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The dissociation of glutamic dehydrogenase by reduced diphosphopyridine nucleotide (DPNH)
voL. 27 (z958)
PRELIMINARY NOTES
43I
A l t h o u g h c r o s s - r e a c t i v i t y of c h y m o t r y p s i n a n d t r y p s i n to c e r t a i n s u b s t r a t e s is n o t w i t h o u t parallel, it w a s n o t a n t i c i p a t e d t h a t m # g a m o u n t s of t r y p s i n w o u l d necessarily p r o d u c e a r a p i d h y d r o l y s i s of C T p N P . H o w e v e r , t h e r a t e c u r v e o b t a i n e d w i t h t r y p s i n w a s c o m p a r a b l e to t h a t o b s e r v e d w i t h c h y m o t r y p s i n a n d velocity w a s p r o p o r t i o n a l to t h e t r y p s i n c o n c e n t r a t i o n (Fig. i, B). T h e m a x i m u m r a t e of h y d r o l y s i s occurred a t a b o u t p H 8. As w a s f o u n d for c h y m o t r y p s i n , t r y p t i c a c t i v i t y t o w a r d C T p N P w a s c o m p l e t e l y i n h i b i t e d b y diiso p r o p y l p h o s p h o r o f l u o r i d a t e . T h u s , t h e u s e of C T p N P p e r m i t s direct a n d rapid m e a s u r e m e n t s of reaction r a t e s utilizing q u a n t i t i e s of c h y m o t r y p s i n as low as 3 m p g / m l a n d a m o u n t s of t r y p s i n as low as 7 m/zg/ml. T h i s w o r k h a s been s u p p o r t e d in p a r t b y research g r a n t s A-727 a n d R G 4624 f r o m t h e U n i t e d S t a t e s Public H e a l t h Service.
Biochemistry Department, School o/Medicine, University o[ Pittsburgh, Pittsburgh, Pa. (U.S.A.)
CHARLES J. MARTIN JULIUS GOLUBOW A. E. AXELROD
1 C. J. MARTIN AND A. E. AXELROO, Biochim. Biophys. Acta, 27 (1958) 52. C. J. MARTIN, Federation Proc., 15 (1956) 455. s W. J. DREYER, R. D. WADE, AND H. ~EURATH, Arch: Biochem. Biophys., 59 (1955) 145. 4 N. M. GREEN AND H. NEURATH, J. Biol. Chem., 204 (1953) 379. N. M. GREEN AND H. NEURATH, in H. NEURATH AND I~. BAILEY', The Proteins, Vol. II, P a r t B, A c a d e m i c Press, Inc., N e w York, 1954, P. 1155. R e c e i v e d O c t o b e r I 4 t h , 1957
The dissociation of glutamic dehydrogenase by reduced diphosphopyridine nucleotide (DPN H) I n 1952, OLSON AND A N F I N S E N 1 s h o w e d t h a t c r y s t a l l i n e g l u t a m i c d e h y d r o g e n a s e e x h i b i t e d a n a n o m a l o u s s e d i m e n t a t i o n b e h a v i o r in t h e u l t r a c e n t r i f u g e . T h e a u t h o r s a t t r i b u t e d t h i s b e h a v i o r to e i t h e r dissociation or u n f o l d i n g of t h e e n z y m e a t low c o n c e n t r a t i o n s . I t h a s n o w been s h o w n t h a t d i s s o c i a t i o n of t h e e n z y m e m a y be o b t a i n e d b y t h e a d d i t i o n of a s u b s t r a t e for t h e e n z y m e , DPNH. C r y s t a l l i n e g l u t a m i c d e h y d r o g e n a s e w a s p r e p a r e d f r o m beef liver b y t h e m e t h o d of STRECKER ~ a n d r e c r y s t a l l i z e d t w i c e w i t h s a t u r a t e d s o d i u m s u l f a t e a t 5 °1. A t p H 7.4 in o.o52]4 p h o s p h a t e buffer, t h e e n z y m e s h o w s a single p e a k in t h e u l t r a c e n t r i f u g e . I n all e x p e r i m e n t s , t h e Spinco Model E a n a l y t i c u l t r a c e n t r i f u g e w a s used*. T h e p a t t e r n , s h o w n in Fig. ia, h a s a h y p e r s h a r p l e a d i n g b o u n d a r y , a n d is s l i g h t l y s k e w e d on t h e t r a i l i n g edge, s i m i l a r to t h a t o b t a i n e d b y OLSON AND ANFINSEN. T h e a d d i t i o n of D P N H to t h i s e n z y m e s o l u t i o n c a u s e s t h e single p e a k to split into t w o d i s t i n c t p e a k s as s h o w n in Fig. l b . I n t h i s p a r t i c u l a r e x p e r i m e n t , t h e m o l a r r a t i o of D P N H to e n z y m e w a s a p p r o x i m a t e l y 200 : I. I n c r e a s i n g a m o u n t s of D P N H decrease t h e a m o u n t
Fig. I. S e d i m e n t a t i o n p a t t e r n s a t 59,78o r . p . m . (a) alone a f t e r (c) in t h e p r e s e n c e of 5" I o - 3 M approximately
of g l u t a m i c d e h y d r o g e n a s e in o . o 5 M p h o s p h a t e buffer, p H 7.4 660 sec, (b) in t h e p r e s e n c e of I. 3" x o - S M D P N H a f t e r 66o sec, o - p h e n a n t h r o l i n e a f t e r 159o sec. T h e p r o t e i n c o n c e n t r a t i o n w a s 4.5 m g / m l . S e d i m e n t a t i o n is f r o m left to right.
