102

Biochimica et Biophysica Acta, 624 ( 1 9 8 0 ) 1 0 2 - - 1 1 0 © E l sev ie r /Nor th -Hol l and Biomedica l Press

BBA 38466

PURIFICATION OF MAIZE ALCOHOL DEHYDROGENASE-1 ALLOZYMES AND COMPARISON OF THEIR TRYPTIC PEPTIDES

J A N I C E K E L L Y and M I C H A E L F R E E L I N G *

Department of Genetics, University of California, Berkeley, CA 94720 (U.S.A.)

(Rece ived D e c e m b e r 5 th , 1979)

Key words: Alcohol dehydrogenase alloenzyme; Peptide mapping; (Maize)

Summary

Two naturally occurring allozymes of alcohol dehydrogenase-1 in maize have been purified to homogeneity. Specific activity, molecular weight and amino acid composition have been determined. The difference between these two allozymes was further studied by comparisons of tryptic peptides using a fingerprinting technique. Excellent maps were obtained which resolved 29 out of the 30 peptides which were maximally possible. These allozymes differ in one peptide, consistent with a single, charged amino acid replacement. These results are related to the differences which have been shown to exist between the genes which specify these two allozymes.

Introduction

There are two alcohol dehydrogenase (EC 1.1.1.1) genes in maize [1,2]. One of these genes, Adhl, specifies the alcohol dehydrogenase of the embryonic seed storage organ called the scutellum. Adhl and its product is the object of this study. The enzyme is a dimer composed of identical polypeptides of about 40 000 daltons [2,3] ; Zn 2÷ is required for reassociation of dimers after chemi- cal dissociation in vitro [4].

Felder et al. [5] published a purification scheme which generated purified alcohol dehydrogenase dimers; it should be noted that their gene and enzyme terminology is different from our own. Our purification procedures are refine- ments of the unpublished methods of D. Schwartz and coworkers.

Among those few higher eukaryotic genes which have proved accessible to

* To whom reprint requests should be addressed. Abbrev ia t ion: SDS, s o d i u m d o d e c y l sul fate .

103

both genetic and biochemical technologies, ry and Adh in Drosophila melano- gaster and maize A d h l are perhaps the best unders tood (several important cita- tions are compiled in Ref. 6). Several studies have utilized two of the electro- phoretically distinguishable A d h l variants, designated Adhl -S and Adhl-F. The 'S' and 'F ' allelic designations denote subunits which confer relatively 'slower' and 'faster' anodal migration rates to native dimers subjected to electrophoresis under our conditions. Being specified by alleles, it is not surprising that these allozymes are composed of polypeptides that are the same size, that there is no size modification after initial translation (Sachs, M., unpublished data) and that S<limer does not differ measurably from F<limer in specific enzyme activ- ity. Specific enzyme activity has been measured as activity units per unit alcohol dehydrogenase-1 cross-reacting material [7] and by activity units per initial rate of [3H]leucine incorporation into alcohol dehydrogenase-1 where both alcohol dehydrogenase-l-S and 1-F polypeptides are synthesized in the same cell (unpublished data; using the methods of Sachs and Freeling [8]). There is one report which claims that these allozymes differ in specific activity [9]. Even though these allozymes are similar, five differences between them have been discovered. (1) The surface of F<limer is 2 units of negative charge greater than that of S~limer [1]; (2) F-dimer is more thermostable [9] and binds Zn 2+ more tightly as a monomer [4] ; (3) Adh- lF and Adhl -S alleles are differently expressed in different maize organs; evidence for some sort of compensatory regulation has been presented [10]; (4 )Adhl -def ic ien t mutants induced in Adhl -S recombine intragenically among themselves at a 10-times higher rate than mutants induced from Adhl -F and tested among themselves [11]; (5) Adh l -F does not recombine intragenically with Adhl-S , but either will recombine with a third allele [11].

