October. 1973[Ind. J. Physiol, Pharmc.

ponse by TPN can be givennvironment in directing theabolic alteration in the cell

-ir respective receptors andn of these amines. Even a

TP and these amines

of our future investigation.he vagosympathetic trunk,al action due to increasednstricting effect of myoge-urrence of tachyphylaxis

ever, involve the release of

p. 65, 1960.

triphosphopyridine nucleotide.

Medical Research, 5th Edn.,

chromaffin in the cells of the

d C.A. Stone., Biochemical

orter c.c. and C.A. Stone, 1967.

Proc.

iated cardiovascu lar effects of

n and nor-epinephrine, Circ.

TEMPERATURE AND pH AB ORMALCY 0 THE ACTIVITIES OF SUCCINATE,GLUTAMATE AND GLYCEROPHOSPHATE DEHYDROGE ASES IN CELL FREE

EXTTRACTS OF SHEEP LIVER

K. V. KEsAVA RAO A 0 KARUMURI S. SWAMI

Department of Zoology, Sri Venkateswara University, Tirupati

Summary: (I) Some kinetic properties of succinate, glutamate and glycerophosphate dehydrogenaseswere compared using cell free extracts of sheep liver as sou rce of enzyme. (2) The enzyme-substratecomplex of succinate dehydrogenase was found to be dependent on pH and temperature. (3) ThepH was found to exert an uncompetitive type of inhibitory regulation on the enzyme activity (4). Theionizing groups essential for succinate dehydrogenase activity at 32°C were found to be imidazolium(histidine) and ammonium (0(, cystine), at 37°C the ionizing groups are mostly ammonium (0:,

cystine) whereas at 45°C the ionizing groups are both imidazoluim (histidine) and ammonium(0(, cystine).

Key words: sheep liver dehydrogenases effect of temperature and pH

Body temperature is the resultant of the heat produced and heat lost to the environment(15). During temperature compensation increase in heat production is known to be contribut-ed by the metabolism of the liver, (15) which in turn is mediated by enzymes. The maximumvelocity (Vmax) state of the enzyme activitie are subjected to inhibitory regulation at physiolog-ical pH, so that the economy of the cell is last disturbed (11). Similarly the temperature andpH dependent kinetics are mediated and regulated by cytoplasmic proteins within the cell (I).In pathological states such as fever and other ailments, the pH and thermal states will be altered(15), which may induce significant changes in the structural and functional aspects of enzymesthereby affecting the general enzyme activity and cell metabolism. Under these circumstancesit is felt highly desirable to study the properties of selected enzymes in the physiological statesand see how the pH and temperature abnorma1cy will alter the catalytic properties of tbe enzymesand the possible role of cytosol proteins in providing biochemical basis for protection of enzymicdisturbance within the healthy cells. The succinate, glutamate and glycerophosphate dehydro-genases were selected in the present investigation to represent central oxidative metabolism, neutra-lisation of ammonia toxicity and fatty infiltration respectively in the liver.

MATERIALS A~'D METHODS

Sheep were killed by decapitation and the livers transferred quickly to a clean dry beakerjacketed with ice in a Thermos flask, thus maintaining a temperature at about 0°. The tissue washomogenized in cold 0.025 M buffer (pH 7.7) for 3 minutes with a motor driven ground glasshomogenizer. The homogenate was made at a concentration of 100 mg per ml (Wet weight)

.i>----------- )d-~_~~ -_ ------

--------

350 Kesava Rao and Swami October 1973Ind. J. Physiol. Pharmac.

and centrifuged at 2,000 r.p.m. for about 10 min to sediment the cellular debris. Appropriatelydiluted supernantant was used as the source of enzyme.

