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Role of DPN+ in the mechanism of action of glyceraldehyde-phosphate dehydrogenase
380
PRELIMINARY NOTES
Activation of tetrahydrofolate dehydrogenase by cations is not restricted to guinea-pig liver; similar results have been obtained with enzyme preparations from mouse leukemia cells grown in culture (L5178Y), from Ehrlich ascites carcinoma cells and from the leukocytes of patients with chronic granulocytic and acute leukemia. The author would like to thank Mrs. B. SIMMONS for her assistance in this study.
Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. (U.S.A.)
J. R. BERTINO
1 C. A. ~NICHOL AND A. D. WELCH, Proc. Soc. Exptl. Biol. Med., 74 (195 o) 4o3. 2 S. FUTTERMAN AND M. SILVERMAN, J . Biol. Chem,, 224 (1957) 31. 3 S. F. ZAKRZEWSKI AND C. A. NICHOL, Biochim. Biophys. Acta, 27 (1958) 425 . 4 M. J. OSBORNE, M. FREEMAN AND F. M. ]-IUENNEKENS, Proc. Soc. Exptl. Biol. Med., 97 (I958) 429. 5 j . R. BERTINO, B. W. GABRIO AND F. M. HUENNEKENS, Biochem. Biophys. Research Communs., 3 (196o) 461. 6 S. F. ZAKRZEWSKI AND C. A. NICHOL, J. Biol. Chem., 235 (196o) 2934. 7 j . R . BERTINO, B. S. SIMMONS AND D. M. DONOHUE, Federation Proc., i n t h e p r e s s . 8 M. J. OSBORNE AND F. M. HUENNEKENS, J. Biol. Chem., 233 (1958) 969. 9 S. F. ZAKRZEWSKI, J. Biol. Chem., 235 (196o) 1776. 10 D. K. MISRA, S. R . HUMPHREYS, 1V[. FRIEDKIN, A. GOLDIN AND E . J. CRAWFORD, Nature,
189 (1961) 39. 11 W . C. WERKHEISER, J. Biol. Chem., 236 (1961) 888.
z~ G. A. FISCHER, Biochem. Pharmacol., i (1961) 75.
Received J a n u a r y I9th, 1962 Biochim. Biophys. Acta, 58 (1962) 377-380
Role of DPN + in the mechanism of action of glyceraldehydephosphate dehydrogenase In a recent report 1, it was shown that contrary to what would be expected on the basis of a mechanism for pyridine nucleotide dehydrogenases proposed by VA~ EYS et al. 2, DPN + was not required for the oxidation of D P N H b y acetaldehyde, pyruvate or ~-ketoglutarate (+NH3) catalysed by the respective dehydrogenase. Treatment of the enzymes and the D P N H with DPN+ nucleosidase [DPN+(TPN +) glycohydrolase, EC 3.2.2.67 led to an increase, not a decrease in the reaction rate. In unpublished experiments, it was found that yeast glyceraldehydephosphate dehydrogenase EI)-glyceraldehyde-3-phosphate : DPN+ oxidoreductase (phosphorylating), EC I. 2. i. 12 acting on acetyl phosphate behaved similarly. It has now been found, using the same methods, that DPN + is necessary for the oxidation of D P N H by acetyl phosphate or 1,3-diphosphoglycerate catalysed by muscle glyceraldehydephosphate dehydrogenase. Fig. I shows that little oxidation of D P N H by acetyl phosphate took place when DPN ÷ was removed from the reaction system. Addition of DPN + brought about a temporary stimulation of the rate of o x i d a t i o n - - t e m p o r a r y because the added DPN + is rapidly decomposed. Similar results were obtained with 1,3-diphosphoglycerate, generated by ATP and 3-phosphoglycerate in the presence of phosphoglycerate kinase (ATP:D-3-phosphoglycerate I-phosphotransferase, EC 2.7.2.3) and Mg 2+, in place of acetyl phosphate. Biochim. Biophys. Acta, 58 /I962) 38o-383
PRELIMINARY NOTES
38I
Thus, t h e reverse of t h e p h o s p h o r y l a t i n g o x i d o r e d u c t i o n r e a c t i o n c a t a l y s e d b y g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e requires D P N ÷, as also do some o t h e r r e a c t i o n s c a t a l y s e d b y this enzyme, viz. the aIsenolysis of a c y l p h o s p h a t e s 3, t h e h y d r o l y s i s of a c e t y l p h o s p h a t e 4, a n d t h e 3 ~ P i - a c e t y l p h o s p h a t e e x c h a n g e reaction 5. I
I
0.75
20
0.7C
~0.6~
0.6(
0.5!
