m u s c l e i n v i v o ( L i n k h a r t and

W i l s o n , 1 9 7 5 ) o r i n v i t r o ( W a lk e r and W i l s o n , 1 9 7 5 ) , a s w e l l a s

AChE c l u s t e r i n g (F ra nk e t aiK, 1 9 7 5 ; L0mo and S l a t e r , 1 9 8 0 ;

B r e n n e r e t a l . , 1 9 8 3 ) .

-112-

E l e c t r i c a l s t i m u l a t i o n o f a mammalian f a s t - t w i t c h m u s c l e a t

a c o n t i n u o u s l o w f r e q u e n c y (10 Hz) w h ic h i s t y p i c a l o f a s l o w -

t w i t c h f i b r e i n d u c e s t h e m u s c l e t o e x p r e s s s l o w - t w i t c h f i b r e t y p e

c h a r a c t e r i s t i c s ( S a lm o n s and V r b o v a , 1 9 6 9 ; C o t t e r e t a l . , 1 9 7 3 ;

P e t t e e t a \_. , 1 9 7 3 ; Romanul e t a K , 1 9 7 4 ; S a l m o n s and S r e t e r ,

1 9 7 6 ) . T h i s t r a n s f o r m a t i o n i s a p r o g r e s s i v e p r o c e s s w h ic h

i n v o l v e s an i n i t i a l c h a n g e i n t h e e n d o p l a s m i c r e t i c u l u m and

c a l c i u m s e q u e s t r a t i o n (H e i lm a n n e t a K , 1 9 8 1 ; H ei lm an n and P e t t e ,

1 9 7 9 ; Mabuchi e t a K , 1 9 8 2 ) . A r a p i d i n c r e a s e i n t h e e n z y m e s f o r

o x i d a t i v e m e t a b o l i s m o c c u r s , a c c o m p a n i e d by a r e c i p r o c a l d e c r e a s e

i n t h e e n z y m e s f o r g l y c o l y t i c m e t a b o l i s m (K lu g e t a K , 1 9 8 3 ) .

A f t e r p r o l o n g e d s t i m u l a t i o n , t h e l i g h t c h a i n m y o s i n f e a t u r e s o f

f a s t - t w i t c h m u s c l e a r e r e p l a c e d by t h o s e o f s l o w - t w i t c h m u s c l e

( R u b i n s t e i n e t a K , 1 9 7 8 ) r e s u l t i n g i n a c o n t r a c t u r e t y p i c a l o f a

s l o w - t w i t c h m u s c l e ( S a lm o n s and V rb o v a , 1 9 6 9 ) . S t u d i e s u s i n g

e l e c t r o n m i c r o s c o p y r e v e a l a l a r g e i n c r e a s e i n t h e number o f

m i t o c h o n d r i a and c a p i l l a r i s a t i o n i n s t i m u l a t e d m u s c l e s ( S a l m o n s

and V rb ova , 1 9 6 9 ; S a lm o n s e t a K , 1 9 7 8 ) . The Z d i s k s , w h ic h a r e

n o r m a l l y t h i c k e r i n t h e s l o w - t w i t c h t h a n t h e f a s t - t w i t c h f i b r e s

( G a u t h i e r and P a d y k u l a , 1 9 6 6 ) a l s o become n o t i c e a b l y t h i c k e r i n

t h e s t i m u l a t e d f a s t - t w i t c h m u s c l e ( E i s e n b e r g and S a l m o n s , 1 9 8 1 ;

• S a lm o n s e t a K , 1 9 7 8 ) . Such a d a p t i v e c h a n g e s a r e a t t r i b u t e d t o

t h e dyna mic and p l a s t i c n a t u r e o f m u s c l e ( P e t t e , 1 9 8 0 ; Sa lm o n s

and H e n r i k s s o n , 1 9 8 1 ) . The t r a n s f o r m a t i o n o f f i b r e t y p e s i s

r e v e r s i b l e ( S a l m o n s 1 9 8 0 ) and r e c e n t e v i d e n c e s u g g e s t s t h a t t h e

c h a n g e s a r e due t o t h e i n c r e a s e d t o t a l a c t i v i t y r a t h e r t h a n t o

t h e p a r t i c u l a r f i r i n g p a t t e r n i m p o s e d on t h e m u s c l e , s i n c e

s t i m u l a t i n g t h e m u s c l e c h r o n i c a l l y a t 2 0 o r 40 Hz p r o d u c e d

s i m i l a r c h a n g e s a s t h a t a t 5 o r 1 0 Hz ( E e r b e e k e t a l . , 1 9 8 4 ) .

-113-

The v a r i o u s e v i d e n c e p r e s e n t e d i n C h a p t e r s 2 a n d 3 c l e a r l y

i n d i c a t e s a s p e c i f i c r e l a t i o n s h i p b e t w e e n t h e d i s t r i b u t i o n o f t h e

v a r i o u s f o rm s o f AChE and t h e f i b r e t y p e c o m p o s i t i o n o f m u s c l e s .

The n e x t s t a g e o f t h i s s t u d y i s t o i n v e s t i g a t e t h e e f f e c t s o f

c h r o n i c s t i m u l a t i o n on t h e d i s t r i b u t i o n o f t h e s e f o r m s , w h ic h may

s h e d f u r t h e r l i g h t on t h e way AChE i s r e g u l a t e d by t h e

c o n t r a c t i l e a c t i v i t y o f t h e m u s c l e .

A f u r t h e r d i m e n s i o n t o t h e s t u d y o f t h e n e u r a l r e g u l a t i o n o f

m u s c l e AChE i s p r o v i d e d by t h e l a r g e c h a n g e s i n t h e d i s t r i b u t i o n

o f t h i s enzyme i n t h e m u s c l e o f a m u t a n t , g e n e t i c a l l y d y s t r o p h i c ,

c h i c k e n . I n t h e s e h o m o z y g o t e s , a l a r g e e x c e s s o f AChE, i n

p a r t i c u l a r t h e L f o r m s , i s s y n t h e s i z e d by t h e d y s t r o p h i c

f a s t - t w i t c h m u s c l e s s u c h a s t h e PLD ( L y l e s e t a K , 1 9 7 9 ) . The

s y n t h e s i s and d i s t r i b u t i o n o f AChE fo rm s i n t h e a d u l t d y s t r o p h i c

m u s c l e s r e t a i n an e m b r y o n ic p r o f i l e . N e v e r t h e l e s s , n e u r o n a l

c o n t r o l i s e x e r t e d s i n c e d e n e r v a t i o n a g a i n r e s u l t s i n a r e d u c t i o n

o f t h e H f o rm s ( L y l e s e t a l^ . , 1 9 8 2 ) . I n t h i s s t u d y e x p e r i m e n t s

w e r e p e r f o r m e d w h er e b y t h e normal and d y s t r o p h i c PLD m u s c l e o f

t h e c h i c k e n was c h r o n i c a l l y s t i m u l a t e d i n v i v o . The a im i s t o

s e e how t h e m u s c l e AChE i s m o d i f i e d u n d e r t h i s c h a n g e o f

e l e c t r i c a l a c t i v i t y . I t h a s b e e n p r e v i o u s l y shown t h a t t h e

m o rp h o lo g y o f t h e d y s t r o p h i c p a t i g i a l i s m u s c l e i n y o u n g c h i c k e n

a p p e a r e d t o b e n o r m a l i s e d by i n c r e a s e d m u s c l e a c t i v i t y (A sh m o re ,

1 9 8 2 ) . F u r t h e r m o r e , c h r o n i c lo w f r e q u e n c y s t i m u l a t i o n o f

d y s t r o p h i c mouse m u s c l e s a l s o s t r e n g t h e n e d t h e t w i t c h t e n s i o n and

p r e v e n t e d t h e d e g e n e r a t i o n o f t h e m u s c l e s ( L u t h e r t e t _ a T , 1 9 8 0 ;

Vrbova and Ward, 1 9 8 1 ) . I t i s t h e r e f o r e i m p o r t a n t t o s e e w h e t h e r

o r n o t t h e d y s t r o p h i c p a t t e r n o f AChE c o n t e n t i n d y s t r o p h i c

c h i c k e n ca n a l s o be n o r m a l i z e d by i n d u c e d a c t i v i t y .

-114-

In a d d i t i o n , a p r e l i m i n a r y stu<fy o f AChE i n a f e w

f a s t - t w i t c h m u s c l e s i n t h e l o w e r l im b o f b o t h normal and

c | y s t r o p h i c c h i c k e n , and t h e e f f e c t o f c h r o n i c s t i m u l a t i o n on t h e

AChE a c t i v i t y and d i s t r i b u t i o n o f fo rm s i n t h e s e m u s c l e s i s a l s o

p r e s e n t e d . Such s t u d y a i m s t o compare b e t w e e n t h e e f f e c t o f

c h r o n i c s t i m u l a t i o n on f u n c t i o n a l l y more a c t i v e m u s c l e s , su ch a s

t h e l e g m u s c l e s , and t h e i n a c t i v e , p u r e l y t y p e I IB , PLD.

4 . 2 MATERIALS AMD METHODS

4 . 2 . 1 N e r v e s t i m u l a t i o n

S i l v e r e l e c t r o d e s w e r e i m p l a n t e d i n c o n t a c t w i t h t h e PLD

n e r v e , and i m p u l s e s a t 10 Hz w e r e d e l i v e r e d t o them from a

t o t a l l y i m p l a n t e d , e n c a p s u l a t e d p r i n t e d - c i r c u i t s t i m u l a t o r . The

c o n s t r u c t i o n o f t h e s t i m u l a t o r s and t h e s u r g i c a l i m p l a n t a t i o n

p r o c e d u r e s w e r e c a r r i e d o u t by Mr. J . J a r v i s ( B a r n a r d e t a l . ,

1 9 8 5 ) . The c h i c k e n s u s e d w e r e l i n e 4 1 2 ( n o r m a l ) and l i n e 4 1 3

( h o m o z y g o u s f o r m u s c u l a r d y s t r o p h y ; d y / d y ) , b o t h m a i n t a i n e d a t

t h e I m p e r i a l C o l l e g e b r e e d i n g c o l o n y ( W i l s o n e t a K , 1 9 7 9 ) .

The s u c c e s s o f su ch s t i m u l a t i o n r e l i e s upon u n i n t e r r u p t e d

i m p u l s e t r a n s m i s s i o n t o t h e m u s c l e v i a t h e c o n t a c t b e t w e e n t h e

* e l e c t r o d e s and PLD n e r v e . O c c a s i o n a l l y i n t e r r u p t i o n s have a r i s e n

b e c a u s e o f e x c e s s i v e f i b r o u s i n f i l t r a t i o n b e t w e e n t h e e l e c t r o d e s

and t h e n e r v e , w h i c h was d e t e c t a b l e a t a u t o p s y . T h u s , a l t h o u g h

h i s t o c h e m i c a l l y some m u s c l e s m i g h t show a m in o r s h i f t from t y p e

IIB t o IIA f i b r e s , v e r y l i t t l e c h a n g e i n f a c t o c c u r r e d i n t h e

AChE a c t i v i t y . T h e s e c a s e s w e r e d i s c a r d e d , i . e . , o n l y t h o s e

s t i m u l a t e d l i m b s w h ic h showed normal w h o l e - m u s c l e t w i t c h

- 115 -

r e s p o n s e s a t t h e t i m e o f t e r m i n a t i o n ( 2 - 8 w e e k s a f t e r

i m p l a n t a t i o n ) w e r e u s e d . I m p l a n t a t i o n o f e l e c t r o d e s t o t h e PLD

f o r t h e same d u r a t i o n , b u t w i t h o u t a c t u a l s t i m u l a t i o n , was a l s o

i n c l u d e d a s c o n t r o l .

C h r o n i c s t i m u l a t i o n ( 1 0 Hz) o f t h e l o w e r l im b m u s c l e s was

a c h i e v e d by c o n n e c t i n g t h e e l e c t r o d e s t o t h e l o w e r s e c t i o n o f t h e

s c i a t i c n e r v e , a b o u t 2cm a b o v e t h e p a t e l l a . By l e a v i n g t h e u p p e r

l im b m u s c l e s u n i n t e r r u p t e d , su c h s t i m u l a t i o n d i d n o t c a u s e a n y

n o t i c e a b l e d i s c o m f o r t o r r e s t r i c t i v e movement t o t h e c h i c k e n

d u r i n g t h e e x p e r i m e n t a l p e r i o d .

6 - w e e k - o l d normal c h i c k e n s w e r e s t i m u l a t e d f o r 1 and 3 w e e k s

and a 7 - w e e k - o l d d y s t r o p h i c c h i c k e n was s t i m u l a t e d f o r 3 w e e k s

b e f o r e s a c r i f i c e . The EDL, a n t e r i o r t i b i a l i s (AT) and l a t e r a l

g a s t r o c n e m i u s w e r e s e l e c t e d f o r AChE a n a l y s i s .

4 . 2 . 2 E x t r a c t i o n o f AChE

The s t i m u l a t e d PLD m u s c l e and t h e c o n t r a l a t e r a l u n s t i m u l a t e d

m u s c l e w ere e x c i s e d i m m e d i a t e l y a f t e r d e a t h and c o o l e d on i c e . A

s l i c e from t h e c e n t r e o f e a c h m u s c l e was h o m o g e n is e d ( g l a s s /

g l a s s ) i n 10 v o l s o f i c e - c o l d p h o s p h a t e b u f f e r c o n t a i n i n g 1%

T r i t o n X - 1 0 0 , l M - N a C l , 5 mM EGTA and t h e f o l l o w i n g p r o t e a s e

i n h i b i t o r s : b a c i t r a c i n ( l m g / m l ) , c h i c k e n o v o m u c o id t r y p s i n

i n h i b i t o r ( 0 . 2 m g / m l ) , l e u p e p t i n ( 0 . 0 4 m g / m l ) , p e p s t a t i n

( 0 . 0 2 m g / m l ) , and NEM (5nM ). The h o m o g e n a t e s w ere i m m e d i a t e l y

c e n t r i f u g e d a t 4 3 , 5 0 0 x g f o r 15 min t o y i e l d t h e m u s c l e c r u d e

e x t r a c t s . I n some c a s e s , a s a m p le from t h e same m u s c l e s l i c e was

( w h e r e s t a t e d ) m i c r o d i s s e c t e d on an i c e - c o l d p l a t f o r m a s

d e s c r i b e d i n S e c t i o n 2 . 2 . 5 .

- 1 1 6 -

4 . 2 . 3 S e d im e n t a t io n a n a l y s e s

A l i q u o t s o f t h e c r u d e e x t r a c t , o r t h e e x t r a c t o f t h e m i c r o -

d i s s e c t e d s p e c i m e n s w e r e a n a l y s e d on a l i n e a r s u c r o s e d e n s i t y

(5%-20%) g r a d i e n t c o n t a i n i n g b u f f e r e d 1% T r i t o n X - 1 0 0 / lM - N a C l

( s e e s e c t i o n 2 . 2 . 6 ) . The g r a d i e n t f r a c t i o n s w e r e a n a l y s e d f o r

AChE a c t i v i t y a s d e s c r i b e d i n s e c t i o n 2 . 2 . 7 . The a c t i v i t y o f

V'ChE i n t h e m u s c l e c r u d e e x t r a c t s was a l s o m e a s u r e d by t h e same

m e t h o d s , u s i n g 5 X 1 0 “ ^1 BW284C51 t o i n h i b i t AChE. The

r e l a t i v e a c t i v i t y was d e f i n e d a s t h e r a t i o o f t h e AChE a c t i v i t y

o f t h e s t i m u l a t e d m u s c l e and t h e AChE a c t i v i t y o f i t s

c o n t r a l a t e r a l c o n t r o l .

4 . 3 . RESULTS

4 . 3 . 1 . C h a n g es i n AChE a c t i v i t y i n s t i m u l a t e d norm al c h ic k e n PLD

m u s c le

C h r o n i c lo w f r e q u e n c y s t i m u l a t i o n o f t h e normal c h i c k e n PLD

m u s c l e i n d u c e d a r e m a r k a b l e i n c r e a s e i n i t s AChE a c t i v i t y when

com p are d w i t h t h e c o n t r a l a t e r a l , u n s t i m u l a t e d c o n t r o l m u s c l e

( T a b l e 4 . 1 ) . A s i m i l a r i n c r e a s e was o b s e r v e d i n ^ChE a c t i v i t y ,

t o a b o u t 2 - 3 t i m e s t h e u n s t i m u l a t e d m u s c l e . The c o n t r a - l a t e r a l

c o n t r o l PLD u s e d i n t h i s s t u d y c o n t a i n e d i d e n t i c a l AChE a c t i v i t y

and m o l e c u l a r c o m p o s i t i o n a s t h e PLD i n u n t r e a t e d c h i c k e n s .

I m p l a n t e d durnny e l e c t r o d e s had no e f f e c t on t h e h i s t o l o g y , t h e

t o t a l AChE a c t i v i t y o r t h e d i s t r i b u t i o n o f AChE fo rm s o f t h e

m u s c l e .

PLD i n c h i c k e n s i m p l a n t e d w i t h s t i m u l a t o r s b e t w e e n 4 - 6 w e e k s

o f a g e The g r e a t e s t c h a n g e o c c u r r e d a f t e r 2 w e e k s , when

- 117 -

T a b l e 4 . 1 I n c r e a s e s i n AChE c o n c e n t r a t i o n i n t h e normal PLD

a f t e r d i f f e r e n t p e r i o d s o f s t i m u l a t i o n .

Sa m ple

F i n a l

a g e ( w k s )

S t i m u l a t i o n

( w k s )

C o n tr o l [ A C h E ]

( U / g )

R e l a t i v e

a c t i v i t y

A 6 2 0 . 2 5 5 . 5

B 8 3 0 . 3 3 . 2

C 9 5 0 . 4 3 . 1

D 12 3 0 . 2 2 . 8

E 19 8 0 . 3 2 . 6

In T a b l e s 4 . 1 , 4 . 2 a nd 4 . 3 , t h e c o n t r o l o f AChE c o n c e n t r a t i o n i s

shown f o r t h e c o n t r a l a t e r a l m u s c l e i n t h e s t i m u l a t e d b i r d , and

( l a s t c o lu m n ) t h e AChE c o n c e n t r a t i o n i n t h e s t i m u l a t e d m u s c l e

r e l a t i v e t o t h a t i n t h e c o n t r a l a t e r a l m u s c l e i s g i v e n a s t h e

" r e l a t i v e a c t i v i t y " .

*

- 118 -

s t i m u l a t e d PLD m u s c l e e x h i b i t e d a p p r o x i m a t e l y 5 t i m e s t h e normal

AChE a c t i v i t y . T h i s i n c r e a s e i n t o t a l a c t i v i t y was t h e r e s u l t o f

a l a r g e i n c r e a s e (27 f o l d ) i n t h e a c t i v i t y o f t h e L2 f o r m ,

a c c o m p a n i e d by p a r a l l e l b u t s m a l l e r i n c r e a s e s i n t h e o t h e r

m o l e c u l a r fo rm s (M, 7 f o l d ; H, 2 f o l d ) ( c f . F i g 4 . 1A and 4 . I B ) .

F o r s t i m u l a t i o n p e r i o d o f 3 - 5 w e e k s , t h e i n c r e a s e i n t o t a l AChE

a c t i v i t y was r e d u c e d t o o n l y 3 f o l d . T h i s was a c c o m p a n i e d by a

p r o g r e s s i v e r e d u c t i o n o f t h e l_2 a c t i v i t y w h ich was 16 t i m e s

t h e c o n t r o l v a l u e a f t e r t h r e e w e e k s ( F i g . 4 . 1 C ) , and n i n e t i m e s

h i g h e r a f t e r f i v e w e e k s ( F i g . 4 . I D ) . The r e l a t i v e a c t i v i t y o f

t h e M and H fo rm s r e m a i n e d f a i r l y c o n s t a n t o v e r t h e 2 - 5 week

p e r i o d o f s t i m u l a t i o n , a t t h e end o f w h ic h t h e y d i s p l a y e d

r e l a t i v e a c t i v i t i e s o f 6 and 2 . 5 , r e s p e c t i v e l y . A f t e r f i v e w e e k s

s t i m u l a t i o n , t h e r e was a n o t i c e a b l e i n c r e a s e i n t h e H^c form

( F i g . 4 . I D ) , a form w h ic h was a l m o s t a b s e n t i n u n t r e a t e d b i r d s .

