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Affinity Labeling Studies of the ATP Binding Site of Canine Kidney Na,K-ATPase
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19
Affinity Labeling Studies of the ATP Binding Site of Canine Kidney Na,K-ATPase JAMES B. COOPER, CARL.JOHNSON, AND CHARLES G. WINTER Department of Biochemistry University of Arkansas College of Medicine Little Rock, Arkansm
I.
INTRODUCTION
P r e v i o u s work i n this l a b o r a t o r y h a s e s t a b l i s h e d 5'-p-fluorosulfonylbenzoyladenosine (FSEA) a s a n ATPbinding s i t e a f f i n i t y probe f o r t h e c a n i n e kidney N a , K A T P a s e (Cooper and W i n t e r , 1 9 8 0 ) . The enzyme w a s p u r i f i e d by a m o d i f i c a t i o n o f t h e method of J 6 r g e n s e n ( L i a n g and W i n t e r , 1 9 7 6 ) . FSBA and [3H]FSBA were s y n t h e s i z e d a s p r e v i o u s l y d e s c r i b e d (Cooper and W i n t e r , 1 9 8 0 ) . R e a c t i o n of FSBA w i t h N a , K - A T P a s e c a n b e c a r r i e d o u t a t 37OC by a d d i t i o n of t h e compound d i s s o l v e d i n s m a l l amounts of methanol o r d i m e t h y l s u l f o x i d e t o enzyme s u s p e n s i o n s b u f f e r e d a t pH 8 w i t h b a r b i t a l o r pH 8 . 5 w i t h bicine. R e a c t i o n of FSBA w i t h t h e enzyme and s o l v o l y s i s of t h e r e a g e n t o c c u r s i m u l t a n e o u s l y , so t h a t d e p l e t i o n of FSBA i n t h e r e a c t i o n m i x t u r e a f t e r a s i n g l e a d d i t i o n l i m i t s t h e e x t e n t o f r e a c t i o n w i t h t h e enzyme. Repeated a d d i t i o n s of FSBA c a n p r o d u c e e s s e n t i a l l y c o m p l e t e i n h i b i t i o n of enzyme a c t i v i t y . 367
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved.
ISBN 0-12-153319-0
JAMES 6. COOPER et el.
368
11.
RESULTS AND DISCUSSION
R e a c t i o n o f t h e enzyme i n 0 . 1 M K C 1 w i t h FSBA i s s l i g h t l y more r a p i d t h a n i n 0 . 1 M N a C 1 . Inactivation of N a , K - A T P a s e a c t i v i t y by FSBA c a n be p r e v e n t e d by i n c l u s i o n of ATP i n t h e i n c u b a t i o n medium. When 0 . 1 M N a C l i s p r e s e n t , 2.5 pM ATP p r o t e c t s half-maximally a g a i n s t i n a c t i v a t i o n by FSBA. But c a r r y i n g o u t t h e i n c u b a t i o n i n 0 . 1 M K C 1 medium l e a d s t o much l a r g e r ATP r e q u i r e m e n t f o r half-maximal p r o t e c t i o n ( a b o u t 2 0 0 U M ) The a p p a r e n t d i s s o c i a t i o n c o n s t a n t s f o r ATP p r o t e c t i o n a g a i n s t FSBA i n h i b i t i o n t h e r e f o r e a r e t h o s e e x p e c t e d f o r t h e high- and l o w - a f f i n i t y forms of t h e ATP-binding s i t e . A t 1 0 0 pM c o n c e n t r a t i o n s i n 0 . 1 M N a C 1 , b o t h ATP and ADP are e f f e c t i v e p r o t e c t o r s , whereas AMP i s n o t , a f i n d i n g c o n s i s t e n t w i t h t h e known s p e c i f i c i t y of t h e Na,K-ATPase. I n h i b i t i o n o f t h e enzyme by FSBA i s i r r e v e r s i b l e . Addition of p r o t e c t i n g c o n c e n t r a t i o n s of n u c l e o t i d e a f t e r r e a c t i o n w i t h FSBA h a s a l r e a d y o c c u r r e d d o e s n o t r e s t o r e a c t i v i t y but simply prevents f u r t h e r i n a c t i v a t i o n . Study of t h e k i n e t i c s of r e a c t i o n i s c o m p l i c a t e d by s o l v o l