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Isolation and identification of β-mercaptopyruvate desulfurase
VOL. 27 (z958)
PRELIMINARY NOTES
659
It is i n t e r e s t i n g to n o t e t h a t t h e presence of a c t i v i t y in t h e m i c r o s o m a l fraction w i t h r e q u i r e m e n t of a r e d u c e d c o e n z y m e is identical w i t h s y s t e m s described for a d r e n a l h y d r o x y l a t i o n 5. T h e h i g h yields o b t a i n e d are c o n s i s t e n t w i t h a q u a n t i t a t i v e l y significant role for t h e reaction. D e t a i l e d s t u d i e s of t h e m e c h a n i s m s i n v o l v e d are n o w in progress. T h e a u t h o r w i s h e s to e x p r e s s his a p p r e c i a t i o n to Miss ANN FOLEY for t e c h n i c a l assistance.
Research Laboratories ot the Boston Lying-in Hospital and the Department o/ Obstetrics, Harvard Medical School, Boston, Mass. (U.S.A.)
KENNETH J. RYA~r
i A. S. MEYER, Biochim. Biophys. Acta, 1 7 (1955) 441. 2 13. BAGGETT, L, L. ENGEL, K. SAVARD AND R. I. DORFMAN, J, Biol. Chem., 221 (1956) 931. a H. H. WoTIZ, J. W . DAvis, H. M. LEMON AND M. GUT, J. Biol. Chem., 222 (1956) 487 • 4 S. SOLOMON, R. VANDE WIELE ANr~ ~. LIEBERMAN, J. Am. Chem. Soe., 78 (1956) 5453. K. J. RYAN AND L. L. ENGEL, J. Biol. Chem., 225 (1957) lO3. s M. L. SWEAT, Anal. Chem., 26 (1954) 773. 7 L. L. ENGEL, W . R. SLAUNWHITE, P. CARTER AND I. T. NATHANSON, J. Biol. Chem., 185 (195o} 255. Received N o v e m b e r iSth, 1957
Isolation and identification of p-mercaptopyruvate desulfurase* Earlier e x p e r i m e n t s of MEISTER et al. 1 d e m o n s t r a t e d t h a t liver e x t r a c t s d e c o m p o s e f l - m e r c a p t o p y r u v a t e to s u l f u r a n d p y r u v a t e . More r e c e n t l y S6RBO2 r e p o r t e d t h a t h o m o g e n a t e s of r a t liver a n d k i d n e y as well as blood cells c a t a l y s e t h e t r a n s f e r of sulfur f r o m f l - m e r c a p t o p y r u v a t e to sulfite a n d sulfinates. T h e s e o b s e r v a t i o n s s u g g e s t t h a t t h e r e are a t least t w o t y p e s of e n z y m e s w h i c h a t t a c k f i - m e r c a p t o p y r u v a t e a n d r e s u l t in either " d e s u l f u r a t i o n " or " t r a n s s u l f u r a t i o n " . I n t h e course of o u r s t u d i e s dealing w i t h e n z y m i c r e a c t i o n s of c y s t e i n e a n d f l - m e r c a p t o pyruvateS,4,5, 6 we h a v e isolated a p r o t e i n f r o m r a t liver w h i c h in t h e a b s e n c e of a sulfur a c c e p t o r cleaves f l - m e r c a p t o p y r u v a t e to sulfur a n d p y r u v a t e . I n a g r e e m e n t w i t h t h e o b s e r v a t i o n s o f MEISTER et al. 1 we find t h a t f l - m e r c a p t o e t h a n o l a c t i v a t e s t h i s reaction. Sulfite in t h e a b s e n c e of m e r c a p t o e t h a n o l also i n c r e a s e s t h e r a t e of e n z y m e - c a t a l y z e d p y r u v a t e f o r m a t i o n f r o m f l - m e r c a p t o p y r u v a t e . I n c o n f i r m a t i o n of S6RBO'S r e s u l t s ~ we find t h a t sulfite is c o n v e r t e d to t h i o s u l f a t e in t h e course of t h e l a t t e r reaction. U l t r a c e n t r i f u g a l a n d electrophoretic a n a l y s e s revealed t h a t o u r purified e n z y m e is a t l e a s t 85 to 95 % h o m o g e n e o u s . T h u s , it a p p e a r s t h a t d e s u l f u r a s e a n d t r a n s s u l f u r a s e activities are associa t e d w i t h a single protein. T h e course of r e a c t i o n is d e t e r m i n e d b y e x p e r i m e n t a l c o n d i t i o n s (e.g presence of s u l f u r acceptors). C e r t a i n p h y s i c a l p r o p e r t i e s of t h i s e n z y m e are: isoelectric point, 7.4; $20, 2.7; D, 5.2.io-6cm2sec-X; mol.wt. (from S,0 a n d D), 35,ooo-4o,ooo; a b s o r p t i o n m a x i m a , 28o m/~ a n d 415 ml~, E28o ml,]E415 mu, 15.7; E tl s r ~ , 56; S content, 4 a t o m s / m o l e . S p e c t r o g r a p h i c a n a l y s e s revealed copper as t h e single metallic c o n s t i t u e n t of t h i s protein. T h e p r o t e i n in its purified f o r m easily loses copper w i t h c o n c o m i t a n t loss of e n z y m i c a c t i v i t y . Q u a n t i t a t i v e copper a n a l y s e s are for t h i s r e a s o n u n c e r t a i n . I t is e s t i m a t e d t h a t t w o a t o m s of coppez m a y be a s s o c i a t e d w i t h one molecule of e n z y m e . P r e l i m i n a r y a n a l y s e s indicate t h a t t w o s u l f h y d r y l g r o u p s are p r e s e n t p e r e n z y m e molecule as m e a s u r e d b y t h e s p e c t r o p h o t o m e t r i c m e t h o d of BOYER 7. F u r t h e r biochemical p r o p e r t i e s of t h i s e n z y m e as well as m e t h o d s for its isolation will be p u b l i s h e d in detail.