PLD i n c h i c k e n s i m p l a n t e d a t 9 - 1 1 w e e k s o f a g e The AChE

i n t h e s e s t i m u l a t e d m u s c l e s was a b o u t 3 t i m e s t h e normal AChE

a c t i v i t y a t , o r a f t e r 3 w e e k s s t i m u l a t i o n ( T a b l e 4 . 1 ) . I t was

c l e a r from t h e m o l e c u l a r p r o f i l e s o b t a i n e d from t h e s e m u s c l e s

( F i g 4 . I F , 4 . 1 G ) t h a t t h e H form s w e r e a l w a y s p r e d o m i n a n t , a s i n

t h e c o n t r o l m u s c l e ( F i g . 4 . I E ) , b u t t h e H^c form a g a i n

* i n c r e a s e d g r e a t l y . T h e s e o l d e r b i r d s , h o w e v e r , showed a more

r a p i d p r o g r e s s t o t h e f i n a l p a t t e r n , w h ic h was n o t c o m p l e t e a f t e r

5 w e e k s s t i m u l a t i o n i n t h e 4 - w e e k b i r d . The f i n a l i n c r e a s e s

i n a c t i v i t y f o r e a c h o f t h e m o l e c u l a r form s w e r e q u i t e s i m i l a r

( L , 3 . 7 ; M, 2 . 0 ; H, 2 . 7 t i m e s t h e i r c o n t r o l v a l u e s ) .

E l e c t r o n m i c r o s c o p y r e v e a l e d a r e m a r k a b l e i n c r e a s e i n t h e

number o f m i t o c h o n d r i a and l i p i d g l o b u l e s i n t h e PLD w h ic h had

- 119 -

*

-120-

FIG- 4.1 Sucrose gradient profiles of AChE in normal PLD after

different periods of stimulation. In 6-9 weeks old chickens: A,

contralateral unstimulated control; B-D, after 2, 3 and 5 weeks

stimulation, respectively. In 12-19 weeks old chickens: E,

contralateral unstimulated control; F-G, after 3 and 8 weeks

stimulation, respectively. E and F were from muscles used for

endplate and non-endplate analysis (Fig. 4.3). The amount of

enzyme analysed in each case in this and other figures in this

chapter was arbitrary. Arrow indicates the position of the

catalase (11.4S) marker.

FRACTIONS

-121-

been stimulated for 8 weeks (Fig. 4.2). These ultrastructural

changes correlate with the progressive change of the fibre type

composition from a dominant type I IB (>80%) to a dominant type

IIA (>70%) (Barnard et al., 1985).

4.3.2 S e p a r a t io n o f e n d p l a t e a n d n o n - e n d p la t e fo r m s o f ACME

The non-endplate region of the stimulated fibres in normal

chickens (Fig. 4.3C) contains significant amounts of

extra-junctional H^c and forms which were negligible in

the control samples. In contrast, in the endplates of the

stimulated fibres, there was little change from the normal

profile; the H^c form did, however, become significant,

although still very minor (Fig.4.3A, B).

4.3.3 T h e r e v e r s a l o f t h e e f f e c t o f s t i m u l a t i o n

Mature chickens whose PLD muscles had been stimulated for

4-5 weeks were examined at intervals after the stimulation, had

ceased (Table4.2). After a 1-week rest, the elevation of AChE

content was halved to a value near or (after a two-week rest)

even below that for control muscle. The profile of the

molecular forms in such a muscle in the latter state was

restored to that seen in a control muscle.

4.3.4. C h a n g e s i n AChE a c t i v i t y i n s t i m u l a t e d d y s t r o p h ic c h ic k e n

PLD m u s c le

Stimulators were implanted into dystrophic chickens of 4-6

weeks or 9-10 weeks of age. Chronic stimulation of the PLD nerve

led to a clear reduction in the AChE activity of these muscle by

-122-

- 123 -

FIG- 4.2 Electron micrographs of PLD muscle from a 19 week old

normal chicken. (A) contralateral unstimulated PLD; CB) 8-week-

stimulated PLD. 1, lipid globules; m, mitochondria. Magnifica

tion: 14,600x. Results of J. Jarvis and J. Cavanagh.

W * -- !% x *

' W _i ’ Jk !B ”*« ££ •?»% ..**;.-a--Si

|g&|M v. $»- f l

t xi ,=3b

| | 1 1- -

„., ir >/•-

♦% * -

IH| *#• -*• • - ■

:.v. 1 % *;."- *"''■ w

•(*V •_ ’’ "

■“•4 -

A : . I ; ! :; -':iiPjfe %!ff V ^ w r ^ n...i-r "&• ?KJL ^

i

* T \ : • 4 !

1 i ^2JT " -C ..1 i. r

124

FIG. 4.3 Sucrose gradient profiles of endplate and non-endplate

AChE in control and 3-week-stimulated normal PLD. Endplate AChE:

A, control; B, stimulated. Non-endplate AChE: C, stimulated.

- 125 -

-127-

Table 4.2 Reversal of stimulation-induced changes in AChE after cessation of

the stimulus.

Sample Age(wks)Stimulation

(wks)Control

[AChE] (U/g)Interruption (days)

Relative al1 forms

activity E IT H

F 15 4 0.6 oa 3.916 0.7 7 2.0

G 15 4 0.35 oa 3.017 0.5 14 0.5

H 14 5 0.3 7-14b 0.5 0.5 0.5 0.5

Stimulation was given continuously as shown and was then (where indicated)

interrputed for the period shown (with the electrodes remaining implanted), with

total loss of the induced twitching of the muscle. At the end of that period the PLD

was removed for the measurements. Note that within 2 weeks of cessation of impulses,

the AChE levels in the stimulated muscle regress to within the normal (see Table 4.1)

range, and that all the forms are equally changed.

a. A biopsy sample was taken for the first analysis, for both the PLDs, at the

time of cessation.

b. In this case, the cessation of stimulation and twitching was adventitious

due to fibrous growth over the electrodes contact surface , so that the period of

cessation was not precisely known.

Table 4.3 Decreases in AChE concentration in dystrophic PLD

after different periods of stimulation.

Sample

Final

age(wks)

Stimulation

(wks)

Control [AChE]

(U/g)

Relative

activi ty

A 6 2 2.8 0.3

B 8 3 3.4 0.6

C 9 4 3.2 0.6

D 14 5 2.2 0.4

E 17 8 0.7 1.0

Each value (except A and E) is the mean for 2 or 3 birds.

♦

- 128 -

40% to 70% (Table 4.3). The AChE activity of some of the

stimulated dystrophic muscles however was still about twice that

of the stimulated normal muscles. 0ChE activity, which was

greatly elevated in the dystrophic birds (Lyles et_a]_., 1979),

also declined with stimulation but remained on average 1.5 times

higher than that in the stimulated normal PLD (not shown).

All the molecular forms present in the dystrophic muscle

were reduced upon stimulation, but the reduction of the L forms

was especially noticeable (Fig. 4.4). This preferential

reduction of the L forms was much greater in the more mature

chickens (Fig. 4.4F). After two weeks chronic stimulation, the

dystrophic PLD showed an AChE molecular form profile similar to

that in the stimulated normal muscle (cf. Fig. 4.4B and 4.IB).

This change in activity involved a 70% reduction in the total

amount of the L and M forms,and a 80% reduction in the

form. Thereafter, there was no futher significant change in the

total AChE activity (Table 4. 3) although the relative amount of

the different forms did change, so that eventually both the

activity and distribution of AChE forms resemble that of a

stimulated normal PLD (Fig. 4.4F). This state was reached

earlier in the 14-week birds than in the younger ones. The

profile of a 17 -week old chicken which had been stimulated for 8

weeks was identical to that of the stimulated 14-week birds- The

reduction in the contribution of the L forms averaged 80%. The M

and H forms in contrast were much less affected (0 and 20%

respectively).

The stimulated dystrophic PLD underwent a remarkable

improvement in the structure of the muscle fibres (Fig. 4.5),

- 129 -

F IG - 4 . 4 S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n d y s t r o p h i c PLD

a f t e r d i f f e r e n t p e r i o d s o f s t i m u l a t i o n . In 6 - 9 week o l d

c h i c k e n s : B , 2 w e e k s ; D , 3 - 4 w e e k s . In 14 week o l d c h i c k e n s : F ,

5 w e e k s , A,C a n d E w e r e c o n t r o l p r o f i l e s c o r r e s p o n d i n g t o B , D

a n d F r e s p e c t i v e l y . E an d F w ere from m u s c l e s u s e d f o r e n d p l a t e

and n o n - e n d p l a t e a n a l y s i s ( F i g . 4 . 6 ) .

- 130 -

F IG . 4 . 5 Frozen cross section of PLD muscles from a 14 week old

dystrophic chicken. (A) contralateral unstimulated PLD; (B)

5-week-stimulated PLD. Sections were stained with haematoxylin

and Sirius red. Magnification: 350x. bar represents 20 pm.

Results of J . Jarvi s.

- 1 3 1 -

w h ich w ere now c l o s e l y p ack ed and p o ly g o n a l i n sh a p e in

c r o s s - s e c t i o n . T h ere was a g e n e r a l r e d u c t i o n in f i b r e

h y p e r t r o p h y and t h e n u c l e i became l a r g e l y p e r i p h e r a l . The number

o f t y p e IIA f i b r e s , a s in s t i m u l a t e d normal PLD, i n c r e a s e d

s u b s t a n t i a l l y (B a r n a r d e t a t , 1 9 8 5 ) .

4 . 3 . 5 . S e p a r a t io n o f e n d p l a t e a n d n o n - e n d p la t e fo r m s o f ACME

F i g . 4 .6 A sh o w s t h e m o l e c u l a r form s p r e s e n t a t t h e e n d p l a t e s

o f a d y s t r o p h i c PLD. T h ere was c l e a r l y an abnorm al p r e s e n c e o f

t h e form a t t h e e n d p l a t e , a s fo u n d p r e v i o u s l y i n d y s t r o p h i c

c h i c k e n s o f a d i f f e r e n t a g e ( J e d r z e j c z y k e t a l . , 1 9 8 1 ) . Upon

s t i m u l a t i o n t h i s c o n t r i b u t i o n o f t h e form w as l a r g e l y

e l i m i n a t e d ( F i g . 4 . 6 B ) . O u t s i d e t h e e n d p l a t e s ( F i g . 4 . 6 C ) , a

l a r g e am ount o f and ^ AChE w ere fo u n d in t h e d y s t r o p h i c

PLD f i b r e s , w h ic h w ere a b s e n t in t h e h e a l t h y PLD m u s c l e . The

s t i m u l a t e d d y s t r o p h i c PLD m u s c le f i b r e s c o n t a i n e d , i n c o n t r a s t ,

v e r y l i t t l e L fo rm s e x t r a j u n c t i o n a l l y ( F i g . 4 . 6 D ) . T h i s r e g i o n

w as i n s t e a d d o m in a te d by t h e Hjc and f o r m s . H ence t h e

c e l l u l a r d i s t r i b u t i o n o f t h e s e m o l e c u l a r form s in a s t i m u l a t e d

d y s t r o p h i c PLD f i b r e was v e r y s i m i l a r t o t h a t in a s t i m u l a t e d

norm al PLD f i b r e ( c f . F i g . 4 .6 B ,D and 4 . 3 B , C ) .

4 . 3 . 6 C h a n g es i n AChE a c t i v i t y i n s t i m u l a t e d c h ic k e n l e g m u s c le s

The p r e d o m in a n t ly f a s t - t w i t c h m u s c l e s su ch a s t h e EDL and

t h e AT e x h i b i t e d s i m i l a r AChE a c t i v i t y a s t h e PLD m u s c le ( T a b l e

4 . 5 ) . C h r o n ic s t i m u l a t i o n i n c r e a s e d t h e t o t a l AChE a c t i v i t y i n

t h e s e m u s c l e s , a l t h o u g h t h e m a g n itu d e o f such i n c r e a s e wasa

v a r i a b l e . Thus t h e AChE a c t i v i t y i n t h e s t im u la te d AT and EDL was

- 132 -

F IG . 4 . 6 S u c r o s e g r a d i e n t p r o f i l e s o f e n d p l a t e and n o n - e n d p l a t e

AChE in c o n t r o l and 5 - w e e k - s t i m u l a t e d d y s t r o p h i c PLD. E n d p la te*

AChE: A , c o n t r o l ; B , s t i m u l a t e d . N o n - e n d p la t e AChE: C , c o n t r o l ;

D , s t i m u l a t e d .

- 1 3 3 -

a b o u t t w i c e t h e c o n t r a l a t e r a l c o n t r o l m u s c l e s a f t e r 1 and 3 w e e k s

o f s t i m u l a t i o n r e s p e c t i v e l y , w h i l e t h a t in t h e g a s t r o c n e m i u s was

a n e a r 5 f o l d i n c r e a s e . T h e s e c h a n g e s w e r e a c c o m p a n ie d by an

i n c r e a s e i n t h e t y p e IIA and t y p e I f i b r e s w h ic h w e r e

p a r t i c u l a r l y d i s t i n c t i n t h e g a s t r o c n e m i u s ( T a b l e 4 . 4 ) .

T h e s e f a s t - t w i t c h m u s c l e s c o n t a i n e d p r e d o m in a n t ly

AChE. T h er e w as l i t t l e d i f f e r e n c e i n t h e p r o p o r t i o n o f t h e

form b e tw e e n t h e t h r e e m u s c l e s s t u d i e d e v e n th o u g h t h e EDL

c o n t a i n e d a l a r g e p r o p o r t i o n o f t y p e I f i b r e s w h ich w e r e t h o u g h t

t o c o n t a i n g r e a t e r a m o u n ts o f L^ AChE ( L y l e s e t a K , 1 9 8 2 ;

B arn ard e t a K , 1 9 8 2 ) . The i n c r e a s e in t o t a l AChE a c t i v i t y i n

b o th t h e AT (1 w eek ) and EDL ( 3 w e e k s ) w as due t o an i n c r e a s e i n

t h e am ount o f fo rm . On t h e o t h e r h an d , t h e l a r g e i n c r e a s e

i n AChE in t h e g a s t r o c n e m iu s was c o n t r i b u t e d by an i n c r e a s e i n

a l l t h e fo rm s ( F i g . 4 . 7 and T a b le 4 . 5 ) .

The AChE a c t i v i t i e s i n t h e s e m u s c l e s in d y s t r o p h i c c h i c k e n s

w e r e o n l y m o d e r a t e l y e l e v a t e d ( T a b le 4 . 5 ) . A n a l y s i s o f t h e AChE

form s show ed t h a t in t h e c o n t r o l a l l t h e m o l e c u l a r fo rm s w e r e

o v e r p r o d u c e d : in t h e AT, L ,8 ; M ,3 ; 3 t i m e s t h e normal

AT ( F i g - 4 . 8 A ) ; i n t h e g a s t r o c n e m iu s , L, 13 • H2 c , 2 t i m e s

t h e normal g a s t r o c n e m iu s ( F i g . 4 . 8 C ) . C h r o n ic s t i m u l a t i o n o f

t h e s e m u s c l e s r e s u l t e d in an i n c r e a s e i n AChE c o n t e n t , c o n t r a r y

t o t h a t o b s e r v e d i n t h e d y s t r o p h i c PLD. I t l e d t o a p r e f e r e n t i a l

i n c r e a s e in t h e H^ AChE i n t h e AT ( F i g . 4 . 8 B ) , b u t i n t h e

g a s t r o c n e m i u s , o n l y t h e L^ AChE was s i g n i f i c a n t l y e l e v a t e d .

4 . 4 DISCUSSION

4 . 4 . 1 C h a n g es o f AChE i n s t i m u l a t e d norm al c h ic k e n PLD

The r e s u l t s o f t h i s c h a p t e r show t h a t c h r o n i c lo w f r e q u e n c y

- 134 -

Table 4.4 Fibre type composition of some normal and stimulated

f a s t - t w i t c h m u s c l e s i n t h e lo w e r l im b o f c h i c k e n .

M u s c le F i b r e t y p e c o m p o s i t i o n [%)

I IIA I IB

EDL C o n tr o l 15 13 72

S t i m u l a t e d ( 3 w eek ) 15 67 18

L a t e r a l C o n tr o l 5 29 66

g a s t r o c n e m i u s S t i m u l a t e d ( 3 w eek ) 19 72 9

AT C o n tr o l 7 7 8 6

S t i m u l a t e d (1 w eek ) 2 82 16

R e s u l t s o f J . J a r v i s

-136

-

Table 4.5 Increases in AChE concentration in leg muscles of normal and dystrophic chickens after

d i f f e r e n t p e r i o d s o f s t i m u l a t i o n .

M u s c le F i n a l

a g e ( w k s )

L e n g th o f

S t i m u l a t i o n ( w k s )

[AChE] ( U / g ) R e l a t i v e a c t i v i t y

C o n tr o l S t im a l l form s L MH1C H2C

AT( M*) 7 1 0 . 3 5 0 . 6 6 1 . 9 2 . 3 - - 1 . 1

EDL(N) 9 3 0 . 4 6 0 . 7 2 1 . 6 2 . 7 2 . 0 - 1 . 1

g a s t r o c n e m i u s ( N ) 9 3 0 . 5 3 2 . 4 2 4 . 6 1 3 . 3 5 . 4 6 . 5 3 . 5

AT(D*) 10 3 1 . 3 3 . 9 3 . 0 1 . 9 2 . 9 - 2 . 4

g a s t r o c n e m i u s ( D ) 10 3 1 . 5 2 . 6 1 . 7 2 . 2 - - 1 . 0

* N = norm al c h i c k e n

D = d y s t r o p h i c c h i c k e n

ConN StimN

AA

FRACTIONS

FIG- 4.7 S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n l e g m u s c l e s o f

norm al c h i c k e n a f t e r c h r o n i c s t i m u l a t i o n . Con N ( c o n t r a l a t e r a l

u n s t i m u l a t e d normal m u s c l e ) ; S t im N ( s t i m u l a t e d normal m u s c l e ) .

A,B: a n t e r i o r t i b i a l i s ; C,D: EDL E,F: g a s t r o c n e m i u s . EDL and

g a s t r o c n e m i u s w ere s t i m u l a t e d f o r 3 w e e k s ; AT was s t i m u l a t e d f o r

1 w e e k .

- 137 -

ConD Sti mD

AA

FR A CTIO N S

F IG - 4 - 8 S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n l e g m u s c l e s o f

d y s t r o p h i c c h i c k e n a f t e r c h r o n i c s t i m u l a t i o n . Con D

( c o n t r a l a t e r a l u n s t i m u l a t e d d y s t r o p h i c m u s c l e ) ; S t im 0

( s t i m u l a t e d d y s t r o p h i c m u s c l e ) . A ,B : a n t e r i o r t i b i a l i s ; C ,D :

g a s t r o c n e m i u s . B oth m u s c l e s w ere s t i m u l a t e d f o r 3 w e e k s .