* T h e a u t h o r is i n d e b t e d to Miss CARMELITA LOWRY for v a l u a b l e t e c h n i c a l assistance.
432
PRELIMINARY NOTES
VOL. 27 (1958)
of e n z y m e in the fast p e a k w i t h a corresponding increase in the concentration of t h e slow m o v i n g c o m p o n e n t . The splitting of t h e enzyme w i t h D P N H m a y be completely reversed u p o n r e m o v a l of t h e D P N H b y dialysis. I n addition to the influence of D P N H concentration, t h e p H of the solution a p p e a r s to affect t h e e x t e n t of dissociation in t h e presence of D P N H , a l t h o u g h t h e a m o u n t of dissociation of e n z y m e alone does not a p p e a r to be influenced b y these s a m e p H changes. No splitting of the enzyme is obtained b y using p-chloromercuribenzoate or a-ketoglutarate, a n o t h e r s u b s t r a t e of the enzyme. I n view of t h e fact t h a t Zn has been r e p o r t e d as a c o n s t i t u e n t of glutamic dehydrogenase 3 a n d of the a p p a r e n t i n v o l v e m e n t of Zn in some e n z y m e s which use D P N H as s u b s t r a t e 4, op h e n a n t h r o l i n e w a s added to the enzyme. The s e d i m e n t a t i o n d i a g r a m which resulted is s h o w n in Fig. ic. This e n z y m e h a s a s e d i m e n t a t i o n c o n s t a n t of slightly greater t h a n half of t h e original material a n d as m a y be seen, the p a t t e r n is s y m m e t r i c a l as c o n t r a s t e d to Fig, ia. The concent r a t i o n of the o-phenanthroline used completely inhibited t h e enzymic reaction. OLSON AND ANFINSEN h a v e r e p o r t e d a molecular weight, from s e d i m e n t a t i o n and diffusion studies, for the e n z y m e of one million. D e t e r m i n a t i o n s of the molecular weight of o-phenanthrolinet r e a t e d e n z y m e were m a d e b y the m e t h o d of BALDWIN5. These d e t e r m i n a t i o n s indicated t h a t the p r o t e i n m a y exist as a p o l y m e r of four e n z y m e subunits. The t e t r a m e r m a y be split either into dimers or m o n o m e r s depending on the c o n c e n t r a t i o n of either o-phenanthroline or D P N H . As a result of splitting, the diffusion c o n s t a n t increases and the axial ratio decreases. The splitting caused b y o-phenanthroline, a p o t e n t Zn binding reagent, is evidence for the role of Zn as a n i m p o r t a n t c o m p o n e n t in the binding of the s u b u n i t s . T h a t b o t h o-phenanthroline and D P N H cause dissociation of the e n z y m e m a y indicate the i m p o r t a n c e of Zn in the binding of D P N H . So far as is known, glutamic dehydrogenase is the only enzyme which is affected in such a w a y b y one of its s u b s t r a t e s . The e n z y m e p h o s p h o r y l a s e m a y also be dissociated into m o n o m e r s , b u t only in the presence of p-chloromercuribenzoate e. I n this case, the i n v o l v e m e n t of sulfhydryl g r o u p s is indicated in t h e c o m b i n a t i o n of the four p h o s p h o r y l a s e subunits. Such a change in the molecular size and shape of glutamic dehydrogenase b y a s u b s t r a t e for the e n z y m e offers a m e t h o d b y which f u r t h e r studies of the m e c h a n i s m of the reaction m a y be made. Such investigations of the molecular and kinetic properties of the enzyme are u n d e r w a y in this laboratory. CARL FRIEDEN
Department o/Biological Chemistry, Washington University School o/Medicine, St. Louis, Mo. (U.S.A.) 1 j . A. 2 H. J. 3 B. L. 4 B. L. 5 R. L. 6 N. B.