Department o] Pharmacology and Experimental Therapeutics, The University o] Cali]ornia School ot Medicine, San Francisco, Cali]. (U.S.A.)
ERNEST KUN * * DEAN W. FANSHIER
1 A. MEISTER, P. E. FRASER AND S. V. TICE: J. Biol. Chem., 206 (1954) 56I, z 13. S6RBO, Biochim. Biophys. Acta, 24 (1957) 324 • s E. KUN AND J. L. BRAOIN, JR., Biochim. Biophys. Acta, i1 (1953) 312. 4 E. KUN, J. L. 13RADIN, JR. AND J. M. DECHARY, Biochim. Biophys. Acta, 19 (1956) 153. s E. KUN, Biochim. Biophys. Acta, 25 (1957) I35, 6 W. D. KUMLER AND E, KUN, Bioehim. Biophys. Acta, 27 (1958) 464 • ? P. D. 13OYER, J. Am. Chem. Soc., 76 (1954) 4331. • S u p p o r t e d b y g r a n t s of t h e U.S. P u b l i c H e a l t h Service ( H e a r t H-2897 a n d C a n c e r C-32I I). "" E s t a b l i s h e d I n v e s t i g a t o r of t h e A m e r i c a n H e a r t Association, Inc. R e c e i v e d D e c e m b e r i 5 t h , 1957
PRELIMINARY NOTES
659
It is i n t e r e s t i n g to n o t e t h a t t h e presence of a c t i v i t y in t h e m i c r o s o m a l fraction w i t h r e q u i r e m e n t of a r e d u c e d c o e n z y m e is identical w i t h s y s t e m s described for a d r e n a l h y d r o x y l a t i o n 5. T h e h i g h yields o b t a i n e d are c o n s i s t e n t w i t h a q u a n t i t a t i v e l y significant role for t h e reaction. D e t a i l e d s t u d i e s of t h e m e c h a n i s m s i n v o l v e d are n o w in progress. T h e a u t h o r w i s h e s to e x p r e s s his a p p r e c i a t i o n to Miss ANN FOLEY for t e c h n i c a l assistance.
Research Laboratories ot the Boston Lying-in Hospital and the Department o/ Obstetrics, Harvard Medical School, Boston, Mass. (U.S.A.)
KENNETH J. RYA~r
i A. S. MEYER, Biochim. Biophys. Acta, 1 7 (1955) 441. 2 13. BAGGETT, L, L. ENGEL, K. SAVARD AND R. I. DORFMAN, J, Biol. Chem., 221 (1956) 931. a H. H. WoTIZ, J. W . DAvis, H. M. LEMON AND M. GUT, J. Biol. Chem., 222 (1956) 487 • 4 S. SOLOMON, R. VANDE WIELE ANr~ ~. LIEBERMAN, J. Am. Chem. Soe., 78 (1956) 5453. K. J. RYAN AND L. L. ENGEL, J. Biol. Chem., 225 (1957) lO3. s M. L. SWEAT, Anal. Chem., 26 (1954) 773. 7 L. L. ENGEL, W . R. SLAUNWHITE, P. CARTER AND I. T. NATHANSON, J. Biol. Chem., 185 (195o} 255. Received N o v e m b e r iSth, 1957
Isolation and identification of p-mercaptopyruvate desulfurase* Earlier e x p e r i m e n t s of MEISTER et al. 1 d e m o n s t r a t e d t h a t liver e x t r a c t s d e c o m p o s e f l - m e r c a p t o p y r u v a t e to s u l f u r a n d p y r u v a t e . More r e c e n t l y S6RBO2 r e p o r t e d t h a t h o m o g e n a t e s of r a t liver a n d k i d n e y as well as blood cells c a t a l y s e t h e t r a n s f e r of sulfur f r o m f l - m e r c a p t o p y r u v a t e to sulfite a n d sulfinates. T h e s e o b s e r v a t i o n s s u g g e s t t h a t t h e r e are a t least t w o t y p e s of e n z y m e s w h i c h a t t a c k f i - m e r c a p t o p y r u v a t e a n d r e s u l t in either " d e s u l f u r a t i o n " or " t r a n s s u l f u r a t i o n " . I n t h e course of o u r s t u d i e s dealing w i t h e n z y m i c r e a c t i o n s of c y s t e i n e a n d f l - m e r c a p t o pyruvateS,4,5, 6 we h a v e isolated a p r o t e i n f