Succinate dehydrogenases activity (SDH) was assayed by the colorimetric method des-cribed by achlas et al., (9). The method was modified as follows; the reaction mixture containedin a final volume of 2 ml; 75 f.L moles sodium succinate (pH 7.7), 100 f.L moles KH2P04--K2HP01

buffer (pH 7.7),4 f.L moles I T, 0.5 p. moles PMS and 0.1 ml of appropriately diluted homogenate.After incubation for 30 min at 37°, the reaction was stopped by the addition of 6 ml glacialacetic acid and the formazan formed due to reduction of the dye was extracted into 6 ml toluene.After overnight extraction in cold, the colour was read in DV spectrophotometer (Hilger andWatts, England) at 500 mu,

The cytochrome linked glycerophospbate dehydrogenase (GPDH) and glutamate dehy-drogenase (GDH) were determined by the colorimetric method described by Lee andLardy (6). The reaction mixture contained in 2 ml volume; 50· p. moles sodiumglutamate of sodium glycerophosphate (pH 7.7), 100 f.L moles KH2P04 - K2HPb4 buffer-(pH 7.7), 4 /1. moles INT, 0.5 f.L moles PMS, 0.1 f.L moles KCN and 0.1 mloi appropriately dilutedhomogenate. 0.01 f.L moles of NAD was added to the reaction mixture when glutamate dehydrogenase activity was measured. After 30 minutes incubation at 37'C, the reaction was stoppedand the colour was measured 2S f'iven before. Individual zerotime controls were maintained forall the samples by the addition of glacial acetic acid to the reaction mixture plus homogenatebefore incubation and the enzyme activities are expressed in f.L moles formazan formed/mgprotein/hr. The standard calibration curve of formazan was prepared by taking I T in differentconcentrations and allowed for reduction in the presence of NADH and the enzyme assaycomponents. The formazan formed was extracted into toluene as described before. The opticaldensities were found to be proportional to the formazan concentrations.

The protein content was determined by the method of Lowry et a!., (7), using bovineserum albumin as standard. The mean values of enzyme activity levels of either duplicates ortriplicates were used for calculation. The maximum velocities (Vmax) and the Michaelis con-stants (Km) were calculated by the method of least squares.

Abbreviations:

INTPMSEnzvmes:SDH

2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium ChloridePhenazine me thos ulpha te.

Succina te dehydrogenase(Succinate: INT - Oxictioreductase E.N. 1.3.99.1)

Glycerophosphate dehydrogenase(Glycerophosphate: Cytochrome C Oxidoreductase, E.N. 1.1.99.5).Glutarna te dehydrogenase (L-glu tama e 7"~ADOxdoreduclas't', E.N.i:4:J .2)

GPDH

GDH

-------------

Volume ]7Number 4

All the assays were I

standardisation.

The pH optimum in Iand GPDH (Fig 1). The pIas electron acceptor is about ~dent upon tbe dye used as eh

a:~e:I---ZWf-

ot!:11...e.•...z«'"<~

~IIIW.Jo~:l

Fi

Enzyme activities areforms in response to shifts ~reported that the Michaelisand similar relationship cou

In the present study,dependent on pH and alscfound to decrease with inct8.5 and 9.0. Thus the 10Wl

Km value was at pH 7.0, SI

the temperature. A generageneral and at each temper:the Km values are depend

lar debris. Appropriately All the assays were made under the conditions measuring initial velocities after dueandardisation.

October 1973lnd. J. Physiol, Pharrnac,

colorimetric method des-reaction mixture containedmoles KH2P04--K2HPO.

riately diluted homogenate.e addition of 6 ml glacialextracted into 6 1711 toluene.ophotometer (Hilger and

OH) and glutamate dehy-described by Lee and50 fL moles sodium

KH2P04 - K2HP04 buffer-ml of appropriately dilutedure when glutamate dehy, the reaction was stoppedtrois were maintained formixture plus homogenateles formazan formed/mgby taking INT in differentH and the enzyme assayibed before. The optical

tions.

et aI., (7), using bovineels of either duplicates or) and the Michaelis con-

99.5).se, E.N.i:4:1.2)

ume J7mber4

Dehydrogenases in Sheep Liver 351

RESULTS AND DISCUSSION

The pH optimum in KH2P04 - K2HP04 buffer is about 8.0 for SDH and 8.5 for GDHd GPDH (Fig 1). The pH optimum without PMS and with triphenyl tetrazolium chlorideelectronacceptor is about 9.0 for SDH and GDH (11) indicating that pH optimum is depen-ntupon the dye used as electron acceptor and this is in accordance with McShan (8).