OLSON AND C. B. ANFINSEN, J. Biol. Chem., 197 (1952) 67. STRECKER, Arch. Biochem. Biophys., 46 (1953) 128. VALLEE, S. J. ADELSTEIN AND J. A. OLSON, J. Am. Chem. Soc., 77 (1955) 5196. VALLEE, Advances in Protein Chem., IO (1955) 317 • BALDWIN, Biochem. J., 65 (1957) 503. MADSEN AND C. F. CORI~ J. Biol. Chem., 223 (1956) lO55. Received October I5th, 1957
Addendum (added in proof, D e c e m b e r I I t h , 1957) The degree of splitting of the e n z y m e b y D P N H , b u t not by o-phenanthroline, has been found to be d e p e n d e n t on the specific activity of t h e enzyme.
A study of the synthesis of catalase in liver of tumor-bearing mice by means of radioactive iron I t is well k n o w n t h a t t h e liver catalase activity is s h a r p l y lowered in t u m o r - b e a r i n g animals. I t has n o t y e t been ascertained, however, w h e t h e r this decrease is due to a n actual decrease in the e n z y m e c o n c e n t r a t i o n or to t h e presence of some inhibitor w h i c h interferes in its d e t e r m i n a t i o n 1, According to PRICE AND GREENFIELD 2, less catalase m a y be p r e p a r e d f r o m livers of t u m o r - b e a r i n g r a t s t h a n f r o m t h e h e a l t h y control animals. However, their technique, a l t h o u g h accurate, is a p r e p a r a t i v e one, and therefore involves t h e possibility of losses; f u r t h e r m o r e , it clearly c a n n o t distinguish b e t w e e n the catalase already p r e s e n t at t h e m o m e n t of t u m o r i m p l a n t a t i o n a n d t h a t formed thereafter. I n an a t t e m p t to give an a n s w e r to this problem, we studied b y m e a n s of radioactive iron the s y n t h e s i s of liver catalase after t u m o r i m p l a n t a t i o n . Male albino mice of an inbred strain (ALAL) were i m p l a n t e d w i t h S.i8o and divided into t w o groups. Respectively 5 a n d io days after the implantation, the t u m o r - b e a r i n g mice and the
PRELIMINARY NOTES
43I
A l t h o u g h c r o s s - r e a c t i v i t y of c h y m o t r y p s i n a n d t r y p s i n to c e r t a i n s u b s t r a t e s is n o t w i t h o u t parallel, it w a s n o t a n t i c i p a t e d t h a t m # g a m o u n t s of t r y p s i n w o u l d necessarily p r o d u c e a r a p i d h y d r o l y s i s of C T p N P . H o w e v e r , t h e r a t e c u r v e o b t a i n e d w i t h t r y p s i n w a s c o m p a r a b l e to t h a t o b s e r v e d w i t h c h y m o t r y p s i n a n d velocity w a s p r o p o r t i o n a l to t h e t r y p s i n c o n c e n t r a t i o n (Fig. i, B). T h e m a x i m u m r a t e of h y d r o l y s i s occurred a t a b o u t p H 8. As w a s f o u n d for c h y m o t r y p s i n , t r y p t i c a c t i v i t y t o w a r d C T p N P w a s c o m p l e t e l y i n h i b i t e d b y diiso p r o p y l p h o s p h o r o f l u o r i d a t e . T h u s , t h e u s e of C T p N P p e r m i t s direct a n d rapid m e a s u r e m e n t s of reaction r a t e s utilizing q u a n t i t i e s of c h y m o t r y p s i n as low as 3 m p g / m l a n d a m o u n t s of t r y p s i n as low as 7 m/zg/ml. T h i s w o r k h a s been s u p p o r t e d in p a r t b y research g r a n t s A-727 a n d R G 4624 f r o m t h e U n i t e d S t a t e s Public H e a l t h Service.
Biochemistry Department, School o/Medicine, University o[ Pittsburgh, Pittsburgh, Pa. (U.S.A.)
