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Deuterium isotope effects on beef-heart lactate dehydrogenase
It is possible, therefore, that the significantly higher values of the mitochondrial glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in young animals as compared with the adults, are related to the intensive lipid synthesis in the brain 9 during the early post-natal development period.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, and National Research Institute of Mother and Child, Warsaw (Poland)
G. BAGDASARIAN D. HULANICKA
I M. R. V. MURTHY AND D. A. RAPPOPORT, Biochim, Biophys. Acta. 74 (1963) 328. 2 M. R. V. MURTHY AND D. A. RAPPOPORT, Biochim, Biophys. Acta, 74 (1963) 51. 3 O. H. LOWRY, N. J _ ROSEBROUGH. A. L. FARR AND R. J. RANDALL, J. Bioi. Chem., 193 (195 1) 26 5. 4 K. YAMADA AND N. SHIMAZONO. Biochim, Biophys. Acta, 54 (1961) 205 . 5 K. BLOCH, Lipid Metabolism, Wiley, New York, 1960. 6 S. V. PANDE, R. PARVIN KHAN AND T. A. VENKITASUBRAMANIAN, Biochim, Biophys. Acta, 84 (1964) 239. 7 E. J. MASORO, j. Lipid Res., 3 (1962) 149. 8 B. JEAURENAND, Metabolism, 10 (1961) 535. 9 H. McILWAIN, Biochemistry and the Central Nervous System, end ed., Churchill. London, 1959, p. 186.
Received December end, 1964 Biochim. Biophys. Acta, 99 (1965) 367-369
sc 63099 Deuterium isotope effects on beef-heart lactate dehydrogenase A recent communication from this laboratory- described the effects of substitution of deuterium for the transferable and non-transferable hydrogens in the 4-position of the pyridine ring of NADH 2 on the kinetics of skeletal-muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27). One of the more interesting observations was that in Hp the coenzyme with deuterium in the transferable position ([A-4-2H]NADH2) was bound to and released from the enzyme more rapidly than was NADH 2 , whereas the stereoisomer with non-transferable deuterium ([B-4-2H]NADH2) was bo-ctnd more slowly. This behavior was in contrast to that observed with other dehydrogenases'
v=
(/) 0
-I-
fli ,
fli.
c-l- 5
-I-
sc Biochim, Biophys. Acta, 99 (1965) 369-371
370
SHORT COlllMUNICATIONS
where Eo, C, and 5 are the initial con centrat ions of enzyme, coenzyme, and pyruvate, respectively, and v is the initial velocity. Beef-heart lactate dehydrogenase h as been shown to obey a mechanism of ordered binding with the formation of one or more kinetically sign ificant ternary complexes", For this mechanism, the forwa rd and reverse rates of binding of the coenzyme and enzyme, k1 and k2 (and the equilibrium constant for th e enzymecoenzyme complex, K 1 ) , are the only rate constants that can be obtained directly from the k inetic analysis. The ratios of the 0 constants gives the isotope effect when the syst em is saturated with both coenzyme an d substrate, t he
2 rat ios : a significant normal effect was observed with both of the deuteriated coenzymes with the heart enzyme, where as a slight inverse effect was noted wit h the muscle enzy me. The ratio of ([>n constants for {B-4-2H)N ADH2 was essentially unity with heart lactate dehydrogenase, but slightly lower with m uscle lactate dehydrogenase. The difference between these ratios is marginally significant . The effects on the k1 and k 2 ratios are in the same direction for both enzymes, slightly greater with muscle lactate dehydrogenase than with heart lact at e deh ydrogenase for (A-4-2H)NADH., and somewhat smaller for (B-4-2H)NADH2. TABLE I ISOTOPE EFFECTS OF D E U T E RIUM ON KINETIC CONSTANTS OF MUSCLE AND HEART LACTATE DEHYDROGENAS E S
Data are p r esented as ratios (with sta ndard errors) of cons tants measur ed wit h deuterated coenzyme to those measured with NADH•.