0
5
10
15
Time (rain)
Fig. i. DPN + requirement for oxidation of DPNH by acetyl phosphate catalysed by glyceraldehydephosphate dehydrogenase. Reaction mixture contained o. 15 M Tris-HC1 buffer (pH 7.4), o.122 mM DPNH, 4 mM dilithium acetyl phosphate, 1.8 mg rabbit-muscle glyceraldehydephosphate dehydrogenase (prepared in the presence of I mM EDTA) 3, in a volume of 2. 5 ml. The reference cell contained all additions except the DPNH. The reaction was begun by addition of DPNH. A, not treated with DPN + nucleosida~se; B, the enzyme and the DPNH solutions were previously incubated with 12oo units 1 DPN + nucleosida~e for 2o min, after 5 and 8 min, 2.5/*moles and 20/~moles DPN +, respectively, were added. These findings s u p p o r t a m e c h a n i s m of action of this e n z y m e i n v o l v i n g two a d j a c e n t D P N molecules, as suggested b y VAN EYS et al. 2 for t h e a l c o h o l - t y p e d e h y d r o g e n a s e s , a l t h o u g h these workers specifically e x c l u d e d g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e from t h e i r m e c h a n i s m . S t u d i e s of t h e course of t h e r e d u c t i o n of D P N + b y g l y c e r a l d e h y d e , a s u b s t r a t e which r e a c t s m u c h more slowly t h a n t h e physiological s u b s t r a t e b u t which can be used to s t u d y t h e e n z y m e in "slow m o t i o n ''8, has given evidence for t h e f o r m a t i o n of two D P N c o m p o u n d s , different from D P N + or D P N H . Fig. 2 shows t h a t , a f t e r 7 min, when t h e a b s o r b a n c y at 340 m/z has a l m o s t reached a c o n s t a n t value, t h e a m o u n t of D P N H found in alkali e x t r a c t s of t h e reaction m i x t u r e was o n l y a b o u t one half of t h e a m o u n t of D P N + which d i s a p p e a r e d , as m e a s u r e d on acid e x t r a c t s of t h e reaction m i x t u r e . However, the increased a b s o r p t i o n at 340 m/z, c a l c u l a t e d as D P N H , agreed fairly closely w i t h t h e a m o u n t of D P N + which d i s a p p e a r e d . I t a p p e a r s , then, t h a t a c o m p o u n d a b s o r b i n g a t 340 m/z, which is n o t D P N H , is formed a t t h e b e g i n n i n g of t h e reaction. Over t h e following 35 min, this c o m p o u n d is l a r g e l y c o n v e r t e d to D P N H , w i t h l i t t l e f u r t h e r change in t h e a b s o r p t i o n a t 34o m/z. I n Biochim. Biophys. Acta, 58 (I962) 380-383
382
PRELIMINARY NOTES
addition to this new 34o-mtz compound and D P N H , the alkali extracts contain a compound which analyses like DPN + in the enzymic assay, but which cannot be DPN+ since the latter is completely destroyed under the conditions used to prepare the alkali extract. This alkaline-stable DPN + compound yields DPN +, in less than
(
.