CHARLES J. MARTIN JULIUS GOLUBOW A. E. AXELROD
1 C. J. MARTIN AND A. E. AXELROO, Biochim. Biophys. Acta, 27 (1958) 52. C. J. MARTIN, Federation Proc., 15 (1956) 455. s W. J. DREYER, R. D. WADE, AND H. ~EURATH, Arch: Biochem. Biophys., 59 (1955) 145. 4 N. M. GREEN AND H. NEURATH, J. Biol. Chem., 204 (1953) 379. N. M. GREEN AND H. NEURATH, in H. NEURATH AND I~. BAILEY', The Proteins, Vol. II, P a r t B, A c a d e m i c Press, Inc., N e w York, 1954, P. 1155. R e c e i v e d O c t o b e r I 4 t h , 1957
The dissociation of glutamic dehydrogenase by reduced diphosphopyridine nucleotide (DPN H) I n 1952, OLSON AND A N F I N S E N 1 s h o w e d t h a t c r y s t a l l i n e g l u t a m i c d e h y d r o g e n a s e e x h i b i t e d a n a n o m a l o u s s e d i m e n t a t i o n b e h a v i o r in t h e u l t r a c e n t r i f u g e . T h e a u t h o r s a t t r i b u t e d t h i s b e h a v i o r to e i t h e r dissociation or u n f o l d i n g of t h e e n z y m e a t low c o n c e n t r a t i o n s . I t h a s n o w been s h o w n t h a t d i s s o c i a t i o n of t h e e n z y m e m a y be o b t a i n e d b y t h e a d d i t i o n of a s u b s t r a t e for t h e e n z y m e , DPNH. C r y s t a l l i n e g l u t a m i c d e h y d r o g e n a s e w a s p r e p a r e d f r o m beef liver b y t h e m e t h o d of STRECKER ~ a n d r e c r y s t a l l i z e d t w i c e w i t h s a t u r a t e d s o d i u m s u l f a t e a t 5 °1. A t p H 7.4 in o.o52]4 p h o s p h a t e buffer, t h e e n z y m e s h o w s a single p e a k in t h e u l t r a c e n t r i f u g e . I n all e x p e r i m e n t s , t h e Spinco Model E a n a l y t i c u l t r a c e n t r i f u g e w a s used*. T h e p a t t e r n , s h o w n in Fig. ia, h a s a h y p e r s h a r p l e a d i n g b o u n d a r y , a n d is s l i g h t l y s k e w e d on t h e t r a i l i n g edge, s i m i l a r to t h a t o b t a i n e d b y OLSON AND ANFINSEN. T h e a d d i t i o n of D P N H to t h i s e n z y m e s o l u t i o n c a u s e s t h e single p e a k to split into t w o d i s t i n c t p e a k s as s h o w n in Fig. l b . I n t h i s p a r t i c u l a r e x p e r i m e n t , t h e m o l a r r a t i o of D P N H to e n z y m e w a s a p p r o x i m a t e l y 200 : I. I n c r e a s i n g a m o u n t s of D P N H decrease t h e a m o u n t
Fig. I. S e d i m e n t a t i o n p a t t e r n s a t 59,78o r . p . m . (a) alone a f t e r (c) in t h e p r e s e n c e of 5" I o - 3 M approximately
of g l u t a m i c d e h y d r o g e n a s e in o . o 5 M p h o s p h a t e buffer, p H 7.4 660 sec, (b) in t h e p r e s e n c e of I. 3" x o - S M D P N H a f t e r 66o sec, o - p h e n a n t h r o l i n e a f t e r 159o sec. T h e p r o t e i n c o n c e n t r a t i o n w a s 4.5 m g / m l . S e d i m e n t a t i o n is f r o m left to right.
* T h e a u t h o r is i n d e b t e d to Miss CARMELITA LOWRY for v a l u a b l e t e c h n i c a l assistance.