Some of the differences enumerated above could be explained either at the primary sequence level or at the gene regulation level. The studies reported here provide a methodological foundation for comparing different alcohol dehydro- genase-1 polypeptides and show that alcohol dehydrogenase-l-F and 1-S differ in bu t one of 29 tryptic peptides, at our limits of resolution.

Materials and Methods

Inbred lines. S-dimer was purified from corn meal from the Std-S inbred line and F<limer was purified from the Std-F inbred line. The alleles specifying these homodimers are designated Adhl -S and Adhl -F , respectively.

Purification o f active alcohol dehydrogenase, modified from D. Schwartz and coworkers. Approx. 1.6 kg dry seeds were ground three times in a Hobart coffee grinder and the fine meal was extracted with 50 mM potassium phos- phate buffer, pH 7.5, with 0.1 M 2-mercaptoethanol, using three parts meal to four parts buffer (v/v). The extract was left to stand for 15 min at room tem- perature. The slurry was squeezed through a pad made up of one layer Mira- cloth (Sigma) and three layers of cheese cloth. The extract was heated to 51°C, very slowly with stirring, and the temperature maintained for 15 min. The extract was then cooled to 10--5°C, spun at 20 000 Xg for 15 min and the supernatant was filtered through a layer of Miracloth into a prechilled beaker, on ice. Hereafter, all alcohol dehydrogenase solutions were maintained at 4°C.

104

The supernatant was fractionated in two steps by 0--40 and 40--60% (NH4)2SO4 saturation cuts with the slow addition of an appropriate volume of unbuffered, 4°C saturated (NH4)2SO4 solution. After each addition, the pre- cipitate was stirred for 15 min before spinning. The final 60% pellet was resuspended in the smallest possible volume (approx. 30 ml) of equilibration buffer consisting of 5 mM potassium phosphate buffer, pH 7.2, with I mM DL~lithiothreitol (Sigma), and dialysed against 4 1 of the same buffer for 5.5 h. The dialysate was applied to a Sephacryl L-S-200 Superfine (Pharmacia) column (90 cm × 2.5 cm) equilibrated with equilibration buffer. Those frac- tions containing the peak (approx. 80%) alcohol dehydrogenase activity were pooled and applied to a DEAE-cellulose column (Whatman DE-52; 14 cm × 2.5 cm) which had been equilibrated with equilibration buffer. The protein was eluted with a 300 ml linear gradient from 5--300 mM potassium phosphate buffer, pH 7.2, each with 0.003 M dithiothreitol. The peak (about 95%) of alcohol dehydrogenase activity was pooled and applied to a hydroxyapatite (Bio-Gel, DNA grade) column (7 × 2.5 cm) which had been equilibrated with equilibration buffer; the protein was eluted with a 200 ml gradient identical to that used with the DE-52 column. Pure fractions, usually the entire alcohol dehydrogenase activity peak, were pooled, dialysed against 50 mM NH4HCO3, pH 8.4, and lyophilised. If dimeric alcohol dehydrogenase is freeze~lried in the presence of potassium phosphate, insoluble polypeptides may be generated. Storage of the enzymes was in liquid nitrogen.

Alcohol dehydrogenase activity and protein assay. Alcohol dehydrogenase activity was measured spectrophotometrically by following the linear increase in absorbance at 340 nm due to ethanol-dependent reduction of NAD ÷ to NADH, using the reaction conditions and volumes detailed by Freeling and Schwartz [2]. Specific activity is expressed in IU/mg protein. Protein content was estimated by the Bradford Coomassie blue procedure [12] with bovine serum albumin (Sigma, fraction IV) as a standard.

Native and SDS-polyacrylamide gel electrophoresis. The purity of alcohol dehydrogenase-l-S and 1-F subunits was evaluated by means of electrophoresis in slab, native (non-denaturing) and sodium docecyl sulfate (SDS, BDH Chem- icals) polyacrylamide gels. When an SDS gel was used, samples were treated with SDS prior to loading.