- 138 -

s t i m u l a t i o n b r i n g s a b o u t a l a r g e i n c r e a s e i n t h e a c t i v i t y o f AChE

i n normal PLD m u s c l e . T h is may b e a t t r i b u t e d t o i n c r e a s e d

b i o s y n t h e s i s i n t h e s t i m u l a t e d m u s c le i n r e s p o n s e t o i n c r e a s e d

m u s c le a c t i v i t y . T h i s d e d u c t io n i s b a s e d on o t h e r o b s e r v a t i o n s

on e l e c t r i c a l l y - s t i m u l a t e d mammalian m u s c l e s . T h u s , H e i l i g and

P e t t e ( 1 9 8 3 ) show ed t h a t s t i m u l a t i o n a t 10 Hz f o r 4 w e e k s o f

r a b b i t f a s t - t w i t c h m u s c l e s i n c r e a s e d t h e t o t a l RNA i n t h e m u s c le

an d t h e s lo w m y o s in l i g h t c h a i n mRNAs. S i m i l a r s t u d i e s by

M a s t r i , S a lm on s and Thomas ( 1 9 8 2 ) showed t h a t t h e c o n t e n t o f

p r o t e i n s y n t h e s i s - a s s o c i a t e d p o ly a m in e s and o f t h e en zy m es

p r o d u c in g them w as a l s o m a rk ed ly e l e v a t e d by c h r o n i c s t i m u l a t i o n .

H o w ev er , t h e e f f e c t o f s t i m u l a t i o n on p r o t e i n s y n t h e s i s i s

d e p e n d e n t upon t h e t y p e s o f m u s c le p r o t e i n : t h e l e v e l s o f enzym es

o f o x i d a t i v e m e t a b o l i s m a r e c o n s i d e r a b l y i n c r e a s e d i n s t i m u l a t e d

mammalian m u s c l e s , b u t a r e r e d u c e d f o r some o t h e r p r o t e i n s , e . g .

t h o s e r e s p o n s i b l e f o r c a l c i u m s e q u e s t r a t i o n and pumping a s w e l l

a s en zy m es o f a n a e r o b i c m e t a b o l i s m (K lug e t al^., 1 9 8 3 ) . In

a c c o r d a n c e w i t h t h i s , an abnorm al i n c r e a s e in t h e s y n t h e s i s o f

t h e "slow " i s o f o r m s o f v a r i o u s c o n t r a c t i l e p r o t e i n c h a i n s o c c u r s

w h i l e t h e p r o d u c t io n o f t h e c o r r e s p o n d i n g " f a s t " i s o f o r m s

d e c l i n e s , in r e s p o n s e t o l o n g p e r i o d s o f s t i m u l a t i o n o f f a s t -

t w i t c h mammalian m u s c l e s a t f r e q u e n c i e s (1 0 Hz) c h a r a c t e r i s t i c o f

t h e n a t u r a l a c t i v i t y o f s l o w - t w i t c h f i b r e s ( P e t t e e t a K , 1 9 7 6 ;

R u b i n s t e i n e t a K , 1 9 7 8 ) . In g e n e r a l , t h e r e s p o n s e t o su ch

a p p l i e d s t i m u l a t i o n , o r t o n a t u r a l s t i m u l a t i o n i n s u s t a i n e d

e x e r c i s e (S a lm o n s and H e n r i k s s o n , 1 9 8 1 ; G reen e t , 1 9 8 3 ) , i s a

' s w i t c h - o n ' o f t h e p r o t e i n s r e q u i r e d f o r s l o w - f i b r e a c t i v i t y and

a ' s w i t c h - o f f ' o f t h o s e f o r f a s t - f i b r e a c t i v i t y .

- 139 -

A v ia n m u s c l e s have n o t p r e v i o u s l y b e e n c h r o n i c a l l y

s t i m u l a t e d and a n a l y s e d w i t h r e s p e c t t o p r o t e i n s y n t h e s i s o r mRNA

c h a n g e s , b u t t h e o v e r a l l e f f e c t s o f in d u c e d a c t i v i t y on c h i c k e n

m u s c l e s a r e l i k e l y t o be s i m i l a r . We h a v e o b s e r v e d i n a p a r a l l e l

s t u d y (B a r n a r d e t a ^ . , 1 9 8 5 ) u s i n g f i b r e - t y p i n g t e c h n i q u e t h a t in

t h e s t i m u l a t e d c h i c k e n PLD m u s c l e s u s e d h e r e , a s i m i l a r

t r a n s f o r m a t i o n o f g l y c o l y t i c t y p e IIB f i b r e s t o h i g h l y o x i d a t i v e

t y p e IIA f i b r e s h a s o c c u r r e d . The i n c r e a s e o b s e r v e d i n t h e

m it o c h o n d r i a and l i p i d c o n t e n t r e s e m b l e s c l o s e l y t h e

u l t r a s t r u c t u r a l m o rp h o lo g y o f a p r e d o m in a n t ly t y p e I IA m u s c l e ,

nam ely t h e a d d u c t o r s u p e r f i c i a l i s ( J . C avan agh , p e r s o n a l

c o m m u n ic a t io n ) . T h er e was no s u b s t a n t i a l c h a n g e , h o w e v e r , i n t h e

t i m e - t o - p e a k o f i s o m e t r i c t w i t c h c o n t r a c t i o n o r t h e h a l f -

r e l a x a t i o n t im e o f t h e m u s c l e , up t o 8 w e e k s o f c o n t i n u o u s

s t i m u l a t i o n . T h i s i s i n c o n t r a s t t o t h e b e h a v i o u r o f s i m i l a r l y

s t i m u l a t e d mammalian f a s t - t w i t c h m u s c l e s , w h ose c o n t r a c t i l e

c h a r a c t e r i s t i c s and c y t o c h e m i s t r y a r e s i m i l a r t o t h o s e o f

s l o w - t w i t c h - o x i d a t i v e t y p e I f i b r e s ( R u b i n s t e i n e t a K , 1 9 7 8 ;

H eilm an n and P e t t e , 1 9 7 9 ) . In t h i s t r a n s f o r m a t i o n from t y p e I IB

t o IIA i n t h e c h i c k e n m u s c l e , i t i s c l e a r t h a t t h e o x i d a t i v e

enzym e s y n t h e s i s i s a c t i v a t e d and i t i s now shown h e r e t h a t

* m u s c le AChE p r o d u c t io n a l s o g r e a t l y i n c r e a s e s . T h is i s

i n t e r e s t i n g , s i n c e normal a v i a n t y p e IIA f i b r e s do n o t c o n t a i n , o n

a v e r a g e , more AChE th a n do t h e t y p e I IB f i b r e s , w h e r e a s t h e

c o n t e n t i n a v i a n m u s c l e s r i c h i n t y p e I f i b r e s i s a b o u t 1 0 - f o l d

g r e a t e r (B a r n a r d e t a K , 1 9 8 2 , 1 9 8 4 ) . H en ce , i t a p p e a r s t h a t t h e

t r a n s f o r m e d f i b r e s have a t t a i n e d some b u t n o t a l l o f t h e v a r i o u s

s p e c i f i c p r o p e r t i e s o f t y p e I f i b r e s .

- 140 -

M a s t r i e t a]_. ( 1 9 8 2 ) show ed t h a t t h e b i o s y n t h e t i c a c t i v i t i e s

o f a s t i m u l a t e d f a s t - t w i t c h mammalian m u s c le a r e t r a n s i e n t l y

e l e v a t e d i n a p h a s i c m an n er , many t im e s a b o v e n o r m a l , b e f o r e

l e v e l l i n g o f f t o a new s t e a d y s t a t e t y p i c a l o f a s l o w - t w i t c h

m u s c l e . A s i m i l a r t i m e - d e p e n d e n t c h a n g e i n t o t a l mRNA c o n t e n t o f

su ch m u s c le h a s a l s o b een o b s e r v e d ( P e t t e , 1 9 8 4 ) . In t h i s s t u d y ,

i t w as fo u n d t h a t AChE f o l l o w e d a s i m i l a r p h a s i c i n c r e a s e d u r in g

t h e c o u r s e o f s t i m u l a t i o n . A f t e r two w eek s o f c h r o n i c

s t i m u l a t i o n , t h e PLD m u s c le e x h i b i t e d t h e h i g h e s t l e v e l o f AChE

a c t i v i t y , a b o u t 5 t i m e s t h a t o f t h e c o n t r o l , w h ic h t h e r e a f t e r

m a in t a i n e d a t a l o w e r l e v e l o f 3 t i m e s t h e c o n t r o l v a l u e . T h is

p h a s i c p a t t e r n was b e t t e r i l l u s t r a t e d by t h e r e m a r k a b le i n c r e a s e

i n t h e L fo rm s o b s e r v e d o v e r t h e 8 w e e k s o f s t i m u l a t i o n . As t h e

e l e v a t e d l e v e l o f t h e H AChE w a s , o n c e a t t a i n e d , c o n s t a n t

t h r o u g h o u t t h e s t i m u l a t i o n p e r i o d , t h e l a t e r p r o g r e s s i v e

r e d u c t i o n in t h e L form s f o l l o w i n g t h e i n i t i a l i n c r e a s e was n o t a

r e s u l t o f a p r o g r e s s i v e i n c r e a s e in t h e a s s e m b ly o f t h e H f o r m s ,

b u t i s l i k e l y t o r e f l e c t a r e a l r e d u c t i o n i n t h e s y n t h e s i s o f t h e

L f o r m s . M i c r o d i s s e c t e d e n d p l a t e s and n o n - e n d p l a t e r e g i o n s o f

t h e 3 -w eek s t i m u l a t e d PLD i n d i c a t e d t h a t a t l e a s t p a r t o f t h e

e x c e s s H AChE i s l o c a t e d e x t r a - j u n c t i o n a l l y ( F i g . 4 . 3 ) . T h e s e

* o b s e r v a t i o n s s u g g e s t a b u i l d - u p o f c o l l a g e n - t a i l e d AChE i n an

e x t r a j u n c t i o n a l p o o l w h ic h i s n o t im m e d ia t e ly t r a n s p o r t e d t o t h e

e n d p l a t e . The H2 c form r e m a in e d t h e o n l y e n d p l a t e - s p e c i f i c

fo r m , w h i l e t h e H^c , l a r g e l y e x t r a j u n c t i o n a l , c o u l d be t h e

im m e d ia te p r e c u r s o r o f t h e H2 c fo r m , and n o t i t s breakdow n

p r o d u c t a t t h e e n d p l a t e s . The am ount o f H AChE a t e a c h e n d p l a t e

- 141 -

w as n o t s i g n i f i c a n t l y g r e a t e r th a n t h a t i n t h e u n s t i m u l a t e d

m u s c l e , s o we d e d u c e t h a t t h e r e a r e a f i x e d number o f s i t e s f o r

i t s a t t a c h m e n t i n t h e b a s a l la m in a t h e r e , l e a d i n g t o t h e e x c e s s H

AChE a c c u m u la t in g in i n t e r n a l m em branes. On t h e o t h e r h a n d ,

t h e s e e x t r a j u n c t i o n a l H AChE may r e s e m b le t h e e x t r a j u n c t i o n a l H

AChE fo u n d in mammalian t y p e I f i b r e s , w h ic h s u g g e s t s some

p o s s i b l e l o c a l i s a t i o n o r f u n c t i o n o f t h e s e form s o u t s i d e t h e

e n d p l a t e i n r e s p o n s e t o i n c r e a s e d m u s c le a c t i v i t y .

T h e r e i s now c o n s i d e r a b l e e v i d e n c e t o i n d i c a t e t h a t t h e

b i o s y n t h e s i s and a s s e m b ly o f AChE fo rm s i s p r o g r e s s i v e ,

com m encing w i t h t h e s y n t h e s i s o f L form s in t h e s a r c o p l a s m i c

r e t i c u l u m ( s e e G en era l I n t r o d u c t i o n ) . The L form s a r e

s u b s e q u e n t l y a s s e m b le d i n t o M and H f o r m s . By f o l l o w i n g t h e

g l y c o s y l a t i o n o f t h e a c t i v a t e d AChE m o l e c u l e s , R otundo ( 1 9 8 4 a )

h a s shown t h a t t h e M form i s a s s e m b le d in t h e rough e n d o p la s m ic

r e t i c u l u m (RER), t r a n s p o r t e d t o t h e G o lg i a p p a r a t u s w h ere i t i s

a s s e m b le d i n t o t h e c o l l a g e n - t a i l e d H f o r m s , and f i n a l l y e x p o r t e d

t o t h e e x t r a c e l l u l a r m a t r i x a t t h e n e u r o m u s c u la r j u n c t i o n . In

t h e u n s t i m u l a t e d s t a t e , t h e r e t h u s a p p e a r s t o be a b a l a n c e

b e tw e e n t h e s y n t h e s i s o f t h e L form s and t h e i r a s s e m b ly i n t o H

fo rm s su ch t h a t o v e r 80% o f t h e AChE i s p r e s e n t a s t h e j u n c t i o n a l

H2C form i n t h e PLD m u s c l e . A b u i l d - up o f H fo rm s was

o b s e r v e d when a new s t e a d y d i s t r i b u t i o n o f AChE fo rm s had b een

r e a c h e d in t h e s t i m u l a t e d PLD. The t u r n o v e r o f e n d p l a t e H AChE

i s in o t h e r s i t u a t i o n s known t o be v e r y s lo w com p ared t o t h a t o f

i n t e r n a l L AChE ( R o tu n d o , 1 9 8 4 ; B r i m i j o i n , 1 9 8 3 ; Newman e t a l . ,

1 9 8 4 ) , so t h i s new s t e a d y s t a t e o f H2c AChE ( a t 3 t i m e s t h e

normal l e v e l , o v e r a l l ) can be u n d e r s t o o d in t e r m s o f t h e s e

d i f f e r e n t i a l l i f e t i m e s and t h e i n i t i a l f a s t and l a t e r s lo w

s y n t h e s i s o f t h e L p r e c u r s o r .

- 1 4 2 -

T h a t t h e e f f e c t s o f s t i m u l a t i o n a r e n o t p e r m a n en t and

r e q u i r e t h e c o n t i n u o u s a c t i v i t i e s o f t h e n e r v e i s d e m o n s t r a t e d by

t h e r e v e r s a l i n AChE a c t i v i t y and t h e r e t u r n o f i t s m o l e c u l a r

p r o f i l e , upon c e s s a t i o n o f t h e e l e c t r i c a l i n p u t ( T a b l e 4 . 2 ) ,

P r o d u c t i o n o f t h e L AChE r a p i d l y d e c l i n e s and t h e a s s e m b ly i n t o

H£C v i a H^c i s d i m i n i s h e d t o i t s normal lo w l e v e l . The

h i s t o c h e m i c a l c h a n g e s a l s o u n d er w en t a s i m i l a r r e v e r s a l , a l t h o u g h

o v e r a som ew hat s l o w e r t im e c o u r s e ( J . J a r v i s , p e r s o n a l

c o m m u n i c a t i o n ) . The r e v e r s a l o f t h e e f f e c t o f

s t i m u l a t i o n - i n d u c e d c h a n g e s i n c o n t r a c t i l e a c t i v i t i e s o f

mammalian t y p e I I m u s c le h a s a l s o b e e n r e p o r t e d ( S a lm o n s , 1 9 8 0 ) .

The PLD AChE a c t i v i t y g r a d u a l l y a p p r o a c h e d a l e v e l s i m i l a r t o

t h a t o f t h e c o n t r o l PLD a f t e r 14 d a y s ( T a b l e 4 . 2 ) .

O v e r a l l , t h e e f f e c t s s e e n i n normal a v i a n m u s c le show t h a t

t h e p r o d u c t i o n o f m u s c le AChE i s r e g u l a t e d by t h e a c t i v i t y o f t h e

m o to r n e r v e . The e f f e c t s on m u s c le AChE o f d e n e r v a t i o n and o f

c o n t r a c t i o n b l o c k a d e , a s r e v ie w e d b r i e f l y i n t h e I n t r o d u c t i o n ,

co m b in ed w i t h t h i s e v i d e n c e on s t i m u l a t i o n e f f e c t s , s e r v e t o show

t h a t e l e c t r i c a l a c t i v i t y p e r S £ i s an i m p o r t a n t c o n t r o l l i n g

f a c t o r f o r t h i s e n z y m e . On t h e o t h e r h a n d , a s n o t e d e a r l i e r

c e r t a i n e f f e c t s o f n e r v e e x t r a c t s on m u s c le AChE show t h a t

* t r o p h i c f a c t o r s can a l s o c o n t r i b u t e t o i t s r e g u l a t i o n .

4 . 4 . 2 C h a n g es o f AChE i n s t i m u l a t e d d y s t r o p h ic c h ic k e n PLD

In c o n t r a s t t o t h e i n c r e a s e i n AChE a c t i v i t y i n s t i m u l a t e d

normal m u s c l e s , c h r o n i c low f r e q u e n c y s t i m u l a t i o n o f d y s t r o p h i c

PLD m u s c l e s u n e q u i v o c a l l y l e d t o a r e d u c t i o n o f AChE a c t i v i t y in

t h e s e m u s c l e s . One o f t h e s a l i e n t f e a t u r e s o f a v i a n d y s t r o p h y i s

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t h e l a r g e i n c r e a s e in AChE a c t i v i t y i n a f f e c t e d m u s c l e s ( W i l s o n

e t a K , 1 9 7 9 ) ; t h i s i n c r e a s e i s due t o t h e e x c e s s i v e s y n t h e s e s o f

a l l m o l e c u l a r f o r m s , b u t i n p a r t i c u l a r t h e L fo rm s ( c f . F i g .