9·0

a::!l 8~OClI-Z 7·0

'"....Cl 6.a:U-.. 5·0E--Zq; 4·0N<t

~a::3·0-Cl

LL

'"w 2.0..J

0~~ 1·0

6'0 6'5 7'0 7·5 8·0 8.5 9'''-l>H

Fig. 1: Effect of pH on dehydrogenases,

Enzyme activities are known to undergo reversible transitions between active and inactiveormsin response to shifts in pH and temperature (2, 13, 14, 16) Ghosh and Fishman (4) havereportedthat the Michaelis constant of rat intestinal alkaline phosphatase was pH dependentandsimilar relationship could be envisaged for the succinate dehydrogenase.

In the present study, the Michaelis constant of succinate dehydrogenase was found to bedependenton pH and also temperature (Table I). At 321> and 37°C the Km values werefound to decrease with increase in pH value up to 8.0 pH and started increasing at pH values.5 and 9.0. Thus the lowest Km values were obtained at pH 8.0 while at 451>C the lowest

Km value was at pH 7.0, suggesting that the enzyme efficiencyand the pH dependency vary withIhe temperature. A general effect of temperature increase reflects a decrease in Km values ingeneraland at each temperature, further, the variations in Km values are pH dependent. ThusIhe Km values are dependent upon pH and temperature. These variations in Km value are

---)------

-------------

352 Kesava Rao and Swami October 1973Ind. J. Physiol Pharmac,

having a metabolic significance, since, the pathological states such as fever and other ailmentsmay induce changes in the pH and thermal states and these alterations in phy iological stagemay alter the Km values for the substrate which reflects the efficiency of the enzymes in thehydrolysis of endogenous substrates.

TABLE I: Apparent michaelis constants (mM) of succinate dehydrogenase at different temperature and pH.

pH 32°e 37°e 45°C

1.932 1.678 0.56101.916 0.9625 0.51061.204 0.8243 0.1032

0.9162 0.6246 0.1391

0.5102 0.4129

0.5828 0.44830.7013 0.4513 0.3009

6.06.57.07.58.08.59.0

The double reciprocal plots of (l/V) versus (l/S) showed somewhat parallel relationshipat all the three temperatures studied (Fig. 2), indicating that pH will exert a phenomenonsimilar to that of uncompetitive type of inhibitory regulation. Similar type of inhibitory regula-tion was observed for succinate dehydrogenase, aspartate and alanine amonotransferases by thealterations in the protein environment (10,12). Grazi and Magri (5) reported similar type of some-what parallel curves with arginase activity using arginine as substrate at different pH values.Thus it appears that the pH alterations will exert a phauomenon similar to that of uncompetitivetype of inhibitory regulation.

If the Michaelis constant was found to be dependent on pH and temperature, it is likelythat Vmax may also be dependent on these factors and it was found to be the case in the presentstudy (Fig. 3). At temperature of 32°C, the optimal Vmax values were found between pH 7.0and 8.0 giving a broad curve covering these points. At 37°C, the curve shows a relatively narrowoptimal range at pH 8.0 whereas at 45°, the narrow optimal range is at pH 8.0 (Fig. 3). Thedouble reciprocal plots of (l/S) versus (l/V) indicate a similar trend (Fig. 2). At temperaturesof 32°, the plots from pH values 7.0 to 8.0, at 37°, the plot at pH 8.0 and at45° the plot at pH7.0 represents the tendency of shift towards the "x" axis, thereby suggesting the hyper activityat these pH values in the respective temperature environment. This is in consonance with thepH-Log Vmax relationship as evinced in the Fig.3.

Dixon and Webb (3) have shown that a plot of log Vmax against pH hould exibit 2.

horizontal segment of the curve over the pH range in which the enzyme exists in the optimumactive form and that. the ionization of groups to form inactive enzyme species should appear onsuch plot a lines of integral slope running down from the horizontal. From the position

------------ --------

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\.0

0'6

0·6

0·4

0·2

\.0

0·6X..:~ 0'6>Cl 0'40.J

0·2

\.0

0·6

0-6

0'4

0..2

Fig. 2: Double recija eries of dil

of the bends in the graph, thactivity of the group which f

be obtained (3). Since a chaionization of each of the e gl

in each pK with temperature

October 1973Ind. J. Physiol Pharmae,

h as fever and other ailmentserations in physiological stageciency of the enzymes in the

t different temperature and Hp .