o 0
15 Time (min) 30
45
Fig. 2. ] ' h e f o r m a t i o n of D P N compounds d u n n g the reaction between glyceraldehyde and D P N +, c a t a l y s e d b y g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e . T h e r e a c t i o n m i x t u r e c o n t a i n e d in a t o t a l v o l u m e of IO ml, i m m o l e N a 2 H P O 4, i m m o l e T r i s - H C l buffer, 2 5 / * m o l e s DL-glyceraldehyde, 5.04 /*moles D P N +, IO.8 m g r a b b i t - m u s c l e g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e , c o n t a i n i n g o . 1 9 4 / * m o l e b o u n d D P N +. T h e final p H w a s 9.o. T h e r e a c t i o n w a s b e g u n b y a d d i t i o n of glycera l d e h y d e . A t v a r i o u s t i m e i n t e r v a l s , acid a n d alkali e x t r a c t s of s a m p l e s were m a d e b y t h e p r o c e d u r e of PURVtS7, a n d t h e D P N + a n d D P N H c o n t e n t s of t h e e x t r a c t s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h alcohol d e h y d r o g e n a s e a n d alcohol or a c e t a l d e h y d e . - - A D P N +, d i s a p p e a r a n c e of D P N + m e a s u r e d in acid e x t r a c t s ; +AA340mg, c a l c u l a t e d as D P N H f r o m t h e increase of a b s o r b a n c y a t 34 ° m/z of t h e r e a c t i o n m i x t u r e i m m e d i a t e l y before t a k i n g t h e s a m p l e s for p r e p a r a t i o n of t h e acid a n d alkali e x t r a c t s ; + A D P N H , a m o u n t of D P N H f o u n d in alkali e x t r a c t s ; alkali-stable D P N +, a m o u n t of D P N + f o u n d in alkali e x t r a c t (note t h a t s o m e alkali-stable D P N + w a s f o u n d a t zero t i m e : t h i s is d e r i v e d f r o m t h e e n z y m e ) ; A34o-m/* c o m p o u n d , AA34o my (calc. as D P N H ) minus A D P N H .
lOO% yield, during preparation of the acid extract. The compound absorbing at 34o mF, like D P N H itself, is broken down in acid to a compound which does not estimate as DPN +. No increase of absorption at 34 ° mF was obtained in the absence of enzyme. The experiment shown in Fig. 2 was carried out with catalytic amounts of enzyme and DPN + added in an amount about 25 times greater than the amount of DPN + in the enzyme preparation. Table I shows that ~ather similar results were obtained when only the DPN + of the enzyme preparation was studied. In this case, however, the conversion of the 34o-m~ compound to D P N H shown in Fig. 2 was not observed and - - A D P N + exceeded AA34 o my' More of the 34o-mF compound was found in the presence of phosphate than in the presence of arsenate, but even with arsenate all the enzyme-bound DPN+ was not reduced to D P N H or converted to the 34o-m/z compound. This is to be expected on the basis of a VAN EYS type of mechanism in which one DPN + molecule binds substrate, thereby facilitating the reduction of a second DPN+ molecule without itself becoming reduced. No 34o-m~ compound was Biochim. Biophys. Acta, 58 (1962) 38o-383
PRELIMINARY NOTES
383
TABLE I FORMATION
OF DPN
DPN +
DURING REACTION BETWEEN GLYCERALDEHYDE A N D GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE
COMPOUNDS
BOUND
TO
Phosphate experiment: o.I M I~Ia2HPO 4, 15 m M EDTA, 0.3 m M DL-glyceraldehyde, 46. 5 m g rabbit-muscle glyceraldehydephosphate dehydrogenase containing 47 ° m/~moles bound DPN+; p H 8.9; reaction volume, 7-5 ml. - A r s e n a t e experiment: o.i M Tris-HC1 buffer, o.8 m M EDTA, o.o 4 M NasHAsO4, 2 m M DL-glyceraldehyde, 25.9 m g rabbit-muscle glyceraldehydephosphate dehydrogenase containing 238 m/~moles bound DPN+; p H 9.o; reaction volume, 2. 5 ml. The reactions were started b y addition of glyceraldehyde, and followed spectrophotometrically a t 34 ° m/~. Samples were withdrawn a t various times and D P N + a n d D P N H determined on acid and alkali extracts. Values given are in m/zmoles. P ho sphage T i m e (rain)