4 . 1 A, 4 . 4 A ) . C h r o n ic s t i m u l a t i o n in t u r n p r e f e r e n t i a l l y

s u p p r e s s e d t h e s y n t h e s i s o f L f o r m s ; t h i s e f f e c t w as m o s t c l e a r l y

i l l u s t r a t e d i n t h e PLD o f t h e 1 4 - w e e k - o l d c h i c k e n w h ic h had b e e n

s t i m u l a t e d f o r 5 w e e k s ( F i g . 4 . 4 3 , F ) . The AChE a c t i v i t y a s w e l l

a s i t s m o l e c u l a r p r o f i l e i n t h e s t i m u l a t e d d y s t r o p h i c m u s c le

r e s e m b le d c l o s e l y t h a t o f a s i m i l a r l y s t i m u l a t e d normal PLD

( F i g . 4 . 1 F ) . The same i s t r u e f o r t h e norm al and d y s t r o p h i c

m u s c l e s w h ic h had b e e n s t i m u l a t e d f o r 2 w e e k s ( c f . F i g . 4 . 1 B and

4 . 4 B ) . Over t h e 5 -w eek s t i m u l a t i o n a t 1 0 Hz, t h e r e was c l e a r l y a

p r o g r e s s i v e r e d u c t i o n o f t h e p r o p o r t i o n o f L fo rm s i n t h e

d y s t r o p h i c m u s c l e . T h e s e o b s e r v a t i o n s a r e i n t e r e s t i n g , a s t h e y

show t h a t c h r o n i c s t i m u l a t i o n b r i n g s a b o u t c o m p a r a b le

d i s t r i b u t i o n o f AChE fo rm s in normal and d y s t r o p h i c PLD d e s p i t e

t h e p a t h o l o g i c a l c h a n g e s t h a t had t a k e n p l a c e i n t h e d y s t r o p h i c

m u s c le p r i o r t o s t i m u l a t i o n . The d i f f e r e n t i a l l o c a l i s a t i o n o f

AChE i n and o u t s i d e t h e n e u r o m u s c u la r j u n c t i o n s u b s t a n t i a t e s

t h e s e r e s u l t s . H ence i t sh ow s t h a t a l t h o u g h t h e d y s t r o p h i c

m u s c le c o n s i s t e d o f abnorm al am ou n ts o f L an d H2 C form o u t s i d e

t h e e n d p l a t e s , s t i m u l a t i o n t r a n s f o r m e d t h i s d i s t r i b u t i o n i n t o a

p r o f i l e w i t h p r e d o m in a n t ly H^c and H2 C fo r m s , a s i n t h e

s t i m u l a t e d normal m u s c le ( c f . 4 .6 D and 4 . 3 0 . H i s t o c h e m i c a l and

m o r p h o lo g i c a l f i n d i n g s (B a r n a r d e t a l^ . , 1 9 8 5 ) p r o v i d e f u r t h e r

i n f o r m a t i o n f o r t h e s i m i l a r i t i e s b e tw e e n t h e r e s p o n s e s o f t h e

normal and d y s t r o p h i c PLD t o s t i m u l a t i o n ; t h e y i n c l u d e i n c r e a s e d

f a t i g u e r e s i s t a n c e and t y p e I IB t o 11A t r a n s f o r m a t i o n o f f i b r e

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t y p e . I n d e e d , t h e r e m a r k a b le im p ro v em en t in t h e s t r u c t u r e and

i n t e g r i t y o f t h e d y s t r o p h i c m u s c le f i b r e s upon s t i m u l a t i o n

d e m o n s t r a t e s t h e e f f e c t o f c h r o n i c s t i m u l a t i o n n o t s e e n w i t h

norm al m u s c l e s .%

T h e r e a r e , t h e r e f o r e , two d i s t i n c t i v e a s p e c t s w i t h r e g a r d t o

t h e c h a n g e s i n AChE i n t h e s t i m u l a t e d d y s t r o p h i c PLD. F i r s t l y ,

t h e s y n t h e t i c m a c h in e r y f n t h e d y s t r o p h i c m u s c le r e s p o n d e d t o

I n c r e a s e s 1n c o n t r a c t i l e a c t i v i t y In much t h e same way a s a

norm al m u s c l e d i d . S t i m u l a t i o n m a i n t a i n e d l a r g e am ou n ts o f t h e

e x t r a - j u n c t i o n a l H AChE a f t e r an I n i t i a l a c c u m u l a t i o n o f t h e L

f o r m s . S e c o n d l y , t h e e x c e s s i v e s y n t h e s i s o f t h e L fo r m s w as

r a p i d l y s u p p r e s s e d . T h e s e s i m u l t a n e o u s b u t o p p o s i n g e f f e c t s a r e

c o n s i s t e n t w i t h t h e Id e a t h a t t h e d y s t r o p h i c a v i a n m u s c l e I s

a r r e s t e d a t an e a r l y d e v e lo p m e n t a l s t a g e (L AChE b e i n g v e r y h ig h

In norm al e m b r y o n ic m u s c l e : L y l e s e t a K , 1 9 7 9 ) b u t I s s t i l l

c a p a b l e o f r e s p o n d i n g t o Im posed r e g u l a t o r y f a c t o r s .

An abnorm al e x c e s s o f AChE o c c u r s 1n b o t h t h e e n d p l a t e

and n o n - e n d p l a t e r e g i o n s o f d y s t r o p h i c m u s c l e ( F 1 g . 4 . 6 A ,C ) , an d

I s e l i m i n a t e d by Im posed m u s c le a c t i v i t y ( F 1 g . 4 . 6 B , D ) . T h i s

sh ow s t h a t t h e e l e c t r i c a l e f f e c t s a r e n o t e x e r t e d o n l y on t h e

e x t r a - j u n c t i o n a l membrane a s s e e n 1n s t i m u l a t e d normal PLD. The

e l e c t r i c a l l y s t i m u l a t e d d y s t r o p h i c PLD m a i n t a i n s m o s t o f I t s

e x c e s s AChE a s c o l l a g e n - t a i l e d m o l e c u l e s w h ic h a r e l o c a t e d

e x t r a - j u n c t l o n a l l y ( F 1 g . 4 . 6 D ) , w h ic h 1 s t h e same s t a t e a s 1 s

fo u n d 1n t h e e l e c t r i c a l l y s t i m u l a t e d normal PLD.

T h e s e p a r a l l e l s show t h a t a l t h o u g h t h e o v e r p r o d u c t i o n o f

AChE 1 s g e n e t i c a l l y d e t e r m in e d In t h e d y s t r o p h i c a v i a n f a s t

m u s c l e s , I t i s n e v e r t h e l e s s n o t e n t i r e l y rem oved from r e g u l a t o r y

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i n f l u e n c e s . I t i s b r o u g h t a b o u t by s u s t a i n e d e l e c t r i c a l a c t i v i t y

t o t h e same s t a t e , i n a m ou n t , fo rm s and l o c a t i o n s , a s i n t h e

s i m i l a r l y s t i m u l a t e d normal m u s c l e . The n a t u r e o f t h e p a r t i c u l a r

c o n t r o l o f AChE w h ic h i s abnorm al i n t h e d y s t r o p h i c p h e n o t y p e ,

an d w h ich may be a r e f l e c t i o n o f an e x t e n d e d im m atu re s t a t e o f

t h e s e a n i m a l s (Bandm an, 1 9 8 4 ) , a w a i t s f u r t h e r s t u d y , s i n c e i t i s

l i k e l y t o th ro w new l i g h t on r e g u l a t o r y c o n t r o l s i n m u s c le

d e v e lo p m e n t .

4 . 4 . 3 C h a n g es o f AChE i n l e g m u s c le o f norm al a n d c jy s t r o p h ic

c h i c k e n s

AChE i n t h e f a s t t w i t c h l e g m u s c l e s p r e s e n t e d i n t h i s s t u d y ,

l i k e t h e f a s t - t w i t c h PLD m u s c l e , c o n s i s t s l a r g e l y o f HgQ*

C h r o n ic s t i m u l a t i n g a v e r i s e t o a s u b s t a n t i a l e l e v a t i o n o f t h e

L2 f o r m , when t h e m u s c le f i b r e s becom e p r e d o m in a n t ly t y p e I I A .

U n l i k e t h e s t i m u l a t e d norm al PLD, t h e s e m u s c l e s (EDL an d AT) do

n o t a p p e a r t o u n d er g o a p h a s i c i n c r e a s e in AChE a s t h e r e was

no d i f f e r e n c e b e tw e e n 1 week o r 3 w eek s s t i m u l a t i o n . T h is

p r e f e r e n t i a l i n c r e a s e i n t h e Lg AChE i s t h u s s i m i l a r b u t

som ew hat l e s s m arked a s t h a t o b s e r v e d i n t h e s t i m u l a t e d PLD. On

t h e o t h e r h a n d , t h e g a s t r o c n e m i u s r e s p o n d e d t o c h r o n i c

s t i m u l a t i o n w i t h a r e m a r k a b le i n c r e a s e i n t o t a l AChE a c t i v i t y .

A f t e r 3 w e e k s c o n t i n u o u s s t i m u l a t i o n , t h e AChE a c t i v i t y

a p p r o a c h e d t h a t o b s e r v e d i n a h i g h l y o x i d a t i v e m u s c le su c h a s t h e

r e d s a r t o r i u s o r t h e m ed ia l g a s t r o c n e m i u s (B a r n a r d e t al^., 1 9 8 2 ) .

The i n c r e a s e in t h e number o f t y p e I f i b r e s i n t h e

s t i m u l a t e d g a s t r o c n e m i u s s u g g e s t s t h a t c h r o n i c s t i m u l a t i o n a t 10

Hz ca n in d u c e a f a s t t o s lo w t r a n s f o r m a t i o n in c h i c k e n m u s c l e s .

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The f a c t t h a t m u s c l e s r e s p o n d d i f f e r e n t l y t o t h e same s t i m u l a t i o n

s c h e d u l e s u g g e s t s f a c t o r s o t h e r th a n t h e i m p u ls e f i r i n g p a t t e r n

d e t e r m in e t h e f i b r e t y p e c o m p o s i t i o n o f a m u s c l e . I t a l s o

s u g g e s t s t h a t p r e f e r e n t i a l u t i l i s a t i o n o f t h e g a s t r o c n e m i u s may

h a v e o c c u r r e d d u r in g s t i m u l a t i o n , s i n c e t h e p r o p o r t i o n o f t y p e I

f i b r e s in t h e EDL o r AT was n o t a f f e c t e d .

In t e r m s o f i n c r e a s e d l e v e l s o f AChE, l e g m u s c l e s in

d y s t r o p h i c c h i c k e n s a r e l e s s a f f e c t e d when com p ared t o t h e

d y s t r o p h i c PLD, w h ich c o r r e l a t e s w i t h t h e i n e x t e n s i v e e f f e c t o f

d y s t r o p h y on t h e s e m u s c l e s . The t o t a l AChE a c t i v i t y i s i n c r e a s e d

m o d e r a t e l y , b u t i s h i g h e r th a n p r e v i o u s l y o b s e r v e d ( L y l e s e t a l . ,

1 9 8 2 ; B arn ard e t a l^ . , 1 9 8 2 ) . AChE a c t i v i t i e s i n c r e a s e d i n t h e s e

l e g m u s c l e s in d y s t r o p h i c c h i c k e n s upon s t i m u l a t i o n . In t h e

s t i m u l a t e d AT, H2c and a s i g n i f i c a n t H^c c o m p o n e n t now

p r e d o m in a te ( F ig 4 . 8 B ) . The i n c r e a s e in t h e s e fo rm s and t o t a l

AChE a r e a b o u t 5 t i m e s t h a t e x h i b i t e d by t h e normal AT,

s u g g e s t i n g t h a t t h e m u s c le b e h a v e s more l i k e a normal m u s c le th a n

a d y s t r o p h i c m u s c le a s i s e x p e c t e d from i t s m o r p h o l o g i c a l l y

normal c h a r a c t e r i s t i c s (B a r n a r d e t a l . , 1 9 8 2 ) . H ow ever , s i n c e

b o th t h e AChE a c t i v i t y and i t s m o l e c u l a r d i s t r i b u t i o n i n d i c a t e s

s i g n s o f a b n o r m a l i t y , i t i s l i k e l y t h a t t h e m u s c le d o e s c o n t a i n

p a t h o l o g i c a l IIB f i b r e s w h ich a c c o u n t f o r t h e i n c r e a s e d L an d M

s y n t h e s i s , b u t t h e c h a n g e s t h a t o c c u r i n t h e s e f i b r e s a r e

o b s c u r e d by t h e c h a n g e s t h a t t a k e s p l a c e i n t h e m a j o r i t y o f

normal f i b r e s . A v e r y d i f f e r e n t p a t t e r n o f c h a n g e , h o w e v e r ,

t a k e s p l a c e i n t h e g a s t r o c n e m i u s o f t h e d y s t r o p h i c c h i c k e n . T h is

m u s c l e , l i k e t h e d y s t r o p h i c PLD, c o n t a i n s a l a r g e L c o m p o n e n t ,

e v e n th o u g h i t d o e s n o t e x h i b i t s e v e r e d y s t r o p h i c sym p tom s. When

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i t was s t i m u l a t e d , t h e s y n t h e s i s o f t h e L form was f u r t h e r

i n c r e a s e d , w h i l e l i t t l e i n c r e a s e in t h e H c o m p o n e n t i s o b s e r v e d .

Such c h a n g e s in t h e g a s t r o c n e m iu s i s t h e r e f o r e i n c o n s i s t e n t w i t h

t h e o b s e r v a t i o n s i n e i t h e r s t i m u l a t e d normal o r d y s t r o p h i c

m u s c l e s ; i n v ie w o f t h e l i m i t e d number o f e x p e r i m e n t s c a r r i e d o u t

f o r t h i s s t u d y , f u r t h e r e x p e r i m e n t a t i o n i s n e c e s s a r y t o v e r i f y

t h e p r e s e n t r e s u l t s .

T h e s e p r e l i m i n a r y f i n d i n g s w i t h c h i c k e n l e g m u s c l e s have

p r o v i d e d more s u b s t a n t i a l i n f o r m a t i o n on t h e e f f e c t s o f

s t i m u l a t i o n on AChE a c t i v i t y and d i s t r i b u t i o n i n f a s t m u s c l e s .

I t i s c l e a r t h a t c h r o n i c lo w f r e q u e n c y s t i m u l a t i o n i n d u c e s an

i n c r e a s e i n t h e o x i d a t i v e c a p a c i t y o f t h e m u s c l e s , a c c o m p a n ie d by

an i n c r e a s e i n AChE s y n t h e s i s . H ow ever, t h e e x t e n t o f i n c r e a s e

i n AChE and t h e p a t t e r n o f c h a n g e i n ter m s o f t h e m o l e c u l a r

c o m p o s i t i o n a p p e a r s t o v a r y b e tw e e n m u s c l e s . S t i m u l a t i o n c a n

a l s o i n d u c e a f a s t t o s lo w t r a n s f o r m a t i o n in c h i c k e n m u s c l e s , !I

w h ic h i s i n d i c a t e d by t h e i n c r e a s e d number o f t y p e I f i b r e s and

t h e l e v e l o f AChE a c t i v i t y c h a r a c t e r i s t i c o f t h e s e f i b r e s . I t

seem s t h a t t h e c o n t r a c t i l e a c t i v i t y a s s o c i a t e d w i t h l e g m u s c l e s

d o e s f a c i l i t a t e t h e f a s t t o s lo w t r a n s f o r m a t i o n . H o w ev er , t h e r e

i s y e t no c l e a r e v i d e n c e w h ich s u g g e s t s t h e f i b r e t y p e

c o m p o s i t i o n o f a m u s c le c o r r e l a t e s w i t h i t s t e n d e n c y o f f a s t t o

s lo w t r a n s f o r m a t i o n . The s e e m i n g ly c o m p le x r e s p o n s e o f l e g

m u s c l e s t o s t i m u l a t i o n may in p a r t be due t o t h e t e c h n i c a l

l i m i t a t i o n o f t h e p r e s e n t s t i m u l a t i o n p r o t o c o l when d e a l i n g w i t h

t h e a n a t o m i c a l l y much m ore co m p le x m u s c u la t u r e i n t h e l e g .

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CHAPTER 5

MONOCLONAL ANTIBODIES TO PURIFIED CHICK BRAIN AChE

*

- 149 -

5 . 1 INTRODUCTION

The p h y s i o l o g i c a l e v i d e n c e p r e s e n t e d i n t h e p r e v i o u s

c h a p t e r s s u g g e s t s t h a t t h e s y n t h e s i s and d i s p o s i t i o n o f t h e

m u l t i p l e m o l e c u l a r fo rm s o f AChE i s r e l a t e d t o t h e f u n c t i o n o f

s k e l e t a l m u s c l e s . The l o c a l i s a t i o n o f t h e s e fo rm s i s d e t e r m in e d

by t h e i r c o m p a r t m e n t a l i s a t i o n d u r in g b i o s y n t h e s i s ( R o t u n d o , 1 9 8 4 )

and by t h e i r f i n a l d e s t i n a t i o n w i t h i n t h e m u s c le f i b r e s . The

m o l e c u l a r s t r u c t u r e s o f t h e s e form s a p p e a r t o be a d a p t e d t o t h e i r

s p e c i f i c l o c a l i s a t i o n , w e l l i l l u s t r a t e d by t h e membrane bound

d i m e r i c form o f AChE from human e r y t h r o c y t e s , T o rp ed o e l e c t r i c

o r g a n and human b r a i n , and t h e c o l l a g e n - t a i l e d form s i n m u s c l e s

( s e e g e n e r a l i n t r o d u c t i o n ) . I f i n d e e d t h e l o c a l i s a t i o n o f AChE

fo rm s i s f u n c t i o n a l l y r e l a t e d , and su ch l o c a l i s a t i o n d e p e n d s on

t h e s t r u c t u r a l h e t e r o g e n e i t y o f t h e AChE f o r m s , t h e e l u c i d a t i o n

o f t h e m o l e c u l a r s t r u c t u r e s o f t h e v a r i o u s e n z y m ic fo rm s s h o u l d

p r o v i d e im p o r t a n t i n f o r m a t i o n on t h e r e g u l a t i o n o f s y n t h e s i s ,

l o c a l i s a t i o n , and f u n c t i o n o f t h e s e fo r m s .

The p u r i f i c a t i o n o f AChE fo rm s from s k e l e t a l m u s c le h a s b een

ham pered by t h e i r lo w c o n c e n t r a t i o n . H o w ev er , m o n o c lo n a l

a n t i b o d i e s r a i s e d a g a i n s t p u r i f i e d mammalian AChE from an

a b u n d a n t s o u r c e su ch a s b r a i n t i s s u e s and e r y t h r o c y t e s h a s shown

t h a t t h e s e a n t i b o d i e s c r o s s - r e a c t e d w i t h AChE from o t h e r t i s s u e s

o f t h e same s p e c i e s (Fam brough e t , 1 9 8 2 ) , o r AChE from o t h e r

mammalian s p e c i e s ( B r i m i j o i n e t a l 1 9 8 3 ) The e x p l o i t a t i o n o f

t h i s r e a c t i v i t y f o r a n t i g e n p u r i f i c a t i o n from a d i f f e r e n t t i s s u e

s o u r c e h a s r e c e n t l y b e e n d e m o n s t r a t e d by M in tz and B r i m i j o i n

- 150 -

( 1 9 8 5 ) . Such im m u n o a f f i n i t y c h r o m a t o g r a p h ic t e c h n i q u e s may t h u s

b e u s e d t o p u r i f y m u s c l e AChE. F u r t h e r m o r e , m o n o c lo n a l

a n t i b o d i e s may a l s o be u s e d i n t h e im m u n o h is to c h e m ic a l

l o c a l i s a t i o n o f AChE, p a r t i c u l a r l y a t t h e e l e c t r o n m i c r o s c o p i c

l e v e l , u s i n g c o l l o i d a l g o l d - p r o t e i n A (R o th e t a l . , 1 9 7 8 ) o r

g o l d - l a b e l l e d s e c o n d a n t i b o d y ( S t e r n b e r g e r , 1 9 7 9 ) t e c h n i q u e .

S i m i l a r a p p r o a c h h a s r e c e n t l y b een a p p l i e d t o t h e l o c a l i s a t i o n o f

T o rp ed o a c e t y l c h o l i n e r e c e p t o r and t h e Mr 4 3 , 0 0 0 p r o t e i n

( S e a l o c k e t a l . , 1 9 8 4 . ) T h i s c h a p t e r d e s c r i b e s t h e p u r i f i c a t i o n

o f c h i c k b r a i n AChE and t h e c h a r a c t e r i s a t i o n o f t h e m o n o c lo n a l

a n t i b o d i e s w h ic h w e r e r a i s e d a g a i n s t t h e p u r i f i e d e n z y m e . The

c u r r e n t a p p l i c a t i o n o f t h e s e m o n o c lo n a l a n t i b o d i e s i s a l s o

a s s e s s e d .

5 . 2 MATERIALS AND METHODS

5 . 2 . 1 M a t e r i a l s

C h e m ic a l s f o r p u r i f i c a t i o n ; C o n c a n a v a l in A - A g a r o s e

(C o n A -A g a r o se ) was o b t a i n e d from BRL; a - m e t h y lm a n n o s id e from

F lu k a ; D E A E -Sephacel from S ig m a ; d eca m eth o n iu m from K o c h - L ig h t .

The a f f i n i t y r e s i n , N - m e t h y l a c r i d i n i u m - S e p h a r o s e ( 0 . 3 5 p m o le s

l i g a n d / m l S e p h a r o s e ) w as a g i f t from P r o f e s s o r J . M a s s o u l i e .

C h e m ic a l s f o r SDS PAGE: H igh m o l e c u l a r w e i g h t p r o t e i n s t a n d a r d s

w e r e o b t a i n e d from P h a r m a c ia ; NaDoDSO^ ( S D S ) , d i t h i o t h r e i t o l

from S igm a; p o l y a c r y l a m i d e , t e t r a m e t h y l e t h y l e n e d i a m i n e and

ammonium p e r s u l p h a t e from BDH.