The native gels were prepared by a modification of previous recipes [3,8]. They contained a 7.5% acrylamide (Eastman, electrophoresis grade) separating gel (acrylamide/bisacrylamide was 30 : 0.8) with a final buffer concentration of 375 mM Tris-HC1, pH 8.7, and a stacking gel of 5% acrylamide (acrylamide/ bisacrylamide was 30 : 0.4) with a final buffer concentration of 125 mM Tris- HC1, pH 6.8. The gels were prepared and run on an apparatus described by Ames [13]. After electrophoresis was complete, one half of the slab was stained for protein [8] while the other half was stained for alcohol dehydro- genase activity [1 ].

The SDS-polyacrylamide gels, prepared and run according to Laemmli and Ames (in Ref. 13), contained an 8.75% acrylamide separating gel and a 5% acrylamide stacking gel with acrylamide/bisacrylamide ratios as given above.

Molecular weight markers were as follows: rabbit phosphorylase a (Sigma, Mr 94 000), bovine serum albumin (Sigma, Mr 68 000), bacterial a-amylase

105

(Sigma type II-A, Mr 48 000), horseradish peroxidase (Sigma, type IV, M r 40 000), carbonic anhydrase (Worthington, Mr 30 000), trypsin (Worthington, tosylphenylalaninechloromethylketone treated, M r 24 000), and horse heart cytochrome c (Sigma, type VI, Mr 12 400).

Preparation ofpeptide maps. Aliquots of the desalted proteins were oxidized with performic acid [14] and digested with tosylphenylalaninechloromethyl- ketone-treated trypsin (Worthington), at a 1 : 100 enzyme/prote in ratio, in 10 mM NH4HCO3, pH 8.5, at 27°C for 24 h. The samples were diluted and lyophilised. The digest was redissolved in 0.1 N NH3 and a sample correspond- ing to 50--80 #g alcohol dehydrogenase in 1--5 pl was spotted repetitively in 1 pl aliquots on a 20 × 20 cm thin-layer cellulose plate (Eastman, 160 pm thick without fluorescent indicater) to give a final spot no larger than 2 mm diam- eter. The placement of the spot can be seen in Fig. 2. The first dimension was ascending chromatography in butan-l-ol/acetic acid/H20/pyridine (15 : 3 : 12 : 10, by vol. [15] and the second dimension was electrophoresis at pH 3.5 in pyridine/acetic acid/H20 ( 1 : 1 0 : 8 9 , by vol.). After chromatography, the plate was air<lried overnight in a well-ventilated hood. For electrophoresis, 0.5 /11 of a marker solution containing lysine, histidine, glycine, and aspartic acid, each at 1 mg/ml in 50 mM NH3, was spotted onto the chromatographed plate. The cellulose sheets were prepared for electrophoresis according to the method of Bates and Perham [16], and electrophoresis was carried out at 700 V for 2 h on a Desaga flat bed electrophoresis apparatus maintained at 2--5°C. The sheets were dried for 40 min in a l l 0 ° C oven and allowed to each room temperature before staining.

Peptide detection and photography. Dry plates were stained with 0.025% (v/v) fluorescamine in acetone followed by 5% (v/v) tr iethylamine in acetone [17]. The fluorescent peptides were visualized under ultraviolet light. Our photographic methods are as follows. Fluorescent spots were photographed using Kodak Direct Positive Panchromatic film 5246 and an Edmund Scientific Co., No. 805, light straw colored, filter. When printed, a fluorescent spot appears as dark on a light background.

Amino acid analyses. Amino acid analyses were performed on a Beckman Model 121-M Amino Acid Analyser. Sample loadings were 0.1 nmol protein (by weight)/column. The analyses were on protein samples which had been oxidized with performic acid [14]. The best value for each amino acid was calculated as the amount of amino acid recovered (pmol/pg protein (by weight)).