D PN+ --zJDPN + ADPNH
AA34o m~ A34o-m/* compound Alkali-stable D P N +
o
xo
470 o o
o o 21
2 IO 260 115 189 74 78
Arsenate ao
163 307 -250 -19
30
o
zo
20
137 333 18I 253 72 i8
238 o o o o o
---139 ---
72 166 116 I41 25 o
f o u n d w h e n T r i s o r p y r o p h o s p h a t e b u f f e r s w e r e u s e d i n s t e a d of p h o s p h a t e o r a r s e n a t e . T h a t t h e d i s c r e p a n c i e s b e t w e e n - - A D P N + o r AA34 o mu a n d A D P N H a r e n o t c a u s e d b y a n a l y t i c a l a r t e f a c t s is s h o w n b y : (I) a d d e d D P N + w a s r e c o v e r e d i n t h e a c i d extract and added DPNH in the alkaline extract in more than 9° % yield (no alkalis t a b l e D P N + w a s f o u n d ) ; (2) t h e r e w a s l i t t l e d i s c r e p a n c y a t t h e e n d o f t h e r e a c t i o n i n F i g . 2; (3) t h e r e w a s a l s o n o d i s c r e p a n c y i n e x p e r i m e n t s w i t h e n z y m e - b o u n d D P N +, w h e n "Iris o r p y r o p h o s p h a t e b u f f e r s w e r e u s e d , o r h i g h c o n c e n t r a t i o n s o f g l y c e r a l d e h y d e w e r e u s e d i n p h o s p h a t e b u f f e r ; (4) w h e n t h e b o u n d D P N + w a s reduced by ethanol and yeast alcohol dehydrogenase, there was no discrepancy b e t w e e n t h e AAv, o ~ a n d t h e D P N H f o u n d i n t h e a l k a l i n e e x t r a c 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 a m o u n t of D P N + d i s a p p e a r i n g a n d t h e a m o u n t o f D P N H a p p e a r i n g d u r i n g t h e a c t i o n of g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e r e c a l l s t h e o b s e r v a t i o n s of PURVIS s o n t h e a d d i t i o n of s u b s t r a t e t o r a t - l i v e r m i t o chondria. W e w i s h t o t h a n k P r o f e s s o r E . C. SLATER f o r h i s i n t e r e s t a n d h e l p f u l c r i t i c i s m a n d Miss G. HEDGES ABRAHAMSZ f o r h e r s k i l l f u l t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d i n p a r t b y a g r a n t f r o m t h e Life I n s u r a n c e M e d i c a l R e s e a r c h F u n d .
Laboratory of Physiological Chemistry, University of Amsterdam, Amsterdam (The Netherlands)
A . G . HILVERS J.H.M. WEENEN
1 A. G. HILVERS, Biochim. Biophys. Acta, 54 (1961) 186. 2 j . VAN EYS, A. SAN PIETRO AND N. O. KAPLAN, Science, 127 (I958) 1443. a E. RACKER AND I. KRIMSNY, J. Biol. Chem., I98 {1952) 721, 731. 4 j . HAETING, in W. D. MCELROY AND ]3. GLASS, The Mechanism of Enzyme Action, The Johns Hopkins Press, Baltimore, 1954, p. 536. 5 j . HARTING AND S. F. VELICK, J . Biol. Chem., 207 (1954) 867. s E. RACKER AND I. KRIMSKY, Federation Proc., 17 (1958) 1135. 7 j . L. PUEVIS, Biochim. Biophys. Acta, 38 (I96o) 4358 j . L. PUEVIS, Nature, 182 (1958) 711. Received January
2 9 t h , 1962
Biochim. Biophys. Acta, 58 (1962) 380--383
PRELIMINARY NOTES
Activation of tetrahydrofolate dehydrogenase by cations is not restricted to guinea-pig liver; similar results have been obtained with enzyme preparations from mouse leukemia cells grown in culture (L5178Y), from Ehrlich ascites carcinoma cells and from the leukocytes of patients with chronic granulocytic and acute leukemia. The author would like to thank Mrs. B. SIMMONS for her assistance in this study.
Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. (U.S.A.)
J. R. BERTINO
1 C. A. ~NICHOL AND A. D. WELCH, Proc. Soc. Exptl. Biol. Med., 74 (195 o) 4o3. 2 S. FUTTERMAN AND M. SILVERMAN, J . Biol. Chem,, 224 (1957) 31. 3 S. F. ZAKRZEWSKI AND C. A. NICHOL, Biochim. Biophys. Acta, 27 (1958) 425 . 4 M. J. OSBORNE, M. FREEMAN AND F. M. ]-IUENNEKENS, Proc. Soc. Exptl. Biol. Med., 97 (I958) 429. 5 j . R. BERTINO, B. W. GABRIO AND F. M. HUENNEKENS, Biochem. Biophys. Research Communs., 3 (196o) 461. 6 S. F. ZAKRZEWSKI AND C. A. NICHOL, J. Biol. Chem., 235 (196o) 2934. 7 j . R . BERTINO, B. S. SIMMONS AND D. M. DONOHUE, Federation Proc., i n t h e p r e s s . 8 M. J. OSBORNE AND F. M. HUENNEKENS, J. Biol. Chem., 233 (1958) 969. 9 S. F. ZAKRZEWSKI, J. Biol. Chem., 235 (196o) 1776. 10 D. K. MISRA, S. R . HUMPHREYS, 1V[. FRIEDKIN, A. GOLDIN AND E . J. CRAWFORD, Nature,
189 (1961) 39. 11 W . C. WERKHEISER, J. Biol. Chem., 236 (1961) 888.
z~ G. A. FISCHER, Biochem. Pharmacol., i (1961) 75.
Received J a n u a r y I9th, 1962 Biochim. Biophys. Acta, 58 (1962) 377-380
Role of DPN + in the mechanism of action of glyceraldehydephosphate dehydrogenase In a recent report 1, it was shown that contrary to what would be expected on the basis of a mechanism for pyridine nucleotide dehydrogenases proposed by VA~ EYS et al. 2, DPN + was not required for the oxidation of D P N H b y acetaldehyde, pyruvate or ~-ketoglutarate (+NH3) catalysed by the respective dehydrogenase. Treatment of the enzymes and the D P N H with DPN+ nucleosidase [DPN+(TPN +) glycohydrolase, EC 3.2.2.67 led to an increase, not a decrease in the reaction rate. In unpublished experiments, it was found that yeast glyceraldehydephosphate dehydrogenase EI)-glyceraldehyde-3-phosphate : DPN+ oxidoreductase (phosphorylating), EC I. 2. i. 12 acting on acetyl phosphate behaved similarly. It has now been found, using the same methods, that DPN + is necessary for the oxidation of D P N H by acetyl phosphate or 1,3-diphosphoglycerate catalysed by muscle glyceraldehydephosphate dehydrogenase. Fig. I shows that little oxidation of D P N H by acetyl phosphate took place when DPN ÷ was removed from the reaction system. Addition of DPN + brought about a temporary stimulation of the rate of o x i d a t i o n - - t e m p o r a r y because the added DPN + is rapidly decomposed. Similar results were obtained with 1,3-diphosphoglycerate, generated by ATP and 3-phosphoglycerate in the presence of phosphoglycerate kinase (ATP:D-3-phosphoglycerate I-phosphotransferase, EC 2.7.2.3) and Mg 2+, in place of acetyl phosphate. Biochim. Biophys. Acta, 58 /I962) 38o-383
PRELIMINARY NOTES
38I
Thus, t h e reverse of t h e p h o s p h o r y l a t i n g o x i d o r e d u c t i o n r e a c t i o n c a t a l y s e d b y g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e requires D P N ÷, as also do some o t h e r r e a c t i o n s c a t a l y s e d b y this enzyme, viz. the aIsenolysis of a c y l p h o s p h a t e s 3, t h e h y d r o l y s i s of a c e t y l p h o s p h a t e 4, a n d t h e 3 ~ P i - a c e t y l p h o s p h a t e e x c h a n g e reaction 5. I
I
0.75
20
0.7C
~0.6~
0.6(
0.5!