- 151 -

C h e m ic a l s f o r i m m u n i s a t i o n , c u l t u r e and c l o n i n g : F r e u n d s

a d j u v a n t was o b t a i n e d from D i f c o l a b s ( D e t r o i t M i c h . ) ; DMEM,

f e t a l c a l f serum and p e n i c i l l i n / s t r e p t o m y c i n from G ib c o ; s o f t

a g a r o s e from M i l e s S c i e n t i f i c ; a z a s e r i n e , h y p o x a n t h in e from

S ig m a .

C h e m ic a l s f o r s c r e e n i n g and c h a r a c t e r i s a t i o n o f m o n o c lo n a l

a n t i b o d i e s :

R a b b i t a n t i - m o u s e Ig w as o b t a i n e d from M i l e s S c i e n t i f i c ;

FITC and h o r s e r a d i s h p e r o x i d a s e (HRP) c o n j u g a t e d r a b b i t

a n t i - m o u s e I g from DAKO PATTS; p r o t e i n A - S e p h a r o s e , b o v i n e serum

a lb u m in (B S A ), d ia m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e , N o n id e t

P - 4 0 ( N P - 4 0 ) w e r e from S ig m a ; h y d r o g e n p e r o x i d e (HgOg) and

n i c k e l s u l p h a t e (N iSO ^) w e r e from BRL.

E q u ip m en t

Amicon u l t r a f i l t r a t i o n u n i t f i t t e d w i t h D i a f l o YM30 f i l t e r s

was o b t a i n e d from A m icon ; m i c r o - t i t r e t r a y s and t i s s u e c u l t u r e

t u b e s from G ib c o (N u n c lo n D e l t a ) ; p o l y e t h y l e n e m i c r o t i t r e d i s h

from F a lc o n ( m i c r o t e s t I I I ) ; e l e c t r o b l o t t i n g u n i t from B io r a d and

n i t r o c e l l u l o s e p a p e r from S c h i n d l e r and S c h u e l l . O th e r c h e m i c a l s

a r e a s l i s t e d i n s e c t i o n 2 . 2 . 3 .

5 . 2 . 2 P u r i f i c a t i o n o f c h i c k b r a in ACHE

One day o l d w h i t e l e g h o r n c h i c k s w ere s a c r i f i c e d by

d e c a p i t a t i o n , and t h e b r a i n s rem oved and k e p t on i c e . A b o u t 60g

o f t i s s u e was p o o l e d and h o m o g e n is e d , u s i n g a p o l y t r o n , i n 10 v o l

o f 0.4M N a C l / 1 2 .5 mM p h o s p h a te b u f f e r , pH 7 . 5 c o n t a i n i n g ( a s

- 152 -

p r o t e a s e i n h i b i t o r s ) 5 nM EGTA and b a c i t r a c i n ( l m g / m l ) . The

h o m o g e n a te w as t h e n c e n t r i f u g e d a t 3 0 ,0 0 0 * g f o r 3 0 m in . The

s u p e r n a t a n t w as d e c a n t e d and t h e p e l l e t s w e r e p o o l e d and

h o m o g e n is e d i n 8 v o l o f 0.5M N aC l/0 .5% T r i t o n X - 1 0 0 / 5 0 mM

p h o s p h a t e b u f f e r , pH 7 . 5 c o n t a i n i n g b a c i t r a c i n ( l m g / m l ) , th e n

c e n t r i f u g e d a t 1 0 0 OOOxg f o r 6 0 m in . S u p e r n a t a n t s w e r e th e n

p o o l e d and p a s s e d t h r o u g h a p r e - e q u i l i b r a t e d 5 0 ml Con A -A g a r o s e

colum n o v e r n i g h t a t 4 ° C . The co lum n w as t h e n w ash ed w i t h 8

co lu m n v o l o f 0.5M N a C l /0 .5 % T r i to n X - 1 0 0 / 1 0 nM p h o s p h a t e b u f f e r ,

pH 7 . 5 , f o l l o w e d by 5 co lu m n v o l o f 0 . 5 M a - m e t h y lm a n n o s i d e ( i n

wash b u f f e r ) t o e l u t e t h e bound enzym e o v e r n i g h t a t 4 °C . The

e l u a t e was t h e n d i a l y s e d a g a i n s t 2 x 4 l i t o f 0.5% T r i t o n X - 1 0 0 / 1 0

mM p h o s p h a t e b u f f e r , pH 7 . 5 f o r a t o t a l o f 16 h o u r s , b e f o r e

p a s s i n g t h e d i a l y s a t e th r o u g h a 4 0 ml D E A E -Sephacel c o lu m n ,

p r e - e q u i l i b r a t e d w i t h d i a l y s i s b u f f e r , a t a b o u t 6 0 m l / h o u r . The

enzym e w as e l u t e d w i t h a 0 - 2 5 0 mM NaCI g r a d i e n t i n 18 0 ml o f

d i a l y s i s b u f f e r , and 2 ml f r a c t i o n s w ere c o l l e c t e d . F r a c t i o n s

c o n t a i n i n g t h e peak AChE a c t i v i t y w e r e p o o le d and d i a l y s e d w i t h

t h e T r i t o n / p h o s p h a t e b u f f e r a s a b o v e . The d i a l y s a t e was t h e n

s u b j e c t e d t o a f f i n i t y c h r o m a to g r a p h y u s i n g a 20 ml

N - m e t h y l a c r i d i n i u m - S e p h a r o s e co lu m n ( V a l l e t t e e t a l . , 1 9 8 3 ) ,

w a sh e d w i t h 10 mM N aC l/0 .05% T r i t o n X - 1 0 0 / 1 0 mM p h o s p h a t e , pH 7 . 5 ,

and e l u t e d w i t h t h e same b u f f e r c o n t a i n i n g 10 mM d e c a m e th o n iu m .

F r a c t i o n s w h ic h c o n t a i n e d t h e peak AChE a c t i v i t y w e r e p o o l e d and

d i a l y s e d a g a i n s t 2 x 4 l i t o f wash b u f f e r a t 4°C f o r 2 4 h o u r s . The

d i a l y s a t e o b t a i n e d w as c o n c e n t r a t e d , u s i n g an Ami con

u l t r a f i l t r a t i o n c e l l , t o a b o u t 0 . 5 m l. F r e s h p r e p a r a t i o n

o b t a i n e d t h i s way w as e i t h e r u s e d f r e s h f o r im m u n is a t io n o r

s t o r e d f r o z e n a t -2 0 °C f o r f u r t h e r a n a l y s i s o f p u r i t y .

- 153 -

5 . 2 3 E n zy u e a s s a y

AChE a c t i v i t y w as a s s a y e d by t h e r a d i o m e t r i c m ethod a s

d e s c r i b e d in s e c t i o n 2 . 2 . 7 . A l l a s s a y s w e r e p e r fo r m e d i n t h e

p r e s e n c e o f 1 0 “ S l isoOMPA. P r o t e i n w as m ea su red by t h e m ethod

o f Lowry e t a l . ( 1 9 5 1 ) . BSA w as u s e d a s t h e p r o t e i n s t a n d a r d .

5 . 2 . 4 i H u n i s a t i o n a n d c e l l c u l t u r e

6 - 8 w e e k s o l d f e m a le BALB/c m ic e w e r e im m unised i n t r a -

p e r i t o n e a l l y ( i p . ) , w i t h a p p r o x i m a t e ly 15 pg o f p u r i f i e d AChE i n

c o m p le t e F r e u n d s a d j u v a n t . T h e s e m ic e w ere b o o s t e d w i t h s i m i l a r

a m o u n ts o f p u r i f i e d enzym e i n c o m p le t e F r e u n d s a d j u v a n t e v e r y 2

w eek s u n t i l a p o s i t i v e t i t r e was d e t e c t e d i n t h e serum ( b l o o d

sa m p le w as drawn from t h e t a i l v e i n and t h e serum t e s t e d f o r

a n t i b o d y t i t r e by t h e s o l i d p h a s e m eth od , s e e b e l o w ) . A f i n a l

i n j e c t i o n ( 1 5 pg p u r i f i e d en zy m e) i p . w as g i v e n 4 d a y s b e f o r e

h y b r i d i s a t i o n w as p e r fo r m e d .

S p l e e n c e l l s w e r e h y b r i d i s e d by a m o d i f i c a t i o n ( K e n n e t t e t

a ] . , 1 9 7 8 ) o f t h e K o h l e r - M i l s t e i n p r o c e d u r e ( 1 9 7 5 ) . The s p l e e n

w as g e n t l y d i s s o c i a t e d m e c h a n i c a l l y in 2 ml o f DMEM and t h e c e l l

s u s p e n s i o n was rem o v ed , w a sh ed i n 18 ml o f DMEM and p e l l e t e d .

E x p o n e n t i a l l y g r o w in g inyeloma c e l l s (SP 2/0A G 14 n o n - s e c r e t i n g c e l l

l i n e ) w e r e s i m i l a r l y w a sh ed and 10^ myeloma c e l l s w e r e

c o m b in e d , m ixed w i t h t h e s p l e e n c e l l s and p e l l e t e d . F u s io n was

i n i t i a t e d by a d d in g t o t h e p e l l e t 0 .3 m l o f a 3 3 $ s o l u t i o n o f

p o l y e t h y l e n e g l y c o l 150 0 (BDH) in DMEM. The s u s p e n s i o n was

m ix e d , c e n t r i f u g e d f o r 5 min a t 7 0 0 x g , and t h e p e l l e t w as

a s p i r a t e d d r y and g e n t l y r e s u s p e n d e d i n 1 0 0 ml o f s e l e c t i v e

medium (DMEM, 15$ f e t a l c a l f s e ru m , lp g /m l a z a s e r i n e , 1 0 0 pM

- 154 -

h y p o x a n t h i n e , 1 0 0 u n i t s / m l p e n i c i l l i n / s t r e p t o m y c i n ) . C e l l s w e r e

d i s t r i b u t e d i n t o t e n 96 w e l l m i c r o t i t r e t r a y s and f e d w i t h 1 0 0 pi

o f g r o w th medium ( w i t h o u t a z a s e r i n e and h y p o x a n t h i n e ) a f t e r 7

d a y s o f i n c u b a t i o n a t 3 7 ° C in a h u m i d i f i e d 5 % CO^/95% O2

a t m o s p h e r e . Hybridoma s u p e r n a t a n t s w e r e a s s a y e d f o r

i m m u n o r e a c t i v i t y a s so o n a s t h e c e l l s r e a c h e d c o n f l u e n c e .

H ybridom as w h o se m ed ia w ere p o s i t i v e f o r AChE b i n d i n g ( s e e

b e lo w ) w e r e ex p a n d e d and c l o n e d in s o f t a g a r o s e by t h e m eth od o f

C i v i n an d B a n q u e r in g o ( 1 9 8 3 ) . B r i e f l y , h ybridom a c e l l s t o b e

c l o n e d w e r e s u s p e n d e d in DMEM s u p p le m e n te d medium and f i l t e r e d

t h r o u g h n y lo n mesh t o rem ove c lu m p s o f c e l l s . V i a b l e c e l l c o u n t s

w e r e p e r fo r m e d w i t h a h a e m o c y to m e te r and s e r i a l 1 0 f o l d d i l u t i o n s

5w e r e made t o o b t a i n 2 s e t s o f c u l t u r e t u b e s c o n t a i n i n g 10 ,

1 0 ^ , 10^ and 10^ hybridom a c e l l s i n 150 pi o f medium p e r

c l o n i n g t u b e . F or e a c h s e t o f 8 hybridom a c l o n i n g t u b e s , 9 m is

o f 2 x DMEM s u p p le m e n t e d medium w e r e m ix ed w i t h 9 m is o f 3%

a g a r o s e a t 3 7 ° C. Two m is o f t h i s m ix t u r e w e r e a d d ed t o e a c h

c l o n i n g t u b e , and t h e c o n t e n t m ixed w e l l by t r i t u r a t i o n . T h e s e

t u b e s w e r e t h e n h e l d a t 4°C f o r 45 min t o a l l o w t h e a g a r o s e t o

g e l , a f t e r w h ic h t h e y w e r e i n c u b a t e d a t 37°C i n a h u m i d i f i e d

5% C02/95$ 0^ a t m o s p h e r e u n t i l m a c r o s c o p ic g r o w th o f c l o n e s

a p p e a r e d .

I n d i v i d u a l c l o n e s w as c a r e f u l l y rem oved from t h e a g a r o s e

medium w i t h a p a s t e u r p i p e t t e and t r a n s f e r r e d t o 24 w e l l c u l t u r e

d i s h e s c o n t a i n i n g 1 ml o f DMEM s u p p le m e n t e d m edium , w i t h

t r i t u r a t i o n t o d i s p e r s e t h e c e l l s . The c e l l s w e r e a l l o w e d t o

e x p a n d t o s u b c o n f l u e n c y a t w h ich c u l t u r e medium w as rem oved and

- 155 -

t e s t e d f o r t h e p r e s e n c e o f AChE b i n d i n g a c t i v i t y by t h e s o l i d

p h a s e m ethod ( s e e b e l o w ) .

5 . 2 . 5 S c r e e n in g p r o t o c o l

H ybridoma medium w a s s c r e e n e d f o r t h e p r e s e n c e o f AChE

b i n d i n g a n t i b o d i e s by a s o l i d p h a s e im m u n oad sorb an ce a s s a y . 100

pi o f hybridom a medium w as i n c u b a t e d f o r a t l e a s t 3 h o u r s a t room

t e m p e r a t u r e in a 9 6 w e l l p o l y e t h y l e n e m i c r o t i t r e d i s h w h ic h had

b e e n p r e c o a t e d w i t h r a b b i t a n t im o u s e I g (3 0 p g /m l ) and

p r e - a d s o r b e d w i t h 0.5% BSA i n p h o s p h a t e b u f f e r e d s a l i n e (PBS:

150 mM N aC l, 5 0 irM p h o s p h a t e , pH 7 . 4 ) . The w e l l s w e r e th e n

w a sh ed 3 t i m e s w i t h 2 0 0 pi o f PBS and 100 pi o f an AChE s o l u t i o n

from an e x t r a c t o f o n e day o l d c h i c k b r a i n ( h o m o g e n is e d 1 : 1 0 0 i n

PBS + 0.5% T r i t o n X -1 0 0 ) was a d d ed and sh a k e n f o r a t l e a s t 4

h o u r s a t 4 ° C . The w e l l s w e r e w ash ed 3 t i m e s w i t h 2 0 0 pi o f PBS +

0.05% N P-40 and AChE i m m o b i l i z e d on t h e s u r f a c e was a s s a y e d

d i r e c t l y in t h e w e l l i n a vo lum e o f 1 0 0 p i . T y p i c a l l y t h e

n o n - s p e c i f i c b i n d i n g was l e s s th a n 2% and t h e s i g n a l t o n o i s e

r a t i o o f medium from p o s i t i v e c l o n e s w as a b o u t 7 0 : 1 . T h i s s o l i d

p h a s e a s s a y was a d d i t i o n a l l y u s e d f o r a f f i n i t y and c o m p e t i t i o n

m ea su r e m e n t w i t h m o d i f i c a t i o n a s d e s c r i b e d i n t h e R e s u l t s

S e c t i o n .

5 . 2 . 6 P u r i f i c a t i o n o f M o n o c lo n a l a n t i b o d i e s

A n t i b o d i e s w ere p u r i f i e d from h yb r id om a medium u s i n g a 1 . 5

ml p r o t e i n A - S e p h a r o s e CL4B c o lu m n . The medium w as r e c y c l e d

t h r o u g h t h e p r o t e i n A c o lu m n , e q u i l i b r a t e d w i t h 0.1M T r i s b u f f e r ,

- 156 -

pH 8 . 0 o v e r n i g h t a t 4 ° C . The colum n w a s t h e n w a sh ed e x t e n s i v e l y

w i t h t h e same b u f f e r , and e l u t e d w i t h 0.1M c i t r a t e b u f f e r a t

l o w e r pH a c c o r d i n g t o (Ey e t a l . , 1 9 7 8 ) . 0 . 9 ml f r a c t i o n s w ere

c o l l e c t e d and t h e p r o t e i n c o n t e n t i n e a c h f r a c t i o n was d e t e r m in e d

by a b s o r b a n c e a t 2 8 0 n m /260 nm. The f r a c t i o n s w h ich c o n t a i n e d

peak p r o t e i n c o n t e n t w e r e p o o l e d and d i a l y s e d a g a i n s t 10 nM T r i s ,

pH 8 . 0 and c o n c e n t r a t e d w i t h an Am icon u l t r a f i l t r a t i o n c e l l . The

p r o t e i n c o n c e n t r a t i o n and t h e p u r i t y o f t h e f i n a l im m u n o g lo b u l in

s o l u t i o n was d e t e r m in e d by t h e m ethod o f Lowry e t al^. ( 1 9 5 1 ) and

SDS PAGE ( s e e b e l o w ) , r e s p e c t i v e l y . The p r o t e i n A co lu m n was

c l e a n e d w i t h 10 0 co lu m n v o l o f 0 . 5 M N a C l/ 0.5% N P - 4 0 / 0 . 1 M

c i t r a t e b u f f e r , pH 3 . 0 , and s u b s e q u e n t l y e q u i l i b r a t e d w i t h 0 . 1 M

T r i s , pH 8 . 0 .

5 . 2 . 7 G el e l e c t r o p h o r e s i s a n d w e s t e r n b l o t t i n g

SDS-PAGE u s i n g 10% S D S - p o ly a c r y l am ide g e l s was c a r r i e d o u t

by t h e m eth od o f Laemmli ( 1 9 7 0 ) . S a m p le s w ere d e n a t u r e d w i t h 1%

SDS and 1% d i t h i o t h r e i t o l a t 96°C p r i o r t o e l e c t r o p h o r e s i s .

P h o s p h o r y l a s e b (M^ 9 7 , 0 0 0 ) , BSA (M^ 6 8 , 0 0 0 ) , c a t a l a s e (M^

6 0 , 0 0 0 ) and o v a lb u m in (M^ 4 5 , 0 0 0 ) w e r e u s e d a s p r o t e i n

s t a n d a r d s , P r o t e i n b a n d s w ere s t a i n e d w i t h C o o m a ss ie B l u e .

P r o t e i n s from t h e p o l y a c r y l a m i d e g e l w e r e e l e c t r o - b l o t t e d t o

n i t r o c e l l u l o s e p a p e r f o r 16 h o u r s by t h e m ethod o f Tow bin e t a l .

( 1 9 7 9 ) . The b l o t w as a d s o r b e d w i t h hybridom a medium a t room

t e m p e r a t u r e f o r 3 h o u r s , w ash ed and i n c u b a t e d w i t h 2 0 ml o f HRP

c o n j u g a t e d r a b b i t a n t i - m o u s e Ig ( d i l u t e d 1 : 3 0 0 ) w h ic h was

d e v e l o p e d u s i n g d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e ( 0 . 2 5 m g / m l )

+ H2 0 2 (0 .05% v / v ) and N iS 0 4 ( 0 .3 % ) .

- 157 -

5 . 2 . 8 i H u n o h l s t o c h a r i s t r y

F r o z e n s e c t i o n s ( 1 0 urn) o r t e a s e d f i b r e s from a d u l t c h i c k e n

PLD w e r e i n c u b a t e d w i t h hybridom a c o n d i t i o n e d medium f o r 3 h o u r s ,

w a sh e d 3 t i m e s i n PBS and i n c u b a t e d w i t h e i t h e r FITC o r

p e r o x i d a s e c o n j u g a t e d r a b b i t a n t i - m o u s e Ig f o r 1 - 2 h o u r s a t room

t e m p e r a t u r e . P r e p a r a t i o n s w e r e t h e n r i n s e d 5 t i m e s i n PBS.