Results and Conclusions

Purification and properties of alcohol dehydrogenase-1 dimer Table I follows a typical purification of active, dimeric alcohol dehydro-

genase from the Std-S inbred line; this recovery of 17% of the initial activity is characteristic. Samples from various points in this purification, along with markers, have been subjected to electrophoresis in a SDS slab polyacrylamide gel. An a t tempt was made to adjust protein concentrations so that the most intensely stained bands of protein would be comparable. A photograph of this gel is shown in Fig. 1. Note that alcohol dehydrogenase eluted from the hy-

106

T A B L E I

P U R I F I C A T I O N A N D R E C O V E R Y OF S-DIMER

Typica l results .

Fraction Enzyme units mg/protein Specific activity ( IU) ( I U / m g )

R e c o v e r y (%)

Purifi- ca t ion (-fold)

Af te r hea t 4 6 7 7 3511 1.3 100 Af t e r 40% (N H 4)2SO 4 3 3 0 0 2447 1.3 71 60% pel le t 2561 719 3.5 54.7 Dialysate 2663 710 3.7 57 Off Sephaery l 1785 292 6.1 38 Off DE-52 1079 32.6 33 23 Off h y d r o x y a p a t i t e 773 6.3 123 16.5

m

2.6 2.8 4.5

25 95

0

o ~,

9 4 K

68 K

~ 4 8 K

40 K

30 K

2 4 K

~ 1 2 . 4 K

Fig. 1. E lec t rophore t i c analyses of a lcohol d e h y d r o g e n a s e f rac t ions a t var ious steps in the pur i f i ca t ion fo l lowed in Table I; this is a slab SDS-po lyac ry l amide gel. Eleven lanes are shown , as n u m b e r e d at the t o p of the gel. 'O ' , the origin, and +, the anode . The a s sumed m o l e c u l a r weights of o u r m a r k e r p ro te ins axe given in uni t s of 1000 , abb rev ia t ed ' K ' ; the p ro te ins are ident i f ied in Materials and Methods . No te t h a t lanes 3 and 4 are ident ica l and t h a t lane 6 is uncharac te r i s t i ca l ly s imilar to lane 5. The 'Of f h y d r o x y - apa t i t e ( H A P ) ' s ample in lane 9 is pure a lcohol d e h y d r o g e n a s e , i sola ted m o n t h s earl ier . AS, a m m o n i u m sulfate; A D H , a lcohol d e h y d r o g e n a s e .

107

droxyapatite column (eighth lane) ran as a single band. The molecular weight of alcohol dehydrogenase-l-S is about 40 000, which is consistent with most previous estimates. When the sample off the hydroxyapatite column was subjected to electrophoresis under conditions which preserve enzyme activity, a single band of activity and of protein was visualized (data not shown). F<limer was also purified off the hydroxyapatite column, this aUozyme also ran as a single band in native and SDS-polyacrylamide gels, but expressed its greater negative charge under native conditions. Multiple molecular forms of alcohol dehydrogenase-l-F have been observed on SDS gels in protein samples which previously ran as a single band. This suggests the presence of trace amounts of a protease in the enzyme preparation. The presence of proteases, active in SDS, have been found in other purified enzyme preparations, e.g., yeast alcohol dehydrogenase [18], Drosophila alcohol dehydrogenase [19], yeast phosphoglucose isomerase [20] and in many other cases. Such multiple band gel patterns have rarely been observed for alcohol dehydrogenase-l-S allozyme. The specific activities of the homogeneous pure allozymes were indistinguishable at approx. 120 IU/mg, which is consistent with our previous estimates using more accurate and reliable methods, as clarified in Introduc- tion. The amino acid compositions of pure alcohol dehydrogenase-l-S poly- peptides in residues per polypeptide of 40 000 daltons, is shown in Table II. Alcohol dehydrogenase F amino acid composition was identical or very similar. Our estimate of 17 lysines and 12 arginines per alcohol dehydrogenase-1 poly- peptide leads to an upper limit expectation of 30 tryptic peptides, assuming

T A B L E I I

A M I N O A C I D C O M P O S I T I O N O F ADHol -S

Best value is an average o f 24, 48 , a n d 72-h recoveries for a m i n o acids other than serine and threonine, which were extrapolated to zero t ime and valine and i so leucine which were extrapolated to inf inite t ime of hydrolys i s .