RatiQ
11>0' a/c;[Jo H
(A-4- 2H) NADH z
(B-4-'H)NADH z
Muscle
M uscle
1.4 I
±
0 .1 7
1.35
0.35
±
0.1
J
0.92
0.15 0.43 0.94 2.18
± ± ± ±
0.08 0 .13
0.64 0.6 9 1.48 2.12
(V B /V, H)
I1>I za/c;[JIB (kIH/kl'H )
k.H/h. 2a J(,H/I{t'a l]),zalc;[J.H
1])1." fiftpI2H
Hearl
O . IO
0. 78
Heart
± 0.03 ± O .IO
0.81
± 0.0 8
1. 10
±
0.04
1.20
±
0.49
1.37
±
0 .15
± 0.08 0.07 ± 0. 04-
I,9 I 1.59 o.qo 0.56
± ±
±
0.85 0 ·59 0 .12 0 .21
2 .7 8 2.02 1.30 0 .64-
± ±
0 .63 0 .4 7 0 .05 0 .13
± ±
0.19
Bi oohim, Biophys. Acta, 99 (1965) 369-371
±
±
±
SHORT COMMUNICATIONS
371
Thus it would appear that the two lactate dehydrogenase systems differ very slightly in their response to deuterium substitution in the coenzymes, at le ast under the experimental condit ions employed here (pH 7-4, 25°). The effect of deuteriation of the coenzymes on the rates of formation and decompo sition of the enzymecoenzyme comp lex ar e similar, in that th ere are inverse isotope effects with the (A-4-2H)NADH z but n orm al isot ope effects wit h (B-4-2H)NADH2 for both enzym es. For the mechani sm which t his enzyme seems to obey , the $2 coefficient is a complex one; in the case of two ternary complexes, cfJ 2 is a function of 5 different rat e constants , including t hose concern ed with binding of pyruvate t o the enzymeNADH z complex. In view of t h e differences between heart and muscle lactate dehydrogenase with respe ct t o the inhibitory effects of pyruvat e" it is perhaps not surprising that this t erm should reflect the most consiste nt difference in isotope effects This work was performed under the auspices of the United States Atomic Energy Commission. Division of Biological and M edical Research, Argonne N ational La boratory, Argonne, Ill. ( U.S.A.)
JOHN
F.
THOMSON
S HAR RON
L.
NANCE
1 J . F . TH OMSO N, J . J . D ARLING AND L . F. BORDNER, Biochim , Biopby s. Acta, 85 (1964) 177 . 2 V. J . SHINER, J R., H . R. MAHLER, R . H. B AKER, JR. AND R. R. H IATT, Ann. N. Y. Aoad , su., 8 4 (19 6o ) 58 3. 3 R. H. B AKER, J R., B iochemistry , I (196 2) 4r. 4 J. F . THOMSON, D . A . B RAY AND J. J. BUMMERT, B iochem, Ph armacol., II (19 6z) 943· 5 W . B. N OVOA AND G. W . S CHEWRT, j. Bioi. Chem., 236 (1961) 215 0. 6 G. PFLEIDERER AND D . ]EC KEL, B iochem . Z ., 3 29 (I 9 57) 37 0 .
Recei ved December roth, 1964 Biochim. Biop hys. Acta , 99 (J:9 65 ) 369-371
SC
63102
An ultraviolet absorption band due to the interaction of NAD and glyceraldehyde-3-phosphate dehyd rogenase It has been known for several years that binding of NAD to glyceraldehyde3-phosphate dehydrogenase (n-glyceraldehyde-g-phosphate :NAD oxidoreductase (phosphorylating), EC 1.2.1.12) results in the appearance of an absorption band around 360 mfl (ref. I) . During a st udy of the absorption spectrum of swine muscle glyce raldehy de-g-phosphate deh ydrogenase (containing tightly bound NAD ) we hav e det ected in the ultravi olet region a new absor ption b and char acteristi c of glyceraldehyde-j-phosphat e dehydrogenase-NAD int eraction. In order to resolve small spectral alterations t he method of differen ce spectrophotomet ry-a h as been applied. P ot ent ial errors due to st ray light! and fluores cencehave been excluded. The difference spectrum which appears when NAD is adde d to glyceraldehyde3-phosphate deh ydrogenase freed from tightly bound NAD by ch arcoal-t reatment can be seen in Fig. I , Curve A. The existence of the broad abs orpt ion band ar ound B iochim. B iophys , A cta, 99 (19 6S) 37 T-3 7S