F I T C - l a b e l l e d s e c t i o n s w ere m ounted i n g l y c e r o l - P B S - j e l l y and

o b s e r v e d u n d er a f l u o r e s c e n c e m i c r o s c o p e . P e r o x i d a s e l a b e l l e d

m u s c le f i b r e s w e r e d e v e lo p e d a s d e s c r i b e d a b o v e and m ounted i n

Aquamount m o u n ta n t (BDH) and o b s e r v e d u n d e r a p h a s e c o n t r a s t

m i c r o s c o p e .

5 . 3 RESULTS

5 . 3 . 1 P u r i f i c a t i o n o f c h i c k b r a in AChE

A b ou t 90% o f t h e c h i c k b r a i n AChE e x t r a c t e d i n t h e h ig h

s a l t / T r i t o n b u f f e r e x i s t s a s an I I S form ( F ig 5 . 1 ) . T h e r e i s

a l s o a m in or f r a c t i o n o f 7S form an d 20S form . S i n c e t h e enzym e

i s a g l y c o p r o t e i n and e x h i b i t s b i n d i n g t o Con-A (R o tu n d o and

Fam brough, 1 9 7 9 ) , t h i s p r o p e r t y i s e x p l o i t e d i n t h e f i r s t s t e p o f

i t s p u r i f i c a t i o n , by a p p l y i n g t h e e x t r a c t t o c h r o m a to g r a p h y on a

Con-A A g a r o s e c o lu m n , a f t e r w h ich t h e bound p r o t e i n s a r e

d i s p l a c e d w i t h 0 . 1 M a - m e t h y lm a n n o s i d e . F o l l o w i n g d i a l y s i s , t h e

enzym e i s s u b j e c t e d t o i o n e x c h a n g e c h r o m a to g r a p h y on a DEAE-

S e p h a c e l colum n w h ic h i s n e c e s s a r y f o r t h e e f f i c i e n t b i n d i n g o f

t h e enzym e t o t h e N - m e t h y l a c r i d i n i u m - S e p h a r o s e (MAc) a f f i n i t y

c o lu m n . S i n c e t h e a f f i n i t y r e s i n e x h i b i t s i o n e x c h a n g e

p r o p e r t i e s ( M a s s o u l i e and B on , 1 9 7 6 ) , t h e i o n i c s t r e n g t h o f t h e

- 158 -

FRACTIONS

FIG. 5.1 S u c r o s e g r a d i e n t p r o f i l e o f AChE from c r u d e e x t r a c t o f

o n e day o l d c h i c k b r a i n . AChE was e x t r a c t e d w i t h 8 v o l o f

0 . 5 M N aC l/0 .5% T r i t o n X - 1 0 0 / 5 0 nfll p h o s p h a t e b u f f e r , pH 7 . 5 ,

a f t e r b r a i n t i s s u e had b e e n p r e - h o m o g e n i s e d and p e l l e t e d w i t h 10

v o l o f 0 . 4 M N a C l / 1 2 .5 mM p h o s p h a t e b u f f e r , pH 7 . 5 ( s e e t e x t ) .

B a c i t r a c i n ( lm g /m l ) and 5 nM EGTA w e r e i n c l u d e d a s p r o t e a s e

i n h i b i t o r s . Arrow i n d i c a t e s t h e p o s i t i o n o f t h e c a t a l a s e

m a rk er .

- 159 -

DEAE colum n e l u a t e m u st be r e d u c e d by d i a l y s i s p r i o r t o i t s

a p p l i c a t i o n t o t h e MAc c o lu m n . Under t h e s e c o n d i t i o n s , m ore th a n

95% o f t h e enzym e i s bound t o t h e a f f i n i t y r e s i n . N ear c o m p le t e

e l u t i o n i s a c h i e v e d by d i s p l a c i n g t h e enzym e w i t h d e c a m e th o n iu m .

U s in g t h i s p u r i f i c a t i o n p r o c e d u r e , an e n r ic h m e n t o f o v e r 2 , 0 0 0

f o l d o f AChE was o b t a i n e d ( T a b l e 5 . 1 ) .

The p u r i f i e d enzym e m i g r a t e s a s a m a jo r band on SDS g e l w i t h

a n a p p r o x im a t e o f 1 1 0 , 0 0 0 ( F i g . 5 . 2 b ) ( c . f . A p p e n d ix 2 ) .

The band i s c h a r a c t e r i s t i c a l l y d i f f u s e due m o s t l i k e l y t o t h e

e x t e n s i v e g l y c o s y l a t i o n o f t h e p r o t e i n . The p u r i f i e d enzym e i so

r e a d i l y l a b e l l e d w i t h [ H]DFP ( F i g . 5 . 2 c ) w h o se b i n d i n g i s

- 5b l o c k e d by p r e - t r e a t i n g and c o - i n c u b a t i n g w i t h 2 x 10 e s e r i n e

( F i g . 5 . 2 d ) .

5 . 3 . 2 C h a r a c t e r i s a t i o n o f M o n o c lo n a l a n t i b o d i e s t o AChE

A. S u b c l a s s i f i c a t i o n and p u r i f i c a t i o n

T h r e e h ybridom a s e c r e t i n g a n t i b o d i e s t h a t b in d c h i c k b r a i n

AChE w ere i s o l a t e d and c l o n e d . Two o f th em , d e s i g n a t e d ACB-1 and

ACB-3 a r e o f s u b c l a s s IgG£a w h e r e a s ACB-2 i s o f s u b c l a s s

IgG ^. ACB-3 b i n d s AChE from b o t h c h i c k b r a i n and m u s c l e , and

r e c o g n i s e s a 1 1 0 , 0 0 0 d p e p t i d e on n i t r o c e l l u l o s e b l o t . H ow ever ,

s i n c e i t i s s e c r e t e d from hybridom a c e l l s a t v e r y lo w q u a n t i t i e s

( < 0 .1 ug/ml o f c o n f l u e n t c e l l s ) , o n l y ACB-1 an d ACB-2 w e r e

f u r t h e r c h a r a c t e r i s e d . A f t e r b i n d i n g a n t i b o d i e s t o t h e p r o t e i n

A - S e p h a r o s e c o lu m n , ACB-1 was e l u t e d from t h e co lu m n a t pH 4 . 5 ,

and ACB-2 a t pH 6 . SDS-PAGE o f t h e p u r i f i e d a n t i b o d i e s showed

t h e c h a r a c t e r i s t i c l i g h t (Nlr 2 2 , 0 0 0 ) and h ea v y (Mr 5 5 , 0 0 0 )

c h a i n s ( F i g . 5 . 3 b ) (C o h e n , 1 9 6 7 ) .

- 160 -

T a b le 5 . 1 P u r i f i c a t i o n o f AChE from day o l d c h i c k b r a i n .

Sam p le V o l .

(m l)

T o t a l

AChE

( p m o l/m in )

P r o t e i n 3

( m g /m l )

S p e c i f i c

a c t i v i t y

(p m o l /m in /m g p r o t e i n )

P u r i f i c a t i o n

f a c t o r

AChE

r e c o v e r y

mc r u d e e x t r a c t 4 3 0 40 2 2 1 0 7 0 . 1 9 10 0

Con A e l u a t e 3 9 5 165 130 1 . 2 7 6 7 41

DEAE e l u a t e 61 91 32 2 . 8 4 1 4 . 9 23

MAc c o n c e n t r a t e 0 . 3 7 1 . 2 0 . 1 6 44 5 2 3 4 2 18

a P r o t e i n c o n t e n t o f s a m p le s w as d e t e r m in e d by t h e m eth od o f Lowry e t a l . ( 1 9 5 1 ) , u s i n g BSA a s

p r o t e i n s t a n d a r d .

a b e d

FIG- 5.2 Analysis of purified chick brain AChE using SDS-PAGE.

a, chick brain crude extract; b, AChE preparation stained with

Coomassie blue; c, [3H]DFP (10_4M DFP) labelling of purified

AChE; d, [3H]DFP C10-4M DFP) labelling of purified AChE

blocked by pre-treatment with 2 x 10”t I eserine. Numbers on

the right indicate the positions and the size (xlOOO) of the high

molecular weight protein markers.

-162-

ab

68k-

A6K~*

FIG. 5.3 SDS-PAGE of purified ACB-1 and ACB-2. a, protein

markers; b, purified ACB-2. Purified ACB-1 showed identical

banding pattern.

- 1 6 3 -

B . A f f i n i t y o f m o n o c l o n a l a n t i b o d i e s t o c h i c k b r a i n AChE

The b i n d i n g a f f i n i t y o f ACB-1 a nd ACB-2 t o c h i c k b r a i n AChE

w e r e t e s t e d i n v a r y i n g d i l u t i o n s o f p u r i f i e d a n t i b o d i e s ( F i g .

5 . 4 ) . A ss u m in g t h a t b i n d i n g i n t h e s o l i d p h a s e a s s a y f o l l o w s

m a s s - a c t i o n l a w s ; w i t h o n e e p i t o p e p e r enzyme monomer and two

r e c o g n i t i o n s i t e s p e r IgG m o l e c u l e , an a p p a r e n t d i s s o c i a t i o n

c o n s t a n t f o r t h e a n t i g e n - I g G c o m p l e x c o u l d be e v a l u a t e d . S i n c e

t h e c o n c e n t r a t i o n o f a n t i g e n was l o w ( 0 . 0 2 nM), b i n d i n g a s s a y s

o b e y s t h e e q u a t i o n

b = 2 [ A b ]

2 [ A b ] + Kd

w h e r e b i s t h e f r a c t i o n o f e p i t o p e bo un d, [A b ] i s t h e m o l a r

c o n c e n t r a t i o n o f a n t i b o d y , and i s t h e d i s s o c i a t i o n c o n s t a n t .

F u r t h e r m o r e , a s i t i s l i k e l y t h a t b i n d i n g o f a m u l t i - s u b u n i t AChE

m o l e c u l e t o an IgG m o l e c u l e r e q u i r e s a minimum o f 1 e p i t o p e i n

t h e s o l i d p h a s e a s s a y , b i n d i n g o f AChE c o u l d t h e n be e x p r e s s e d by

c o r r e c t i n g b f o r t h e number o f e p i t o p e s ( n ) p e r m o l e c u l e o f AChE,

h e n c e :

B = 1 - ( l - b ) n

w h e r e B i s t h e f r a c t i o n o f AChE bo un d, and n = 4 f o r t h e

t e t r a m e r i c c h i c k e n b r a i n form o f AChE. The a p p a r e n t

c a l c u l a t e d from 50% b i n d i n g f o r IgG i s 0 . 8 5 rM f o r ACB-1 a n d 0 . 7 7

nM f o r ACB-2.

C. M o n o c lo n a l a n t i b o d i e s r e c o g n i s e s e p a r a t e a n t i g e n i c s i t e s

I t was f o u n d t h a t t h e b i n d i n g o f s a t u r a t i n g am oun ts o f ACB-2

t o AChE d i d n o t i n h i b i t t h e s u b s e q u e n t b i n d i n g o f ACB-1 t o AChE.

ACB-2-AChE c o m p l e x b i n d s t o i m m o b i l i s e d ACB-1 t h r o u g h o u t a r a n g e

- 164 -

FIG- 5-4 D i l u t i o n c u r v e s o f ACB-1 and ACB-2. 0 . 2 m i l l i u n i t s o f

AChE from on e day o l d c h i c k b r a i n was i n c u b a t e d w i t h a

number o f s e r i a l d i l u t i o n s o f e i t h e r ACB-1 o r ACB-2 i n a t o t a l

v o lu m e o f 100 ul f o r a minimum o f 4 h o u r s a t 4 °C . The s o l u t i o n s

w e r e t h e n t r a n s f e r r e d t o t h e 9 6 w e l l p o l y e t h y l e n e m i c r o - t i t r e

d i s h and i n c u b a t e d o v e r n i g h t a t 4 ° C . The w e l l s w e r e t h e n w a sh ed

3 t i m e s w i t h 2 0 0 ill o f PBS + 0.05% N P -4 0 . I m m o b i l i s e d

e n z y m e - a n t i b o d y c o m p le x was m e a s u r e d by t h e r a d i o m e t r i c a s s a y .

The am ou nt o f enzyme bound i s e x p r e s s e d a s a p e r c e n t a g e o f enzyme

a d d e d . The c u r v e s w ere f i t t e d by e y e and c a l c u l a t i o n s o f

a p p a r e n t d i s s o c i a t i o n c o n s t a n t s f o r t h e a n t i b o d i e s ( s e e t e x t )

w e r e p e r f o r m e d u s i n g 50% b i n d i n g v a l u e .

- 165 -

o f c o n c e n t r a t i o n s ( F i g . 5 . 5 ) . B i n d i n g o f t h i s c o m p le x i s s i m i l a r

t o t h e b i n d i n g o f e q u i v a l e n t am oun ts o f AChE s u g g e s t i n g t h a t t h e

two a n t i b o d i e s r e c o g n i s e d i f f e r e n t e p i t o p e s . F u r t h e r m o r e , ACB-1

r e c o g n i s e s S D S - d e n a t u r e d AChE i n n i t r o c e l l u l o s e b l o t s ( s e e F i g .

5 . 6 a ) , w h i l e ACB-2 o n l y r e c o g n i s e s t h e n a t i v e form o f t h e

e n z y m e .

D. C r o s s - r e a c t i v i t y w i t h AChE from o t h e r t i s s u e s and s p e c i e s

ACB-1 was t e s t e d by t h e s o l i d p h a s e a s s a y a s w e l l a s

b l o t t i n g m et h od f o r i t s b i n d i n g t o AChE from o t h e r t i s s u e s and

s p e c i e s . T h i s a n t i b o d y r e c o g n i s e s AChE from c h i c k b r a i n , p l asm a

and m u s c l e a s w e l l a s AChE from q u a i l b r a i n , b u t d o e s n o t

r e c o g n i s e AChE from any o t h e r a n i m a l s t e s t e d ( T a b l e 5 . 2 ) . ACB-2

w as t e s t e d o n l y by t h e s o l i d p h a s e a s s a y s i n c e i t d o e s n o t b i n d

t o S D S - d e n a t u r e d AChE. The r e s u l t show s t h a t t h i s a n t ib o c t y h a s

s i m i l a r r e c o g n i t i o n f o r c h i c k AChE, b u t d o e s n o t r e c o g n i s e q u a i l

b r a i n AChE, o r AChE from o t h e r s p e c i e s ( T a b l e 5 . 2 ) . N e i t h e r

a n t i b o d y b i n d s t o V>ChE from c h i c k p las ma o r b r a i n . A l l t h e AChE

fo rm s i n c h i c k m u s c l e a r e r e c o g n i s e d by b o t h ACB-1 a nd ACB-2.

ACB-1 r e c o g n i s e s a p o l y p e p t i d e (Mr 1 1 0 , 0 0 0 ) from p u r i f i e d

c h i c k b r a i n AChE ( F i g . 5 . 6 a ) , from b r a i n and m u s c l e c r u d e

e x t r a c t s o f c h i c k ( F i g . 5 . 6 b and c ) and q u a i l ( F i g . 5 . 6 d and e) ,

b u t d o e s n o t b i n d d e t e c t a b l y t o any p e p t i d e i n r a b b i t , r a t o r

g u i n e a p i g b r a i n , T o r p e d o o r E l e c t r o p h o r u s e l e c t r i c o r g a n

e x t r a c t s .

E. I m m u n o h is t o c h e m i c a l a p p l i c a t i o n o f ACB-1 and ACB-2

Bo th ACB-1 and ACB-2 a r e h i g h l y c o n c e n t r a t e d i n t h e e n d p l a t e

- 1 6 6 -

ANTIBODY IN SOLID PHASE (ng)

FIG- 5-5 C o m p e t i t i o n o f ACB-1 and ACB-2 f o r e p i t o p e s o f AChE.

AChE was f i r s t e x p o s e d t o a s a t u r a t i n g c o n c e n t r a t i o n o f ACB-2 and

t h e n AChE-ACB-2 c o m p le x e x p o s e d t o ACB-1 a t t a c h e d t o s o l i d p h a s e .

The d i l u t i o n c u r v e o f AChE-ACB-2 ( # ) c l o s e l y r e s e m b l e s t h e

c o n t r o l c u r v e ( O ) i n w h ic h AChE i s n o t i n i t i a l l y c o m p l e x e d

w i t h ACB-2. The c u r v e i s f i t t e d by e y e . P e r c e n t AChE b i n d i n g i s

e x p r e s s e d a s i n F i g . 5 . 4 .

- 167 -

a b c d e

FIG- 5-6 Reactivity of ACB-1 to avian AChE by western blotting

(see Methods). The position of ACB-1 binding on blot was

visualised by a HRP-conjugated rabbit antimouse second antibody,

a, purified chick brain AChE; b, day old chick brain crude

extract; c, day old chick muscle crude extract; d, quail brain

crude extract; e, quail muscle crude extract. Results of W.R.

Randall.

- 1 6 8 -

TABLE 5 . 2 R e a c t i v i t y ® o f ACB-1 and ACB-2 w i t h (pChE and AChE

from o t h e r s p e c i e s .

AChE S o u r c e 15 ACB-1 ACB-2

C h i c k e n b r a i n + +

m u s c l e + +

Q u a i l b r a i n + -

m u s c l e + -

T o r p e d o e l e c t r i c o r g a n - -

E l e c t r o p h o r u s e l e c t r i c o r g a n - -

R a b b i t b r a i n - -

R a t b r a i n - -

G u in ea p i g b r a i n - -

C h ic k e n <PChE ( b r a i n ) - -

( p l a s m a ) - -

a B i n d i n g o f AChE and ^ChE was m e a s u r e d by t h e s o l i d p h a s e

a s s a y .

k AChE a c t i v i t y was m e a s u r e d i n t h e p r e s e n c e o f 1 X 10"^M

isoOMPA f o r a l l b u t t h e l a s t two s a m p l e s . <pChE was m e a s u r e d i n

t h e p r e s e n c e o f 5 X 10"^M BW284C51 f o r t h e s e s a m p l e s .

- 1 6 9 -

r e g i o n s o f a d u l t PLD m u s c l e , a s HRP o r FITC c o n j u g a t e d r a b b i t

a n t i - m o u s e Ig l a b e l l i n g c l e a r l y sh ow s on t e a s e d f i b r e s ( F i g .

5 . 7 A ) o r f r o z e n t r a n s v e r s e s e c t i o n s ( F i g . 5 . 7 B ) t r e a t e d w i t h

e i t h e r o f t h e a n t i b o d i e s . The d e p o s i t i o n o f a n t i b o d y l a b e l l i n g

r e v e a l s s t r u c t u r e s c h a r a c t e r i s t i c o f t h e en p l a q u e m o to r

e n d p l a t e s a s o b s e r v e d by t h e c o n v e n t i o n a l h i s t o c h e m i c a l s t a i n i n g

f o r e n d p l a t e AChE ( H e s s , 1 9 6 1 ) .

5 . 4 DISCUSSION

AChE from c h i c k b r a i n h a s b een p u r i f i e d , u s i n g a c r i d i n i u m

a f f i n i t y c h r o m a t o g r a p h y , by a b o u t 2 , 0 0 0 f o l d . The e f f i c i e n c y o f

t h i s p r o c e d u r e i s g r e a t l y im p r o v e d by c h r o m a t o g r a p h y on t h e

DEAE-Sephacel co lu mn p r i o r t o a f f i n i t y c h r o m a t o g r a p h y s u g g e s t i n g

t h a t t h e l o w r e t e n t i o n and r e c o v e r y o f t h e c h i c k b r a i n enzyme on

t h e MAc colum n r e p o r t e d by V a l l e t t e e t a l . ( 1 9 8 3 ) may be due t o

i n t e r f e r e n c e by c o n t a m i n a t i n g p r o t e i n s i n t h e e x t r a c t .