Best value Res idues /40 0 0 0 subuni t (pmol//~g protein; b y weight) molecular weight

Lysine 339 .7 17 Histidine 145 7

Arginine 236 12 Aspartic acid * 680 .7 34

Threon ine 3 1 9 16 Serine 421 27 Glutamic acid * 512 26

Proline 253 .7 13

Glycine 584 .7 29

Alanine 629 .7 32 Cysteinc ** 243 13 Va l ine 553 .5 34 Methionine * * 129 .5 7 Iso leucine 3 7 4 24

Leucine 3 4 4 17 Tyros ine 79.7 4 Phenylalanine 261 13

* A m i d a t e d residues were n o t determined and are inc luded in these figures. * * As cyste ic acid and meth ion ine in ox id i sed samples .

108

complete trylJtic cleavage. Additional peptides of tryptic fragments which were not cleaved might be observed as discrete spots or beneath some other spots.

Comparison of peptide maps between alcohol dehydrogenase-l-S and 1-F poly- peptides

The peptide maps are depicted in Fig. 2. We have resolved clearly 29 tryptic peptides out of a possible number of 30. With the exception of peptide 14, see Fig. 2, the maps of alcohol dehydrogenase-l-S and 1-F appear to be identical, both in position of spots and in their intensities of fluorescence. Peptide 14 has been designated 14 s in the alcohol-l-S polypeptide and 14 F in alcohol dehy- drogenase-l-F. Some discrepancy exists between the relative intensities of

Cm

c~" ~ " "~ C'

o ~

A d 7

Or ig in

- v s ~l El iOt r ophore|i s

i

~ve

i~ ~

,, ,,,o 00_o:, (:

"o0 0~. --~

O' 0 o. 0" 0 6

B

2b

0(7

Origin

b o o0O~.o.~ :::, ~o° "o c20, ,, • ~o 'v o'0" ~I', 0

6o" d

@

C Otlgin

Fig. 2. T ryp t i c pep t lde maps . Out l ines of f luorescen t spots are for (A) a lcohol d e h y d r o g e n a s e - l - S ; (B) a lcohol d e h y d r o g e n a s e - l - S and 1-F m i x e d pr io r to digest ion, and (C) a lcohol d e h y d r o g e n a s e - l - F . D. A p h o t o g r a p h of typ ica l a lcohol d e h y d r o g e n a s e - l - S f ingerpr in t , b u t n o t t h a t t r aced in A. Pept ides 1--6 do n o t a p p e a r well s epa ra t ed in this p h o t o g r a p h , b u t t hey were indeed dis t inct , and pep t ide 23, wh ich m o v e s m o r e qu ick ly ca thoda l ly t h a n free lysine dur ing e lec taophoras is , was i n a d v e r t a n t i y r u n o f f the pla te . Pept idas circled in . . . . . . a p p e a r to be l ow quan t i t a t i ve ly b u t are cons i s ten t ly p re sen t on o u r maps . Th e few spots in t he p h o t o g r a p h (D) wh ic h were n o t assigned p ep t i d e n u m b e r s were n o t cons i s ten t ly p re sen t and p r o b a b l y resul t f r o m i n c o m p l e t e d iges t ion or occas ional c leavage a t sites o th e r t han lyaine or arglnlne.

109

fluorescence of peptides 14 s and 14 F. Peptide 14 s shows more fluorescence than peptide 14 F. This was demonstrated very well in a mixture experiment, where equal amounts of alcohol dehydrogenase-l-F and 1-S were digested together and fingerprinted. This observation has caused us some concern, but clearly the maps are identical except for peptide 14. The change in the position of tryptic peptide 14 in the electrophoretic dimension could be most simply explained as a single amino acid replacement involving the appropriate charge change (i.e., a neutral to acidic substitution). Such a replacement would deter- mine the one surface unit charge/subunit difference between the native allo- zymes at pH values between 7 and 9. It should be noted that the fingerprint method used may not detect exchanges of amino acids with similar properties.