0.56100.51060.10320.1391

0.3009

omewhat parallel relationshipwill exert a phenomenon

ilar type of inhibitory regula-me amonotransferases by thereported similar type of some-strate at different pH values.

iIar to that of uncompetitive

and temperature, it is likelyd to be the ea e in the present

s were found between pH 7.0rve shows a relatively narrow

! is at pH 8.0 (Fig. 3). Thed (Fig. 2). At temperatures

18.0 and at 450 the plot at pHsuggesting the hyper activity

'his is in consonance with the

iagainst ,pH ,ShOUld exibit anzyme exists 10 the optimumyme species should appear onrizontal. From the positions

me 17ber4

Dehydrogenases m Sheep Liver 353

1.2r-------------------------------~45°

L-~~--~~~~~~~~~~~('----6.0 6.S 7.0 7·5 8·0 8·5 s-oI'H

Fig. 2: Double reciprocal plots of initial velocity against succinate concentration ata series of different pH values and temperatures.

/'>. _~~ ~ '" -- ~

ofthe bends in the graph, the pKs of the ionizing groups in the ES complex which affect theactivityof the groups which either form part of the active centre of closely associated with it can

obtained (3). Since a change of the temperature will itself produce a change in the stage ofIonization of each of these groups, by determining the curves at different temperatures the changeIII each pK with temperature can be mea ured by the displacement of the bends (3), In the present

-.i->:

------------

1·0

0.4

1.0

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0·2

1·0

0·8

0-6

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r.t.

..--

354 Ke ava Rao and Swami October 1973Ind. J. Physiol. Pharrnac,

Fig. 3: Effect of pH and temperature on the Vmax of succinate dehydrogenase activity at different temperatures.

Volume 17Number 4

2. Behrisch, H.W. and r.w. H);IProperties of rainbow-trout IDixon, M. and E.C. Webb. j

Ghosh, .K. and W.H. FishmlL-Phenylalanine: I. Kinetic stuGrazi. E. and E. Magri. MolLee, Y.L. and H.A. Lardy. IJother dehydrogenases in varioiLowry, O.H., .J. Ro ebrougfreagent. J. Bioi. Chem., 193 21Mc Shan, W.H. In: RespiratoChap VI, 1949.

achlas, M.M., S.l. Marguliedehydrogenase activity. J. BiPrameclamma, Y, K.V.K. Raintracellular fluids in the gas!Rao, K.V.K. and K.S. Swami.Internal. Physiol. Bioch., 77Rao, K.V.K, S.M. Basha andand alanine arninotransferasesunicated).Somero, G. . and P.W. Hochvale kinase of the rainbow I1968.Smith, M.W. Influence of tetivity of gold fish intestinal :Snell, E.S. and E. Atkins. JEdward Bittar and eville BiTaketa, K. and RM. Pogell.phosphate. J. Bioi. Chem., i

experiment, the integral slopes running down were observed at pH 6.6 and 8.2 at 32°C at 7.8and 8.2 at 37°C and at 6.75 and 7.25 at 45°C indicating that at 32° the ionizing groups essentialfor SDH are imidazolium (histidine) and ammonium (a, cystine), at 3r the ionizing groupsare mostly ammonium (a, cystine) whereas at 45° the ionizing groups are both imidazolium(histidins) and ammonium (a, cystine) as suggested by Dixon and Webb (3). Thus at pbysio-logical temperature the emphasis of the active site is towards the ionization of the ammonium(a, cyctine) whereas as under hypothermic and hyperthermic states the ionization of activecentre is shifted also to imidazolium (histidine), indicating the existence of a regulative factorwhich i highly operative at the physiological state, which interferes with the contribution ofionization potential of imidazolium (histidine) in the catalytic centre of succinate dehydrogenase.

0-4532°

Ir-a: .::J 0-.400I---z 0-35w.... •0

./ ~

Q: 0-30Q..../ ~;E 0-25

z<{

'"<J:

4~~a:0~

~~III

LL'-:::-

...J

0~:t

0-05'-I>

One of the authors (K.V. K.R.) is grateful to the Council of Scientific and IndustrialResearch, ew Delhi, for the award of a Senior Research Fellowship, during the tenure of whichthis work was carried out.