0
5
10
15
Time (rain)
Fig. i. DPN + requirement for oxidation of DPNH by acetyl phosphate catalysed by glyceraldehydephosphate dehydrogenase. Reaction mixture contained o. 15 M Tris-HC1 buffer (pH 7.4), o.122 mM DPNH, 4 mM dilithium acetyl phosphate, 1.8 mg rabbit-muscle glyceraldehydephosphate dehydrogenase (prepared in the presence of I mM EDTA) 3, in a volume of 2. 5 ml. The reference cell contained all additions except the DPNH. The reaction was begun by addition of DPNH. A, not treated with DPN + nucleosida~se; B, the enzyme and the DPNH solutions were previously incubated with 12oo units 1 DPN + nucleosida~e for 2o min, after 5 and 8 min, 2.5/*moles and 20/~moles DPN +, respectively, were added. These findings s u p p o r t a m e c h a n i s m of action of this e n z y m e i n v o l v i n g two a d j a c e n t D P N molecules, as suggested b y VAN EYS et al. 2 for t h e a l c o h o l - t y p e d e h y d r o g e n a s e s , a l t h o u g h these workers specifically e x c l u d e d g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e from t h e i r m e c h a n i s m . S t u d i e s of t h e course of t h e r e d u c t i o n of D P N + b y g l y c e r a l d e h y d e , a s u b s t r a t e which r e a c t s m u c h more slowly t h a n t h e physiological s u b s t r a t e b u t which can be used to s t u d y t h e e n z y m e in "slow m o t i o n ''8, has given evidence for t h e f o r m a t i o n of two D P N c o m p o u n d s , different from D P N + or D P N H . Fig. 2 shows t h a t , a f t e r 7 min, when t h e a b s o r b a n c y at 340 m/z has a l m o s t reached a c o n s t a n t value, t h e a m o u n t of D P N H found in alkali e x t r a c t s of t h e reaction m i x t u r e was o n l y a b o u t one half of t h e a m o u n t of D P N + which d i s a p p e a r e d , as m e a s u r e d on acid e x t r a c t s of t h e reaction m i x t u r e . However, the increased a b s o r p t i o n at 340 m/z, c a l c u l a t e d as D P N H , agreed fairly closely w i t h t h e a m o u n t of D P N + which d i s a p p e a r e d . I t a p p e a r s , then, t h a t a c o m p o u n d a b s o r b i n g a t 340 m/z, which is n o t D P N H , is formed a t t h e b e g i n n i n g of t h e reaction. Over t h e following 35 min, this c o m p o u n d is l a r g e l y c o n v e r t e d to D P N H , w i t h l i t t l e f u r t h e r change in t h e a b s o r p t i o n a t 34o m/z. I n Biochim. Biophys. Acta, 58 (I962) 380-383
382
PRELIMINARY NOTES
addition to this new 34o-mtz compound and D P N H , the alkali extracts contain a compound which analyses like DPN + in the enzymic assay, but which cannot be DPN+ since the latter is completely destroyed under the conditions used to prepare the alkali extract. This alkaline-stable DPN + compound yields DPN +, in less than
(
.