Discussion

Since we have purified two differently charged allozymes to single bands on native and SDS gels, and since their tryptic fingerprints appear identical except for peptide 14, we are confident that our alcohol dehydrogenase is homoge- neously pure.

The introduction lists five differences between the products or behaviors of alleles Adh l -S and Adhl-F. These differences include phenomena readily expli- cable at the level of the structural gene (coding) DNA sequences (e4~., net surface charge or thermostabil i ty of product) and phenomena more easily explained by differences in non-coding, regulatory DNA sequences which act on the gene in cis (e.g., organ-specific differences in allelic expression). We now add a sixth difference: alcohol dehydrogenase-l-S and 1-F differ in but one of the 29 tryptic peptides, as resolved by our two-dimensional thin-layer chro- matographic procedures.

One of the more interesting differences between Adh l -S and A d h l - F alleles, difference No. 5 of the introduction, is their total inability to recombine intra- genically. One type of explanation for such recombinational restriction might invoke structural genes which differ in so many sites that homologous pairing at meiosis is impossible mechanically. Our result is that alcohol dehydrogenase- 1-S and 1-F differ very little; a single amino acid substi tution accounts for our data. We conclude that such a small difference could not account for the recombinational restriction, and that some non-homology located near, but not as part of, the coding sequences probably exists.

Our methods should allow further alcohol dehydrogenase-1 polypept ide comparisons where an argument for identi ty is required, or where one desires amino acid sequences of the differences. Further, these methods should extra- polate to the alcohol dehydrogenases of other plants.

Acknowledgements

We would like to thank the following: Tauny Ruymaker and Eve Lednicky for assistance in the purification procedure; Professor Rober ta Palmour for the amino analysis runs and for useful discussions; Marty Sachs for use of his un- published native polyacrylamide gel procedure; and David S.K. Cheng for photography. Supported by U.S.P.H.S. Grant GM21734.

110

References

1 Schwartz, D. (1966) Proc. Natl. Acad. Sci. U.S.A. 56, 1431--1436 2 Freeling, M. and Schwartz, D. (1973) Biochem. Genet. 8, 27--36 3 Ferl, R.J., Dlouhy, S.R. and Schwartz, D. (1979) Mol. Gen. Genet. 169, 7--12 4 Fisher, M. and Schwartz, D. (1973) Mol. Gen. Genet. 127, 33--38 5 Felder, M.R., Scandalios, J.G. and Liu, E.H. (1973) Biochim. Biophys. Acta 318, 149--159 6 Freeling, M. and Woodman, J.C. (1979) in The Plant Seed: Development, Preservation and Germina-

t ion (Rubenstein, I. et al., eds.), Academic Press, New York 7 Schwartz, D. (1973) Genetics 74, 615--617 8 Sachs, M. and Freeling, M. (1978) Mol. Gen. Genet. 161 ,111- -115 9 Felder, M. and Scandalios, J.G. (1971) Mol. Gen. Genet. 111 ,317- -326

10 Schwartz, D. (1971) Genetics 67 ,411 - - 425 11 Freeling, M. (1978) Genetics 89 ,211- -224 12 Bradford, M. (1976) Anal. Biochem. 72, 248--254 13 Ames, G.F.L. (1974) J. Biol. Chem. 249 ,634- -644 14 Hirs, C.H.W. (1956) J. Biol. Chem. 219 ,611 15 Waley, S.G. and Watson, J. (1953) Biochem. J. 55 ,328- -337 16 Bates, D.L. and Perham, R.N. (1975) Anal. Biochem. 68, 175--184 17 Stephens, R.E. (1978) Anal. Biochem. 84, 116--126 18 Grunow, M. and Sch6pp, W. (1978) FEBS Lett. 94, 375--379 19 Thatcher, D.R. (1977) Biochem. J. 163 ,317- -323 20 Kempe, T.D., Gee, D.M., Hathaway, G.M. and Noltmann, E.A. (1974) J. Biol. Chem. 249, 4625--

4633