3.4.

5.6.

7.

8.

9.

10.

11.

n.

13.

14 .

15.

16.

I I I I200 400 600 800

ACKNOWLEDGEME T

REFERE CF.S

I. Basha, S.S. and K.S. Swami. lnhibition Kinetics of succinate dehydrogenase by sodium malonate and coppersulphate. Enzymotogia., (Communicated).

------ -------

October 1973Ind. J. Physiol, Pharmac.

6.6 and 8.2 at 32 C at 7.8he ion'izing group essentialat 37" the ionizing grouPBps are both imidazoliumebb (3). Thu at physio-nization of the ammoniumthe ionization of activece of a regulative factorwith the contribution ofsuccinate dehydrogenase.

•~• 9.°__ -.~___ .-O.~._s _-.

~- 8·0 •-::::::D'==.

r-::- •--- 7·0

lVityat different temperatures,

cientific and Industrialring the tenure of which

odium malonate and copper

'ume 17mber4

Dehydrogenases in Sheep Liver 355

Behrisch, H.W. and P.W. Hochachka. Temperature and regulation of enzyme activity in Poikilotherms:Properties of rainbow-trout fructose diphosphatase. Biochem. J., 111 : 287, 1969.Dixon, M. and E.C. Webb. Enzymes, 2nd Ed. Academic Press, New York, 1964.Ghosh, .K. and W.H. Fishman. On the mechanism of inhibition of intestinal alkaline phosphatase byL-Phenylalanine: J. Kinetic studies. J. BioI. Chem., 241 : 2516, 1966.Grazi. E. and E. Magri. Molecular Characteristics of chicken liver arginase. Biochem. J., 126 : 667, 1972.Lee, Y.L. and H.A. Lardy. Influence of thyroid hormones on L- O(-glycerophosphate dehydrogenase andother dehydrogenases in various organs of the rat. J. BioI. Chem., 240 : 1427, 1965.Lowry, O.H., .J. Rosebrough, A.L. Farr and R.J. Randall. Protein measurements with the Folin phenolreagent. J. BioI. Cltem., 193 265, 1951.Me Shan, W.H. In: Respiratory enzymes, (ed) Lardy, H.A. Burgen Publishing Company, Minneapolis,Chap VI, 1949.

achlas, M.M., S.L Margu1ies, and A.M. Seligman. A Colorimetric method for the estimation of succinicdehydrogenase activity. J. BioI. Chem., 235 : 499, 1960.Prameclamma, Y., K.V.K. Rao and KS. Swami. On the regulation of succinate dehydrogenase activity byintracellular fluids in the gastrocnemius muscle of frog (In preparation).Rao, KV.K. and KS. Swami. Kinetic characteristics of selected oxidases in sheep liver homogenate. Arch.lnternat, Physiol. Bioch., 77 : 812, 1969.

, Rao, K.V.K, S.M. Basha and K.S. Swami. Intracellular electromigration and kinetic properties of aspartateand alanine aminotransferases in the gastrocnemius mu c1e of frog. Arch. internal. Physiol. Bioch., (Comm-unicated).Somero, G. . and P.W. Hochachka. The effect of temperature on catalytic and regulatory functions of pyru-vate kinases of the rainbow trout and the antarctic fish Trematomus bernacchii, Biochem., J., 110 : 395,1968. 'Smith, M.W. Influence of temperat u re acclimatization on the temperature dependence and on ouabain sensi-tivity of gold fish intestinal adenosine triphosphatase. Biochem. J., 105 : 65, 1967.

). Snell, E.S. and E. Atkins. The mechanisms of fever. In: The biological basis of Medicine, ed. by E.Edward Bittar and Neville Bitter, Academic Press, New York, Vol. IT, 397, 1968.

6. Taketa, K. and B.M. Pogell. Allosteric inhibition of rat liver fructos 1, 6-phosphatase by Adenosine 5-mono-phosphate. J. BioI. Chem., 240 : 651, 1965.

,

-------------- )