y s i s of t h e r e a g e n t . However, measurement o f f l u o r i d e release from FSBA w i t h t h e a i f of a f l u o r i d e s p e c i f i c e l e c t r o d e p e r m i t s m o n i t o r i n g of t h e l o s s of r e a g e n t . Repeated small a d d i t i o n s of FSBA t o m a i n t a i n i t s c o n c e n t r a t i o n c o n s t a n t t o w i t h i n 215% a l l o w s d e t e r m i n a t i o n of t h e c o n c e n t r a t i o n dependence of FSBA i n h i b i t i o n . I n a c t i v a t i o n i s e s s e n t i a l l y l i n e a r w i t h FSBA c o n c e n t r a t i o n t h r o u g h 1 mM, t h e approximate s o l u b i l i t y l i m i t of t h e r e a g e n t . This finding i s c o n s i s t e n t with t h e f a c t t h a t FSBA f a i l s t o compete w i t h ATP f o r hydrol y s i s on t h e h i g h - a f f i n i t y ATP s i t e u s i n g Na-ATP ase a s s a y c o n d i t i o n s . T h i s r e s u l t i n d i c a t e s t h a t t h e appar e n t d i s s o c i a t i o n c o n s t a n t f o r FSBA must exceed 5 mM. To e s t a l i s h t h a t FSBA re a c ts c o v a l e n t l y a t t h e ATPb i n d i n g s i t e , t h e a b i l i t y o f [3H]ADP t o b i n d t o t h a t s i t e a f t e r t r e a t m e n t o f t h e enzyme w i t h FSBA w a s determined. A l o s s of ADP-binding c a p a c i t y i n d i r e c t proport i o n t o loss of N a , K - A T P a s e a c t i v i t y was o b t a i n e d . T h i s f i n d i n g i n d i c a t e s t h a t o c c u p a t i o n o f t h e ATP-binding s i t e by FSBA i r r e v e r s i b l y p r e v e n t s s u b s e q u e n t [ 3H] ADP b i n d i n g . I n a d d i t i o n , t h e r e l a t i v e s t o i c h i o m e t r y of s p e c i f i c (ATP-protectable) [ 3H] FSBA a t t a c h m e n t t o t h e enzyme w a s found t o b e 0.94 k 0 . 2 4 moles bound p e r mole of o u a b a i n bound. An a p p r o x i m a t e l y e q u i v a l e n t amount of n o n s p e c i f i c l a b e l i n g w a s a l s o found, b u t t h i s a t t a c h m e n t of FSBA t o t h e enzyme h a s no e f f e c t on Na,K-ATPase act i v i t y . Most of t h e n o n s p e c i f i c l a b e l i n g and a l l of t h e s p e c i f i c l a b e l i n g i s found i n t h e c a t a l y t i c ( a ) s u b u n i t .
.
STUDIES OF THE ATP BINDING SITE OF CANINE KIDNEY
369
One c u r r e n t view o f N a , K - A T P a s e s t r u c t u r e r e l a t i n g t o i t s f u n c t i o n i s t h a t a s i n g l e a c t i v e s i t e on a + d i m e r i c a , B - u n i t c a n c a t a l y z e a l l a c t i v i t i e s of t h e Na pump w i t h o u t any r e q u i r e m e n t f o r a-a s u b u n i t i n t e r a c t i o n i n a n ~ 1 2 ~ 8t e2 t r a m e r i c u n i t (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1 9 8 1 ) . I f t h i s were t r u e , t h e n i n h i b i t i o n of N a , K - A T P a s e a c t i v i t y by FSBA s h o u l d b l o c k p - n i t r o p h e n y l p h o s p h a t a s e (pNPPase) a c t i v i t y i n e q u a l p r o p o r t i o n , s i n c e t h e l a t t e r a c t i v i t y i s c a t a l y z e d by t h e l o w - a f f i n i t y form o f t h e ATP-binding s i t e ( R o b i n s o n , 1 9 7 6 ) . An a , B - u n i t would need t o c y c l e between t h e s e two forms t o c a t a l y z e c o m p l e t e Na,K-ATPase a c t i v i t y . In a c t u a l i t y , l o s s of N a , K - A T P a s e a c t i v i t y (measured w i t h 5 mM ATP) a f t e r FSBA t r e a t m e n t i s r o u g h l y t w i c e a s ext e n s i v e a s l o s s of K+-phosphatase a c t i v i t y , w h e t h e r t h e t r e a t m e n t i s c a r r i e d o u t i n 0 . 