o 0
15 Time (min) 30
45
Fig. 2. ] ' h e f o r m a t i o n of D P N compounds d u n n g the reaction between glyceraldehyde and D P N +, c a t a l y s e d b y g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e . T h e r e a c t i o n m i x t u r e c o n t a i n e d in a t o t a l v o l u m e of IO ml, i m m o l e N a 2 H P O 4, i m m o l e T r i s - H C l buffer, 2 5 / * m o l e s DL-glyceraldehyde, 5.04 /*moles D P N +, IO.8 m g r a b b i t - m u s c l e g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e , c o n t a i n i n g o . 1 9 4 / * m o l e b o u n d D P N +. T h e final p H w a s 9.o. T h e r e a c t i o n w a s b e g u n b y a d d i t i o n of glycera l d e h y d e . A t v a r i o u s t i m e i n t e r v a l s , acid a n d alkali e x t r a c t s of s a m p l e s were m a d e b y t h e p r o c e d u r e of PURVtS7, a n d t h e D P N + a n d D P N H c o n t e n t s of t h e e x t r a c t s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h alcohol d e h y d r o g e n a s e a n d alcohol or a c e t a l d e h y d e . - - A D P N +, d i s a p p e a r a n c e of D P N + m e a s u r e d in acid e x t r a c t s ; +AA340mg, c a l c u l a t e d as D P N H f r o m t h e increase of a b s o r b a n c y a t 34 ° m/z of t h e r e a c t i o n m i x t u r e i m m e d i a t e l y before t a k i n g t h e s a m p l e s for p r e p a r a t i o n of t h e acid a n d alkali e x t r a c t s ; + A D P N H , a m o u n t of D P N H f o u n d in alkali e x t r a c t s ; alkali-stable D P N +, a m o u n t of D P N + f o u n d in alkali e x t r a c t (note t h a t s o m e alkali-stable D P N + w a s f o u n d a t zero t i m e : t h i s is d e r i v e d f r o m t h e e n z y m e ) ; A34o-m/* c o m p o u n d , AA34o my (calc. as D P N H ) minus A D P N H .
lOO% yield, during preparation of the acid extract. The compound absorbing at 34o mF, like D P N H itself, is broken down in acid to a compound which does not estimate as DPN +. No increase of absorption at 34 ° mF was obtained in the absence of enzyme. The experiment shown in Fig. 2 was carried out with catalytic amounts of enzyme and DPN + added in an amount about 25 times greater than the amount of DPN + in the enzyme preparation. Table I shows that ~ather similar results were obtained when only the DPN + of the enzyme preparation was studied. In this case, however, the conversion of the 34o-m~ compound to D P N H shown in Fig. 2 was not observed and - - A D P N + exceeded AA34 o my' More of the 34o-mF compound was found in the presence of phosphate than in the presence of arsenate, but even with arsenate all the enzyme-bound DPN+ was not reduced to D P N H or converted to the 34o-m/z compound. This is to be expected on the basis of a VAN EYS type of mechanism in which one DPN + molecule binds substrate, thereby facilitating the reduction of a second DPN+ molecule without itself becoming reduced. No 34o-m~ compound was Biochim. Biophys. Acta, 58 (1962) 38o-383
PRELIMINARY NOTES
383
TABLE I FORMATION
OF DPN
DPN +
DURING REACTION BETWEEN GLYCERALDEHYDE A N D GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE
COMPOUNDS
BOUND
TO