432
PRELIMINARY NOTES
VOL. 27 (1958)
of e n z y m e in the fast p e a k w i t h a corresponding increase in the concentration of t h e slow m o v i n g c o m p o n e n t . The splitting of t h e enzyme w i t h D P N H m a y be completely reversed u p o n r e m o v a l of t h e D P N H b y dialysis. I n addition to the influence of D P N H concentration, t h e p H of the solution a p p e a r s to affect t h e e x t e n t of dissociation in t h e presence of D P N H , a l t h o u g h t h e a m o u n t of dissociation of e n z y m e alone does not a p p e a r to be influenced b y these s a m e p H changes. No splitting of the enzyme is obtained b y using p-chloromercuribenzoate or a-ketoglutarate, a n o t h e r s u b s t r a t e of the enzyme. I n view of t h e fact t h a t Zn has been r e p o r t e d as a c o n s t i t u e n t of glutamic dehydrogenase 3 a n d of the a p p a r e n t i n v o l v e m e n t of Zn in some e n z y m e s which use D P N H as s u b s t r a t e 4, op h e n a n t h r o l i n e w a s added to the enzyme. The s e d i m e n t a t i o n d i a g r a m which resulted is s h o w n in Fig. ic. This e n z y m e h a s a s e d i m e n t a t i o n c o n s t a n t of slightly greater t h a n half of t h e original material a n d as m a y be seen, the p a t t e r n is s y m m e t r i c a l as c o n t r a s t e d to Fig, ia. The concent r a t i o n of the o-phenanthroline used completely inhibited t h e enzymic reaction. OLSON AND ANFINSEN h a v e r e p o r t e d a molecular weight, from s e d i m e n t a t i o n and diffusion studies, for the e n z y m e of one million. D e t e r m i n a t i o n s of the molecular weight of o-phenanthrolinet r e a t e d e n z y m e were m a d e b y the m e t h o d of BALDWIN5. These d e t e r m i n a t i o n s indicated t h a t the p r o t e i n m a y exist as a p o l y m e r of four e n z y m e subunits. The t e t r a m e r m a y be split either into dimers or m o n o m e r s depending on the c o n c e n t r a t i o n of either o-phenanthroline or D P N H . As a result of splitting, the diffusion c o n s t a n t increases and the axial ratio decreases. The splitting caused b y o-phenanthroline, a p o t e n t Zn binding reagent, is evidence for the role of Zn as a n i m p o r t a n t c o m p o n e n t in the binding of the s u b u n i t s . T h a t b o t h o-phenanthroline and D P N H cause dissociation of the e n z y m e m a y indicate the i m p o r t a n c e of Zn in the binding of D P N H . So far as is known, glutamic dehydrogenase is the only enzyme which is affected in such a w a y b y one of its s u b s t r a t e s . The e n z y m e p h o s p h o r y l a s e m a y also be dissociated into m o n o m e r s , b u t only in the presence of p-chloromercuribenzoate e. I n this case, the i n v o l v e m e n t of sulfhydryl g r o u p s is indicated in t h e c o m b i n a t i o n of the four p h o s p h o r y l a s e subunits. Such a change in the molecular size and shape of glutamic dehydrogenase b y a s u b s t r a t e for the e n z y m e offers a m e t h o d b y which f u r t h e r studies of the m e c h a n i s m of the reaction m a y be made. Such investigations of the molecular and kinetic properties of the enzyme are u n d e r w a y in this laboratory. CARL FRIEDEN
Department o/Biological Chemistry, Washington University School o/Medicine, St. Louis, Mo. (U.S.A.) 1 j . A. 2 H. J. 3 B. L. 4 B. L. 5 R. L. 6 N. B.
OLSON AND C. B. ANFINSEN, J. Biol. Chem., 197 (1952) 67. STRECKER, Arch. Biochem. Biophys., 46 (1953) 128. VALLEE, S. J. ADELSTEIN AND J. A. OLSON, J. Am. Chem. Soc., 77 (1955) 5196. VALLEE, Advances in Protein Chem., IO (1955) 317 • BALDWIN, Biochem. J., 65 (1957) 503. MADSEN AND C. F. CORI~ J. Biol. Chem., 223 (1956) lO55. Received October I5th, 1957
Addendum (added in proof, D e c e m b e r I I t h , 1957) The degree of splitting of the e n z y m e b y D P N H , b u t not by o-phenanthroline, has been found to be d e p e n d e n t on the specific activity of t h e enzyme.
A study of the synthesis of catalase in liver of tumor-bearing mice by means of radioactive iron I t is well k n o w n t h a t t h e liver catalase activity is s h a r p l y lowered in t u m o r - b e a r i n g animals. I t has n o t y e t been ascertained, however, w h e t h e r this decrease is due to a n actual decrease in the e n z y m e c o n c e n t r a t i o n or to t h e presence of some inhibitor w h i c h interferes in its d e t e r m i n a t i o n 1, According to PRICE AND GREENFIELD 2, less catalase m a y be p r e p a r e d f r o m livers of t u m o r - b e a r i n g r a t s t h a n f r o m t h e h e a l t h y control animals. However, their technique, a l t h o u g h accurate, is a p r e p a r a t i v e one, and therefore involves t h e possibility of losses; f u r t h e r m o r e , it clearly c a n n o t distinguish b e t w e e n the catalase already p r e s e n t at t h e m o m e n t of t u m o r i m p l a n t a t i o n a n d t h a t formed thereafter. I n an a t t e m p t to give an a n s w e r to this problem, we studied b y m e a n s of radioactive iron the s y n t h e s i s of liver catalase after t u m o r i m p l a n t a t i o n . Male albino mice of an inbred strain (ALAL) were i m p l a n t e d w i t h S.i8o and divided into t w o groups. Respectively 5 a n d io days after the implantation, the t u m o r - b e a r i n g mice and the