I d e n t i f i c a t i o n o f t h e p u r i f i e d p r o t e i n a s AChE i s b a s e d on i t s

s u b u n i t m o l e c u l a r s i z e , w h ich a g r e e s w e l l w i t h t h a t p r e v i o u s l y

o b s e r v e d ( A p p e n d ix 2 ) ( M a s s o u l i e and Bon , 1 9 8 2 ; R o t u n d o , 1 9 8 4 ) ,

i t s s p e c i f i c i n h i b i t i o n by BW284C51 ( R a n d a l l e t a l . , 1 9 8 5 ) ,

[ H]DFP b i n d i n g w h ic h i s i n h i b i t e d by e s e r i n e , and

p r e c i p i t a t i o n o f AChE, b u t n o t ^ChE, by m o n o c lo n a l a n t i b o d i e s

d i r e c t e d a g a i n s t t h e p u r i f i e d p r o t e i n . I t i s c o n c l u d e d

t h e r e f o r e , t h a t t h e p u r i f i e d p r o t e i n i s AChE. T h a t t h e

a n t i b o d i e s r e c o g n i s e d e g l y c o s y l a t e d AChE (M^ 1 0 0 , 0 0 0 ) show s

t h a t t h e a n t i g e n i c d e t e r m i n a n t s a r e a s s o c i a t e d w i t h t h e

p o l y p e p t i d e and n o t t h e c a r b o h y d r a t e m o i e t y ( R a n d a l l e t a l

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FIG- 5.7 Immunohistochemical localisation of AChE in unfixed

chicken PLD muscle using ACB-1 and ACB-2. A, whole mount of PLD

fibres treated with ACB-2 and stained with HRP-conjugated rabbit

antimouse Ig. Deposits of HRP reaction product reveals

characteristic motor endplate structure. Magnification: 750x;

bar represents 2o urn. B, frozen cross section (10 urn) of PLD

muscle treated with ACB-1 and stained with FITC-conjugated rabbit

antimouse Ig. Fluorescence label (appears white in photograph)

is clustered and limited to the sarcolemma of the fibres.

Magnification: 380x. Plate (B) fromW.R. Randall.

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1 9 8 5 ) . The a n t i b o d i e s show h i g h a f f i n i t y f o r t h e a v i a n form o f

AChE. T h e r e i s no c r o s s - r e a c t i v i t y w i t h $ChE o r AChE from

mammals o r f i s h . T h i s l a c k o f c r o s s - r e a c t i v i t y h a s b e e n

s i m i l a r l y o b s e r v e d i n m o n o c lo n a l a n t i b o d i e s r a i s e d a g a i n s t human

e r y t h r o c y t e AChE (Fambrough e t a l . , 1 9 8 2 ) , w h er e t h e a n t i b o d i e s d i d

n o t r e c o g n i s e a v i a n AChE. T h i s s u g g e s t s l i m i t e d ho m o lo g y b e t w e e n

t h e a n t i g e n i c r e g i o n s o f a v i a n and mammalian o r f i s h AChE. The

f a c t t h a t ACB-2 o n l y b i n d s t o t h e n a t i v e form o f t h e enzyme

s u g g e s t s t h a t i t r e c o g n i s e s a c o n f o r m a t i o n a l d e p e n d e n t a n t i g e n i c

s i t e . S i n c e t h e y b i n d a l l t h e fo rm s e q u a l l y w e l l , t h e y m u s t

r e c o g n i s e a common e p i t o p e on t h e c a t a l y t i c s u b u n i t s . I t d o e s

n o t , h o w e v e r , p r e c l u d e t h e p o s s i b l e d i f f e r e n c e s i n t h e m o l e c u l a r

s t r u c t u r e o f t h e c a t a l y t i c s u b u n i t s b e t w e e n t h e v a r i o u s m o l e c u l a r

f o r m s , a s p r e v i o u s s t u d i e s have s u g g e s t e d ( D o c t o r e t a l ^ . , 1 9 8 3 ;

S o r e n s e n e t a l . , 1 9 8 2 ) .

Two p o l y p e p t i d e c h a i n s , t er m e d a and B, w e r e r e c o g n i s e d by

SDS-PAGE from p u r i f i e d c h i c k e n b r a i n AChE ( R o t u n d o , 1 9 8 4 ) . T h i s

s u b u n i t p a t t e r n w a s , h o w e v e r , n o t o b s e r v e d i n o u r p u r i f i e d

p r e p a r a t i o n o f c h i c k b r a i n AChE ( F i g . 5 . 6 a ) . The d i f f e r e n c e i n

t h i s s u b u n i t b a n d in g p a t t e r n may be a t t r i b u t e d t o t h e g e n o t y p e o f

t h e c h i c k e n s t r a i n s u s e d i n t h e two s t u d i e s ( R a n d a l l e t a l . ,

1 9 8 5 ) . T h i s i s e v i d e n t from t h e s t u d y o f a n o t h e r l i n e o f

c h i c k e n , l i n e 4 1 2 ( D a v i s , C a l i f o r n i a ) , w h er e i n d i v i d u a l s e x h i b i t

v a r y i n g b a n d i n g p a t t e r n s o f AChE. Some c o n t a i n o n l y t h e a c h a i n ,

some c o n t a i n o n l y t h e B c h a i n and some c o n t a i n b o t h . T h i s

v a r i a t i o n i s a l s o i n d e p e n d e n t o f t h e a g e o f t h e b i r d o r t h e

t i s s u e s o u r c e o f t h e e n z y m e . I t i s l i k e l y , t h e r e f o r e , t h a t t h e

h e t e r o g e n e i t y i n t h e s u b u n i t s i z e o b s e r v e d i n b o t h c a s e s i s due

t o t h e a l l e l i c d i f f e r e n c e o f t h e AChE g e n e i n t h e s e s t r a i n s o f

b i r d s .

- 1 7 2 -

The p u r i f i c a t i o n o f t h e AChE form m u s c l e u s i n g t h e s e

m o n o c l o n a l a n t i b o d i e s a s i m m u n o a f f i n i t y l i g a n d s i s c u r r e n t l y

b e i n g u n d e r t a k e n a t t h i s l a b o r a t o r y . I m m o b i l i s d ACB-1 a l s o b i n d s

t h e enzyme w i t h h i g h a f f i n i t y , h o w e v e r , t h e r e c o v e r y o f t h e

enzyme i s much l o w e r t h a n t h a t w i t h ACB-2. H e n c e , ACB-2 w h i c h

r e c o g n i s e s a c o n f o r m a t i o n a l d e p e n d e n t e p i t o p e i s s e l e c t e d f o r t h e

i m m u n o a f f i n i t y p u r i f i c a t i o n o f t h e e n z y m e , w h i l e ACB-1 i s

s u i t a b l e f o r a n a l y t i c a l s t u d i e s o f t h e p u r i f i e d e n z y m e .

I m m u n o a f f i n i t y c h r o m a t o g r a p h y u s i n g i m m o b i l i s e d ACB-2 on

S e p h a r o s e co lum n b i n d s AChE from one day o l d c h i c k m u s c l e

e x t r a c t s u n d e r h i g h i o n i c s t r e n g t h c o n d i t i o n s ( 0 . 5 M N aC l /0 .5%

T r i t o n X - 1 0 0 / 1 0 nM HEPES), w h ic h i s n e c e s s a r y f o r t h e

s o l u b i l i s a t i o n o f t h e a s y m m e t r i c f o r m s . T h e s e c o n d i t i o n s a l s o

a l l o w t h e e l i m i n a t i o n o f t h e l a r g e amount o f c o n t a m i n a n t s , i n

p a r t i c u l a r t h e m y o f i b r i l l a r p r o t e i n s , w h ic h c a n n o t be remove d by

c o n v e n t i o n a l p r o t e i n p u r i f i c a t i o n t e c h n i q u e s ( u n p u b l i s h e d d a t a ) .

The bound AChE i s a l s o e a s i l y e l u t e d w i t h h i g h pH b u f f e r (pH 11)

w i t h a b o u t 80% r e c o v e r y .

In summary, c h i c k b r a i n AChE h a s b e e n e n r i c h e d by o v e r 2 , 0 0 0

f o l d u s i n g a f f i n i t y c h r o m a t o g r a p h y . T h re e m o n o c lo n a l a n t i b o d i e s

w h i c h r e c o g n i s e t h i s enzyme ha v e b e e n o b t a i n e d and two o f th em ,

ACB-1 and ACB-2, h a v e b e e n c h a r a c t e r i s e d . Both a n t i b o d i e s

e x h i b i t s i m i l a r b i n d i n g a f f i n i t y t o t h e enzyme and r e c o g n i s e

s e p a r a t e a n t i g e n i c s i t e s on t h e p o l y p e p t i d e b a c k b o n e . T h e s e

a n t i b o d i e s do n o t c r o s s - r e a c t w i t h c h i c k $C hE ,m am m al ian o r f i s h

AChE, b u t ACB-1 d o e s r e c o g n i s e q u a i l AChE. T h e s e a n t i b o d i e s b i n d

a t h i g h c o n c e n t r a t i o n t o t h e e n d p l a t e s o f a d u l t c h i c k e n m u s c l e .

I m m u n o a f f i n i t y p u r i f i c a t i o n o f t h e AChE u s i n g t h e s e

a n t i b o d i e s i s now i n p r o g r e s s .

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C h a p t e r 6

GENERAL DISCUSSION

- 1 7 4 -

AChE a t t h e n e u r o m u s c u l a r j u n c t i o n : m o l e c u l a r f o r m s and r e g u l a t i o n

It is widely thought that the distribution of AChE forms has

important functional significance. The present study demonstrates

unequivocally that the heavy AChE predominates at the neuromuscular

junction of vertebrate twitch muscle where very little L and M forms are

present. It was found that the technique of isolating the neuromuscular

endplates and the precautionary measures employed to protect the heavy

AChE forms from proteolytic degradation are both crucial factors in the

identification of heavy AChE as the endplate specific forms. Although

H2 C is with no exception the predominant form in all endplates, in some

muscles a significant H^c component is also found. The evidence is as

yet insufficient to determine whether this H^c component is a stable

form at the endplate, or a degradatory product of H2 C . The latter

possibility cannot be ruled out from the present result since some lower

molecular weight forms are always seen in the endplate despite the use

of protease inhibitors. Further studies are therefore required to

establish the nature of this component and whether it has a role in the

neuromuscular endplates.

Reduction of the amount of endplate AChE upon chronic

denervation may be brought about by 3 ways: (a) an impaired synthesis of

the collagen-tail, (b) an increased degradation of the enzyme, and (c) a

reduced capacity for the attachment of these forms at the endplate. (a)

appears unlikely, at least in denervated slow-twitch muscles where the

content of the asymmetric forms actually increases, (b) has been shown

in rat to be involved in the reduction of endplate AChE following

denervation, as a result of increased turnover rate. In denervated type

I fibres, L forms become predominant at the endplate, which may be

readily contributed by the increased degradation of the H forms.

However, this process may not be the only cause of these changes in

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denervated muscles which over-produce AChE. On the other hand, there is

a good deal of evidence that AChE is associated with the basal lamina

where AChE clustering occurs and which is known to have a highly

specific organization at the synapse. An impairment of this specialized

structure in disease (Inestrosa, 1985) or absence of nerve may reduce

the attachment of the H AChE to the endplate region.

Neuromuscular contact is important in maintaining the normal

concentration of H AChE at the endplate. However, the enzyme does not

rapidly disappear upon denervation and the clustering of these forms is

largely maintained. Such persistence of endplate AChE may be attributed

to the increase in AChE content in denervated rabbit and guinea pig

muscles. In rat muscles where AChE content is drastically reduced upon

denervation, the level of endplate AChE disappears more rapidly. The

persistence of AChE at denervated endplates therefore appears to depend

largely on the capacity of the denervated muscle in producing H AChE.

The fact that avian tonic fibres contain a majority of L forms

at the endplate, while twitch fibres contain a majority of H forms

suggests a fundamental difference in the organisation of endplate AChE

in the two fibre types. Furthermore, since embryonic and neonatal tonic

fibres are capable of synthesizing significant amount of H AChE, the

organization of endplate AChE is therefore a highly selective process

which evolves according to the function of the muscle. The failure to

obtain cross-reinnervation between type II and tonic fibres in mature

chickens (Hnik et al., 1967), suggests that the endplate forms of AChE

in mature muscles are not inducible, and that the quaternary association

of the AChE catalytic units within the synapse may be specific to the

type of neural transmission of the muscle (Barnard et al., 1984b). In

this respect, the distribution of AChE forms clearly correlates with

the fibre type composition/function of a muscle. To understand the

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p r e c i s e n a t u r e o f t h e c o n t r o l m ec h a n ism r e q u i r e s s t u d i e s a t t h e

m o l e c u l a r l e v e l , w h ic h w i l l l e a d n o t o n l y t o t h e u n d e r s t a n d i n g o f t h e

r e g u l a t o r y m ec h a n is m u n d e r l y i n g t h e p o ly m o r p h is m o f AChE, b u t may a l s o

p r o v i d e a u s e f u l model f o r t h e s t u d y o f t h e c o n t r o l o f g e n e e x p r e s s i o n

i n m u s c l e s .

Extrajunctional AChE forms: function and regulation

C o n t r a r y t o t h e e n d p l a t e AChE f o r m s w h ic h e x h i b i t an a p p a r e n t

a s s o c i a t i o n w i t h t h e f u n c t i o n a l a c t i v i t y o f t h e m u s c l e , t h e d i s t r i b u t i o n

o f e x t r a j u n c t i o n a l AChE i s much more v a r i a b l e . The l o w e r m o l e c u l a r

w e i g h t fo r m s a r e g e n e r a l l y c o n f i n e d t o t h e e x t r a j u n c t i o n a l r e g i o n i n

t w i t c h m u s c l e s ; t h e a c t i v i t y t h e s e f o rm s c o n t r i b u t e s t o t h e t o t a l AChE

i s sm a l l i n t y p e I m u s c l e s , b u t v a r i e s c o n s i d e r a b l y i n t y p e I I m u s c l e s

o f d i f f e r e n t s p e c i e s . The m o s t s t r i k i n g d i f f e r e n c e was s e e n b e t w e e n t h e

h a m s t e r and r a t f a s t m u s c l e s , w h e r e t h e L and M c o m p o n e n t s a r e s m a l l i n

t h e f o r m e r , b u t a r e d o m in a n t i n t h e l a t t e r . The p h y s i o l o g i c a l

s i g n i f i c a n c e o f t h e s e e x t r a j u n c t i o n a l f o rm s i s n o t w e l l e s t a b l i s h e d . The

e v i d e n c e o f d i s t i n c t m e t a b o l i c p o o l s o f AChE i n c u l t u r e d m u s c l e c e l l s

and t r a n s f o r m e d n e r v e c e l l l i n e r a i s e s s e v e r a l i m p o r t a n t q u e s t i o n s : ( 1 )

Do t h e s e m e t a b o l i c p o o l s o f AChE a l s o e x i s t i n m a t u r e m u s c l e s i n v i v o ?

( 2 ) What i s t h e i r r e l a t i o n s h i p w i t h t h e f u n c t i o n a l a s y m m e t r i c f o rm s a t

t h e e n d p l a t e ? And ( 3 ) What i s t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e s e

m e t a b o l i c p o o l s . D i f f e r e n t am oun ts o f e x t r a j u n c t i o n a l AChE i n d i f f e r e n t

m u s c l e s may i n d i c a t e v a r i a t i o n s i n t h e m e t a b o l i c r e g u l a t i o n o f t h e

enzyme i n d i f f e r e n t s p e c i e s . I t i s g e n e r a l l y a g r e e d t h a t a c t i v e ,

f u n c t i o n a l AChE f o r m s a r e r e l a t i v e l y s t a b l e , w h i l e p r e c u r s o r f o rm s

e x h i b i t more r a p i d t u r n o v e r r a t e . In v i v o s t u d i e s on t h e t u r n o v e r o f

t h e s e v a r i o u s f o r m s s h o u l d p r o v i d e k n o w l e d g e on t h e s i z e o f t h e p o o l

d e s t i n e d f o r t h e s y n t h e s i s o f t h e e n d p l a t e f o r m s .

- 177 -

Heavy form o f AChE a r e f o u n d e x t r a j u n c t i o n a l l y i n t y p e I m u s c l e s

and t h e y a r e t h e r e f o r e n o t e n d p l a t e s p e c i f i c . The p h y s i o l o g i c a l

s i g n i f i c a n c e o f t h e s e a s y m m e t r i c fo rm s i s n o t c l e a r . H ow e v e r , b e c a u s e

t h e y a r e f o u n d g e n e r a l l y i n m u s c l e s w h ic h e x h i b i t a c t i v e c o n t r a c t i l e

a c t i v i t y , i t may be s p e c u l a t e d t h a t a r e l a t i o n s h i p e x i s t s b e t w e e n t h i s

e x t r a j u n c t i o n a l p o p u l a t i o n and t h e p h y s i o l o g i c a l s t a t e o f t h e m u s c l e . In

a c c o r d a n c e w i t h t h i s e v i d e n c e , an e x t r a j u n c t i o n a l p o p u l a t i o n o f AChE

r e c e p t o r ca n a l s o be f o u n d i n t h e t y p e I m u s c l e f i b r e s . T h e s e

e x t r a j u n c t i o n a l ACh r e c e p t o r s and e s t e r a s e enzyme c o u l d p o s s i b l y b e : ( a )

a s u r p l u s p o o l o f p r o t e i n s i n d u c e d by m u s c l e a c t i v i t y f o r r e p l e n i s h i n g

t h e j u n c t i o n a l co m p o n e n t when r e q u i r e d ; o r ( b ) a d i s c r e t e p o p u l a t i o n

w i t h a s p e c i f i c f u n c t i o n a l l o c a l i s a t i o n . The i n t e r e s t i n g q u e s t i o n a s t o

w h ic h o f t h e s e p o s s i b i l i t i e s a r e c o r r e c t r e m a i n s t o be s e t t l e d .

U 1 t r a s t r u c t u r a l l o c a l i s a t i o n u s i n g m o n o c l o n a l a n t i b o d i e s

s p e c i f i c f o r a s y m m e t r i c AChE i s p r o b a b l y t h e m o s t s e l e c t i v e p r o b e f o r

t h i s t a s k . I m m u n o c y t o c h e m i s t r y u s i n g t h e s e m o n o c l o n a l a n t i b o d i e s a l s o

o f f e r s a p o w e r f u l t e c h n i q u e f o r t h e d i r e c t d e m o n s t r a t i o n o f t h e

a s s o c i a t i o n o f h e a v y AChE w i t h t h e b a s e m e n t membrane c o m p o n e n t s o f t h e

s y n a p s e . M o n o c lo n a l a n t i b o d i e s r a i s e d a g a i n s t t h e b r a i n form o f c h i c k

AChE i n t h i s s t u d y i s b e i n g c u r r e n t l y a p p l i e d f o r t h e e n r i c h m e n t o f t h e

a s y m m e t r i c form i n m u s c l e w h ic h i n t u r n ca n be u s e d f o r t h e p r o d u c t i o n

o f m o n o c lo n a l a n t i b o d i e s s e l e c t i v e f o r t h i s fo rm .

The e n h a n c e m e n t o f t h e amount o f e x t r a j u n c t i o n a l H f o r m s i n

normal PLD and t h e r e d u c t i o n o f e x c e s s L f o r m s i n d y s t r o p h i c PLD by

c h r o n i c s t i m u l a t i o n s u g g e s t s t h a t m u s c l e a c t i v i t y i s i m p o r t a n t i n t h e

r e g u l a t i o n o f e x t r a j u n c t i o n a l AChE. The e l u c i d a t i o n o f t h e a s s o c i a t i o n

o f t h i s c o m p o n e n t w i t h t h e e x t r a j u n c t i o n a l ACh r e c e p t o r , and i t s

m e t a b o l i c t u r n o v e r c o m p a r i n g w i t h t h e j u n c t i o n a l AChE p o o l s h o u l d s h e d

f u r t h e r l i g h t on t h e f u n c t i o n o f t h i s e x t r a j u n c t i o n a l H c o m p o n e n t and

- 178 -

i t s r e l a t i o n s h i p w i t h t h e j u n c t i o n a l AChE. How m u s c l e a c t i v i t y r e g u l a t e s

t h e m e t a b o l i s m o f AChE r e m a i n s a p r im e i n t e r e s t f o r f u r t h e r r e s e a r c h .