Phosphate experiment: o.I M I~Ia2HPO 4, 15 m M EDTA, 0.3 m M DL-glyceraldehyde, 46. 5 m g rabbit-muscle glyceraldehydephosphate dehydrogenase containing 47 ° m/~moles bound DPN+; p H 8.9; reaction volume, 7-5 ml. - A r s e n a t e experiment: o.i M Tris-HC1 buffer, o.8 m M EDTA, o.o 4 M NasHAsO4, 2 m M DL-glyceraldehyde, 25.9 m g rabbit-muscle glyceraldehydephosphate dehydrogenase containing 238 m/~moles bound DPN+; p H 9.o; reaction volume, 2. 5 ml. The reactions were started b y addition of glyceraldehyde, and followed spectrophotometrically a t 34 ° m/~. Samples were withdrawn a t various times and D P N + a n d D P N H determined on acid and alkali extracts. Values given are in m/zmoles. P ho sphage T i m e (rain)
D PN+ --zJDPN + ADPNH
AA34o m~ A34o-m/* compound Alkali-stable D P N +
o
xo
470 o o
o o 21
2 IO 260 115 189 74 78
Arsenate ao
163 307 -250 -19
30
o
zo
20
137 333 18I 253 72 i8
238 o o o o o
---139 ---
72 166 116 I41 25 o
f o u n d w h e n T r i s o r p y r o p h o s p h a t e b u f f e r s w e r e u s e d i n s t e a d of p h o s p h a t e o r a r s e n a t e . T h a t t h e d i s c r e p a n c i e s b e t w e e n - - A D P N + o r AA34 o mu a n d A D P N H a r e n o t c a u s e d b y a n a l y t i c a l a r t e f a c t s is s h o w n b y : (I) a d d e d D P N + w a s r e c o v e r e d i n t h e a c i d extract and added DPNH in the alkaline extract in more than 9° % yield (no alkalis t a b l e D P N + w a s f o u n d ) ; (2) t h e r e w a s l i t t l e d i s c r e p a n c y a t t h e e n d o f t h e r e a c t i o n i n F i g . 2; (3) t h e r e w a s a l s o n o d i s c r e p a n c y i n e x p e r i m e n t s w i t h e n z y m e - b o u n d D P N +, w h e n "Iris o r p y r o p h o s p h a t e b u f f e r s w e r e u s e d , o r h i g h c o n c e n t r a t i o n s o f g l y c e r a l d e h y d e w e r e u s e d i n p h o s p h a t e b u f f e r ; (4) w h e n t h e b o u n d D P N + w a s reduced by ethanol and yeast alcohol dehydrogenase, there was no discrepancy b e t w e e n t h e AAv, o ~ a n d t h e D P N H f o u n d i n t h e a l k a l i n e e x t r a c 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 a m o u n t of D P N + d i s a p p e a r i n g a n d t h e a m o u n t o f D P N H a p p e a r i n g d u r i n g t h e a c t i o n of g l y c e r a l d e h y d e p h o s p h a t e d e h y d r o g e n a s e r e c a l l s t h e o b s e r v a t i o n s of PURVIS s o n t h e a d d i t i o n of s u b s t r a t e t o r a t - l i v e r m i t o chondria. W e w i s h t o t h a n k P r o f e s s o r E . C. SLATER f o r h i s i n t e r e s t a n d h e l p f u l c r i t i c i s m a n d Miss G. HEDGES ABRAHAMSZ f o r h e r s k i l l f u l t e c h n i c a l a s s i s t a n c e . T h i s w o r k w a s s u p p o r t e d i n p a r t b y a g r a n t f r o m t h e Life I n s u r a n c e M e d i c a l R e s e a r c h F u n d .
Laboratory of Physiological Chemistry, University of Amsterdam, Amsterdam (The Netherlands)
A . G . HILVERS J.H.M. WEENEN
1 A. G. HILVERS, Biochim. Biophys. Acta, 54 (1961) 186. 2 j . VAN EYS, A. SAN PIETRO AND N. O. KAPLAN, Science, 127 (I958) 1443. a E. RACKER AND I. KRIMSNY, J. Biol. Chem., I98 {1952) 721, 731. 4 j . HAETING, in W. D. MCELROY AND ]3. GLASS, The Mechanism of Enzyme Action, The Johns Hopkins Press, Baltimore, 1954, p. 536. 5 j . HARTING AND S. F. VELICK, J . Biol. Chem., 207 (1954) 867. s E. RACKER AND I. KRIMSKY, Federation Proc., 17 (1958) 1135. 7 j . L. PUEVIS, Biochim. Biophys. Acta, 38 (I96o) 4358 j . L. PUEVIS, Nature, 182 (1958) 711. Received January
2 9 t h , 1962
Biochim. Biophys. Acta, 58 (1962) 380--383