r o m r a t liver w h i c h in t h e a b s e n c e of a sulfur a c c e p t o r cleaves f l - m e r c a p t o p y r u v a t e to sulfur a n d p y r u v a t e . I n a g r e e m e n t w i t h t h e o b s e r v a t i o n s o f MEISTER et al. 1 we find t h a t f l - m e r c a p t o e t h a n o l a c t i v a t e s t h i s reaction. Sulfite in t h e a b s e n c e of m e r c a p t o e t h a n o l also i n c r e a s e s t h e r a t e of e n z y m e - c a t a l y z e d p y r u v a t e f o r m a t i o n f r o m f l - m e r c a p t o p y r u v a t e . I n c o n f i r m a t i o n of S6RBO'S r e s u l t s ~ we find t h a t sulfite is c o n v e r t e d to t h i o s u l f a t e in t h e course of t h e l a t t e r reaction. U l t r a c e n t r i f u g a l a n d electrophoretic a n a l y s e s revealed t h a t o u r purified e n z y m e is a t l e a s t 85 to 95 % h o m o g e n e o u s . T h u s , it a p p e a r s t h a t d e s u l f u r a s e a n d t r a n s s u l f u r a s e activities are associa t e d w i t h a single protein. T h e course of r e a c t i o n is d e t e r m i n e d b y e x p e r i m e n t a l c o n d i t i o n s (e.g presence of s u l f u r acceptors). C e r t a i n p h y s i c a l p r o p e r t i e s of t h i s e n z y m e are: isoelectric point, 7.4; $20, 2.7; D, 5.2.io-6cm2sec-X; mol.wt. (from S,0 a n d D), 35,ooo-4o,ooo; a b s o r p t i o n m a x i m a , 28o m/~ a n d 415 ml~, E28o ml,]E415 mu, 15.7; E tl s r ~ , 56; S content, 4 a t o m s / m o l e . S p e c t r o g r a p h i c a n a l y s e s revealed copper as t h e single metallic c o n s t i t u e n t of t h i s protein. T h e p r o t e i n in its purified f o r m easily loses copper w i t h c o n c o m i t a n t loss of e n z y m i c a c t i v i t y . Q u a n t i t a t i v e copper a n a l y s e s are for t h i s r e a s o n u n c e r t a i n . I t is e s t i m a t e d t h a t t w o a t o m s of coppez m a y be a s s o c i a t e d w i t h one molecule of e n z y m e . P r e l i m i n a r y a n a l y s e s indicate t h a t t w o s u l f h y d r y l g r o u p s are p r e s e n t p e r e n z y m e molecule as m e a s u r e d b y t h e s p e c t r o p h o t o m e t r i c m e t h o d of BOYER 7. F u r t h e r biochemical p r o p e r t i e s of t h i s e n z y m e as well as m e t h o d s for its isolation will be p u b l i s h e d in detail.
Department o] Pharmacology and Experimental Therapeutics, The University o] Cali]ornia School ot Medicine, San Francisco, Cali]. (U.S.A.)
ERNEST KUN * * DEAN W. FANSHIER
1 A. MEISTER, P. E. FRASER AND S. V. TICE: J. Biol. Chem., 206 (1954) 56I, z 13. S6RBO, Biochim. Biophys. Acta, 24 (1957) 324 • s E. KUN AND J. L. BRAOIN, JR., Biochim. Biophys. Acta, i1 (1953) 312. 4 E. KUN, J. L. 13RADIN, JR. AND J. M. DECHARY, Biochim. Biophys. Acta, 19 (1956) 153. s E. KUN, Biochim. Biophys. Acta, 25 (1957) I35, 6 W. D. KUMLER AND E, KUN, Bioehim. Biophys. Acta, 27 (1958) 464 • ? P. D. 13OYER, J. Am. Chem. Soc., 76 (1954) 4331. • S u p p o r t e d b y g r a n t s of t h e U.S. P u b l i c H e a l t h Service ( H e a r t H-2897 a n d C a n c e r C-32I I). "" E s t a b l i s h e d I n v e s t i g a t o r of t h e A m e r i c a n H e a r t Association, Inc. R e c e i v e d D e c e m b e r i 5 t h , 1957