#

- 179 -

APPENDICES

- 180 -

A p p e n d ix 1 . R a p id p r o c e s s i n g o f s k e l e t a l M u s c le c r u d e e x t r a c t s .

To show t h a t AChE c a n b e e x t r a c t e d e f f i c i e n t l y from m u s c l e

t i s s u e s by r a p i d p r o c e s s i n g , t h e s o l e u s and EDL m u s c l e s w e r e

d i s s e c t e d from t h e h i n d l i m b s o f t h e r a t , w e i g h e d and h o m o g e n i s e d

i n 10 v o l o f t h e u s u a l h i g h s a l t / T r i t o n b u f f e r ( s e e s e c t i o n

2 . 2 . 4 ) . A s m a l l a l i q u o t from e a c h h o m o g e n a te was k e p t f o r

s u b s e q u e n t a s s a y . The r e s t was c e n t r i f u g e d a t 4 3 , 5 0 0 x g f o r 15

min and t h e s u p e r n a t a n t s w e r e k e p t . The p e l l e t s w e r e

r e h o m o g e n i z e d i n t h e same b u f f e r t o g i v e t h e same f i n a l d i l u t i o n

a s t h e f i r s t h o m o g e n i s a t i o n , and t h e s u s p e n s i o n s r e c e n t r i f u g e d .

The p e l l e t s o b t a i n e d from t h i s s e c o n d e x t r a c t i o n w e r e a g a i n

r e s u s p e n d e d i n t h e same b u f f e r a s f o r t h e s e c o n d h o m o g e n i s a t i o n

s t e p . The s u p e r n a t a n t s and h o m o g e n a t e s o b t a i n e d w e r e t h e n

a s s a y e d f o r AChE a c t i v i t y by t h e r a d i o m e t r i c a s s a y m ethod

( s e c t i o n 2 . 2 7 ) . The s u p e r n a t a n t s o b t a i n e d from t h e f i r s t and

s e c o n d e x t r a c t i o n w e r e s u b j e c t e d t o d e n s i t y g r a d i e n t a n a l y s i s

( s e c t i o n 2 . 2 . 6 ) .

I t was f o u n d t h a t t h e f i r s t e x t r a c t i o n y i e l d e d 90-100% o f

t h e enzyme a c t i v i t y p r e s e n t i n t h e m u s c l e s ( s e e T a b l e ) . The

d i s t r i b u t i o n o f t h e m o l e c u l a r fo rm s o f AChE o b t a i n e d from t h e

f i r s t e x t r a c t i o n was t y p i c a l o f t h a t o b s e r v e d f o r t h e two

m u s c l e s , w h e r e a s t h e s e c o n d e x t r a c t s c o n t a i n e d r e s i d u a l AChE,

m a i n l y L a nd H f o rm s and c o n s t i t u t e d f o r l e s s t h a n 8% o f t o t a l

AChE a c t i v i t y i n t h e m u s c l e s ( s e e F i g u r e ) . I t i s c l e a r ,

t h e r e f o r e , t h a t a l l t h e m o l e c u l a r f o rm s a r e e f f i c i e n t l y e x t r a c t e d

i n t h e h i g h s a l t / T r i t o n b u f f e r d u r i n g r a p i d p r o c e s s i n g o f t i s s u e

c r u d e e x t r a c t s .

- 1 8 1 -

FRACTIONS FRACTIONS

S u c r o s e g r a d i e n t p r o f i l e s o f AChE i n c r u d e e x t r a c t s o f r a t EDL

and s o l e u s

S o l i d l i n e r e p r e s e n t s s u c r o s e g r a d i e n t p r o f i l e o f AChE from t h e

f i r s t e x t r a c t i o n ; a c t i v i t y i n d i c a t e d by t h e y - a x i s on t h e l e f t

hand s i d e . D o t t e d l i n e r e p r e s e n t s s u c r o s e g r a d i e n t p r o f i l e o f

AChE from t h e s e c o n d e x t r a c t i o n ; a c t i v i t y i n d i c a t e d by t h e y - a x i s

on t h e r i g h t hand s i d e . A l l e x t r a c t s w e r e p r e p a r e d i n t h e

p r e s e n c e o f p r o t e a s e i n h i b i t o r s .

AChE a c t i v i t y i n v a r i o u s e x t r a c t s o f r a t EDL and s o l e u s

[AChE] ( U n i t s / g )

M u s c l e Hom ogenate 1 s t e x t r a c t 2nd e x t r a c t p e l l e t

EDL 1 . 1 2 1 . 2 1 0 . 1 0 0 . 0 6

s o l e u s 0 . 8 9 0 . 9 0 0 . 0 5 0 . 0 5

- 1 8 2 -

A p p e n d ix 2 . M o le c u la r s i z e d e t e r m in a t io n o f ACHE b y r a d i a t i o n

i n a c t i v a t i o n

T a r g e t s i z e a n a l y s i s by h i g h e n e r g y r a d i a t i o n i n a c t i v a t i o n

h a s b e e n u s e d f o r d e t e r m i n i n g t h e minimum m o l e c u l a r w e i g h t

a s s o c i a t e d w i t h a b i o l o g i c a l a c t i v i t y i n a p r o t e i n ( f o r r e v i e w ,

s e e Kempner a n d S c h l e g e l , 1 9 7 9 ) . T h i s t e c h n i q u e h a s b e e n u s e d t o

d e t e r m i n e t h e m o l e c u l a r s i z e o f H2 c AChE i n c h i c k e n m u s c l e

METHOD

The s o u r c e o f H2 c AChE was e i t h e r p r e p a r e d a s a membrane

s u s p e n s i o n o r i n s o l u b l e fo rm . F o r membrane p r e p a r a t i o n s , o n e

day o l d c h i c k p e c t o r a l m u s c l e was h o m o g e n i s e d i n 10 v o l o f 10 mM

^ 4^ 3/ ^ EDTA> PH 7 . 0 c o n t a i n i n g , a s p r o t e a s e i n h i b i t o r s ,

b a c i t r a c i n ( l m g / m l ) , b e n z e m i d i n e ( 2 n t l ) , NEM ( 5 nM) a n d S o y a b e a n

t r y p s i n i n h i b i t o r ( 0 . 1 m g / m l ) , and c e n t r i f u g e d a t 4 3 , 5 0 0 x g f o r

30 m in . The same p r o c e d u r e was r e p e a t e d o n c e and t h e f i n a l

p e l l e t o b t a i n e d w a s r e s u s p e n d e d i n 10 v o l o f 10 mM NH^COg/O.l mM

ntl EDTA, pH 7 . 0 , and f i l t e r e d t h r o u g h g l a s s w o o l . For s o l u b l e

H2 c AChE p r e p a r a t i o n s , p e c t o r a l m u s c l e s w e r e h o m o g e n i s e d w i t h

10 v o l o f 10 nM p h o s p h a t e / l n M EGTA, pH 7 . 0 c o n t a i n i n g p r o t e a s e

i n h i b i t o r s a s a b o v e and c e n t r i f u g e d i n a 45 Ti r o t o r (Beckman) a t

28 K rpm f o r 3 0 m in . T h i s p r o c e d u r e was r e p e a t e d a nd t h e f i n a l

p e l l e t o b t a i n e d w a s e x t r a c t e d w i t h 3 v o l o f 1M N a C l / 2 0 mM sodium

p y r o p h o s p h a t e / 1 mM MgCl2 / l mM EGTA/10 nM p h o s p h a t e , pH 8 . 0 ,

c o n t a i n i n g p r o t e a s e i n h i b i t o r s a s a b o v e . The h o m o g e n a te was

c e n t r i f u g e d a t 26 K rpm i n a Beckman SW 6 0 Ti r o t o r f o r 3 0 min

- 183 -

and i t s s u p e r n a t a n t a p p l i e d t o d e n s i t y g r a d i e n t c e n t r i f u g a t i o n ,

a s d e s c r i b e d i n S e c t i o n 2 . 2 . 6 (5% a nd 20% s u c r o s e s o l u t i o n s w e r e

made up i n 1M N a C l / 1 0 nW p h o s p h a t e , pH 7 . 0 ) . The g r a d i e n t

f r a c t i o n s w h ic h c o n t a i n e d t h e 20 S peak w e r e p o o l e d .

Membrane s u s p e n s i o n o r p o o l e d 20 S f r a c t i o n s w e r e a l i q u o t e d

i n t o P y r e x s a m p le i r r a d i a t i o n t u b e s ( 1 2 x 1 0 0 mm) ( C o r n i n g G l a s s

L t d . ) , ADH (E C . l 1 1 . 1 ) (1 u n i t / s a m p l e t u b e ) and p y r u v a t e k i n a s e

( E . C . 2 . 7 . 1 . 4 0 ) ( 2 0 u n i t s / s a m p l e t u b e ) w e r e r o u t i n e l y u s e d a s

i n t e r n a l p r o t e i n s t a n d a r d s . The s a m p l e s 7 t r i p l i c a t e d f o r e a c h

r a d i a t i o n d o s a g e and n o n - i r r a d i a t e d c o n t r o l s , w e r e f r o z e n i n dry

i c e . f l u s h e d w i t h g a s and t h e t u b e s s e a l e d w i t h an o x y g e n

f l a m e t o r c h . The s a m p l e s w e r e i r r a d i a t e d w i t h 8 MeV e l e c t r o n s a t

t h e Hammersmith H o s p i t a l l i n e a r e l e c t r o n a c c e l e r a t o r , a t a d o s e

r a t e o f 0 . 4 M r a d /m in , w h ic h was m o n i t o r e d w i t h e l e c t r o n

c o l l e c t o r s a b o v e and b e l o w t h e sa m p le a r e a , t o d e t e r m i n e t h e

t o t a l d o s e and t h e d o s e r a t e r e s p e c t i v e l y . I r r a d i a t e d s a m p le

t u b e s w e r e s t o r e d a t - 7 0 ° C u n t i l o p e n e d f o r a s s a y .

A l i q u o t s o f n o n - i r r a d i a t e d c o n t r o l and i r r a d i a t e d membrane

p r e p a r a t i o n w e r e e x t r a c t e d w i t h a 1% T r i t o n / I M NaCl/lmM EDTA/

p h o s p h a t e b u f f e r , pH 7 . 0 , c e n t r i f u g e d a t 4 3 , 5 0 0 x g f o r 30 min and

t h e AChE a c t i v i t y i n t h e s u p e r n a t a n t s m ea s u r e d by t h e m ethod o f

E l l man e t a l . ( 1 9 6 1 ) a s i n S e c t i o n 2 . 2 . 7 . N o n - i r r a d i a t e d

c o n t r o l and i r r a d i a t e d 20S f r a c t i o n s w e r e th aw ed and a l i q u o t s

w ere a s s a y e d f o r AChE a c t i v i t y by t h e r a d i o m e t r i c m eth od ( S e c t i o n

2 . 2 . 7 ) . T h i s work w as c a r r i e d o u t i n c o l l a b o r a t i o n w i t h A. L a i ,

who p e r f o r m e d t h e i r r a d i a t i o n p r o c e d u r e and t h e s u b s e q u e n t a s s a y s

o f t h e i n t e r n a l p r o t e i n s t a n d a r d s .

- 184 -

R e s u l t s

The d o s e - d e p e n d e n t i n a c t i v a t i o n o f t h e 20S fo rm o f AChE i s

shown i n F i g . 1 . The i n a c t i v a t i o n o f o n e o f t h e i n t e r n a l p r o t e i n

s t a n d a r d s i s a l s o sho wn. The m o l e c u l a r s i z e o f AChE d e t e r m i n e d

fr o m t h e c a l i b r a t i o n c u r v e o f A . 1_ v s . m o l e c u l a r w e i g h t o fAo D o se

p r o t e i n s t a n d a r d s was 1 1 0 , 0 0 0 d , w h ic h a g r e e s w i t h t h e s u b u n i t

m o l e c u l a r mass e s t i m a t i o n o f p u r i f i e d c h i c k AChE ( R o t u n d o , 1 9 8 4 ) .

A n a l y s i s o f t h e i r r a d i a t e d membrane p r e p a r a t i o n g a v e i d e n t i c a l

r e s u l t s . T h a t s u c h o b s e r v a t i o n i s n o t due t o d e g r a d a t i o n o f t h e

20S fo rm i s i l l u s t r a t e d i n F i g . 2 . N o n - i r r a d i a t e d c o n t r o l

s a m p l e s o f a membrane p r e p a r a t i o n c o n t a i n s p r e d o m i n a n t l y

AChE. T h u s , t h e s e a n a l y s e s show t h a t t h e minimum m o l e c u l a r s i z e

a s s o c i a t e d w i t h t h e h y d r o l y s i s o f ACh i s i d e n t i c a l t o t h e

m on om er ic c a t a l y t i c u n i t m o l e c u l a r m a s s , s u g g e s t i n g t h a t t h e

monomers a c t a s i n d e p e n d e n t e n z y m i c u n i t s , and t h e r e i s i n s u f f i c i e n t

e n e r g y t r a n s f e r b e t w e e n t h e c a t a l y t i c s i t e s w i t h i n t h e m u l t i m e r

s u c h a s t h e H f o r m . P r e v i o u s o b s e r v a t i o n s on t h e s t r u c t u r e and

s t a b i l i t y o f t h e H2 c ^ ] 2 ^ ^orm E^e c t r °P*10rus S *10W t h a t

l i t t l e i n t e r a c t i o n e x i s t s b e t w e e n t h e t e t r a m e r i c u n i t s l i n k e d t o

a common c o l l a g e n t a i l ( M a s s o u l i e and Bon, 1 9 8 2 ) . The r e s u l t s o f

t h i s s t u d y s u g g e s t t h a t t h e r e a p p e a r s t o be l i t t l e a l l o s t e r i c

i n t e r a c t i o n w i t h i n t h e t e t r a m e r s . The m o l e c u l a r s i z e o f t h e

AChE fo rm i n c h i c k e n m u s c l e , t h e r e f o r e , c a n n o t be r e s o l v e d by

t a r g e t s i z e a n a l y s i s .

- 185 -

FRACTIONS

F I G . 2 S u c r o s e g r a d i e n t p r o f i l e o f AChE e x t r a c t e d from a n o n -

I r r a d i a t e d c o n t r o l s a m p l e o f m u s c l e membrane p r e p a r a t i o n .

-186-

1-0

4 8 12 16Mrad

F I G . 1 D o s e - d e p e n d e n t I n a c t i v a t i o n o f 20S form o f AChE from

c h i c k m u s c l e . A, enzyme a c t i v i t y a f t e r i r r a d i a t i o n ; AQ, o r i g i n a l ( n o n - i r r a d i a t e d ) enzym e a c t i v i t y . PK, p y r u v a t e k i n a s e .

- 187 -

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- 188 -

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Raven P r e s s , N .Y .

- 1 8 9 -

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List of publications

1 . E .A . B a r n a r d , P . J . B a r n a r d , J . J a r v i s , J . J e d r z e j c z y k , J . L a i , J . A . P i z z e y , W. R a n d a l l and I . S i lm a n ( 1 9 8 4 ) M u l t i p l e m o l e c u l a r f o r m s o f a c e t y l c h o l i n e s t e r a s e and t h e i r r e l a t i o n s h i p t o m u s c l e f u n c t i o n . In C h o l i n e s t e r a s e s (M. B r z i n e t a l . e d s . ) , W a l t e r de G r u y t e r , B e r l i n - N e w Y o r k , p p . 4 9 - 7 1 .

2 . J . J e d r z e j c z y k , I . S i l m a n , J . L a i , E .A . B arn ard ( 1 9 8 4 ) M o l e c u l a r f o r m s o f a c e t y l c h o l i n e s t e r a s e i n s y n a p t i c and e x t r a s y n a p t i c r e g i o n s o f a v i a n t o n i c m u s c l e . N e u r o s c i . L e t t . , 46_, 2 8 3 - 2 8 9 .

3 . E .A . B a r n a r d , J . Lai and J . P i z z e y ( 1 9 8 4 ) S y n a p t i c and e x t r a s y n a p t i c fo rm s o f a c e t y l c h o l i n e s t e r a s e i n s k e l e t a l m u s c l e s : v a r i a t i o n w i t h f i b r e t y p e and f u n c t i o n a l c o n s i d e r a t i o n s . In N e u r o m u s c u l a r D i s e a s e s (G. S e r r a t r i c e e t a l . e d s ) , Raven P r e s s , New Y o r k , p p . 4 5 5 - 4 6 3 .

4 . W.R. R a n d a l l , J . Lai and E .A . Bar nar d ( 1 9 8 5 ) A c e t y l c h o l i n e s t e r a s e o f m u s c l e and n e r v e . In M o l e c u l a r B a s i s o f N erveA c t i v i t y ( J . P . Changeux e t a l . e d s . ) , W a l t e r de G r u y t e r , B e r l i n - New Y o r k , p p . 5 9 5 - 6 1 7 .

5 . E .A . B a r n a r d , P . J . B a r n a r d , J . C . J a r v i s and J . Lai ( 1 9 8 5 )C ha nge s i n m o l e c u l a r fo r m s o f a c e t y l c h o l i n e s t e r a s e i nc h r o n i c a l l y s t i m u l a t e d normal and d y s t r o p h i c c h i c k e n m u s c l e . J . P h y s i o l . , s u b m i t t e d f o r p u b l i c a t i o n .

6 . J . L a i , J . J e d r z e j c z y k , J . A . P i z z e y , D. G reen and E . A . Ba rna rd( 1 9 8 5 ) N e u r o n a l c o n t r o l o f t h e fo rm s o f AChE i n s l o w mammalian m u s c l e s . N a t u r e , s u b m i t t e d f o r p u b l i c a t i o n .

7 . W.R. R a n d a l l , J . Lai and E .A . B arn ard ( 1 9 8 5 ) M o n o c lo n a l a n t i b o d i e s t o p u r i f i e d a c e t y l c h o l i n e s t e r a s e from a v i a n b r a i n and t h e d i s c l o s u r e o f tw o a l l e l i c fo rm s o f t h e c a t a l y t i c s u b u n i t . E u r . J . B i o c h e m . , s u b m i t t e d f o r p u b l i c a t i o n .

8 . J . L a i , J . P i z z e y and E .A . Barnar d ( 1 9 8 3 ) AChE i n t h e d i f f e r e n t f i b r e t y p e s o f mammalian m u s c l e . I . R e l a t i o n s h i p o f AChE m o l e c u l a r for m s t o t h e f i b r e t y p e s . In: S e c o n d I n t e r n a t i o n a l M e e t i n g on C h o l i n e s t e r a s e s , B l e d , Y u g o s l a v i a .

9 . J . L a i , J . P i z z e y , D. Green and E .A . B a r n a r d ( 1 9 8 3 ) AChE i n t h ed i f f e r e n t f i b r e t y p e s o f mammalian m u s c l e . I I . E f f e c t s o f d e n e r v a t i o n . In: S e c o n d I n t e r n a t i o n a l M e e t i n g onC h o l i n e s t e r a s e s , B l e d , Y u g o s l a v i a .

10 J . C . J a r v i s , J . Lai and P . J . Ba rn ard ( 1 9 8 3 ) AChE i n s t i m u l a t e d f a s t - t w i t c h a v i a n m u s c l e . In: S eco n d I n t e r n a t i o n a l M e e t i n g on C h o l i n e s t e r a s e s , B l e d , Y u g o s l a v i a .

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