1 M N a + or K+ medium. The same r e s u l t s are o b t a i n e d i f N a + - A T P a s e a c t i v i t y i s measured u s i n g 2 0 pM ATP. T h e r e i s no change i n appar e n t K~ for pNPP €or t h e s u r v i v i n g p h o s p h a t a s e a c t i v i t y . These r e s u l t s imply t h a t t h e f u n c t i o n a l a 2 8 2 pump u n i t s have two s i t e s f o r pNPP h y d r o l y s i s p e r e a c h f u n c t i o n a l ATP s i t e . B l o c k i n g t h e l a t t e r w i t h a n u c l e o t i d e a n a l o g s u c h a s FSBA i n h i b i t s Na-ATPase and Na,K-ATPase a c t i v i t y a l o n g w i t h one of t h e two pNPP s i t e s . The una f f e c t e d s i t e i s u n a b l e t o assume t h e c o n f o r m a t i o n req u i r e d t o p r o d u c e h i g h a f f i n i t y €or n u c l e o t i d e , e v e n i n These r e s u l t s a r e c o n s i s N a + medium c o n t a i n i n g no K + . t e n t w i t h a model o f Na,K-ATPase s t r u c t u r e h a v i n g e i t h e r (1) one pNPPase c a t a l y t i c s i t e on e a c h of t h e two d i f f e r e n t a-subunits i n t h e a282 tetramer, with r e s t r i c t i v e c o n f o r m a t i o n a l i n t e r a c t i o n s between them; or ( 2 ) two pNPPase s i t e s on a s i n g l e - s u b u n i t , w i t h t h e p o s s i b i l i t y t h a t these sites a l t e r n a t e as t h e ATPase c a t a l y t i c s i t e . I n t h e l a t t e r case, t h e o t h e r a - s u b u n i t of t h e ~ 1 2 B 2 t e t r a m e r would b e c a t a l y t i c a l l y i n a c t i v e .
ACKNOWLEDGMENT S u p p o r t e d i n p a r t by N I H g r a n t 5S07RR5350.
REFERENCES
Cooper, J. B . ,
165-174.
a n d W i n t e r , C. G.
(1980).
J . S u p r a n w l . Struct. 1 3 ,
370
JAMES 9. COOPER eta/.
Liang, S. M . , and W i n t e r , C. G ( 1 9 7 6 ) . B i o c h i m . B i o p h y s . A c t a 452, 552-565. Moczydlowski, E . G . , and F o r t e s , P. A . G. (1981). J. B i o l . Chem. 256, 2346-2356. Robinson, J. D. ( 1 9 7 6 ) . B i o c h i m . B i o p h y s . A c t a 429, 1006-1019. Smith, R. L . , Zinn, K . , and C a n t l e y , L. C . (1980). J. B i o l . Chem. 255, 9852-9859.
Affinity Labeling Studies of the ATP Binding Site of Canine Kidney Na,K-ATPase JAMES B. COOPER, CARL.JOHNSON, AND CHARLES G. WINTER Department of Biochemistry University of Arkansas College of Medicine Little Rock, Arkansm
I.
INTRODUCTION
P r e v i o u s work i n this l a b o r a t o r y h a s e s t a b l i s h e d 5'-p-fluorosulfonylbenzoyladenosine (FSEA) a s a n ATPbinding s i t e a f f i n i t y probe f o r t h e c a n i n e kidney N a , K A T P a s e (Cooper and W i n t e r , 1 9 8 0 ) . The enzyme w a s p u r i f i e d by a m o d i f i c a t i o n o f t h e method of J 6 r g e n s e n ( L i a n g and W i n t e r , 1 9 7 6 ) . FSBA and [3H]FSBA were s y n t h e s i z e d a s p r e v i o u s l y d e s c r i b e d (Cooper and W i n t e r , 1 9 8 0 ) . R e a c t i o n of FSBA w i t h N a , K - A T P a s e c a n b e c a r r i e d o u t a t 37OC by a d d i t i o n of t h e compound d i s s o l v e d i n s m a l l amounts of methanol o r d i m e t h y l s u l f o x i d e t o enzyme s u s p e n s i o n s b u f f e r e d a t pH 8 w i t h b a r b i t a l o r pH 8 . 5 w i t h bicine. R e a c t i o n of FSBA w i t h t h e enzyme and s o l v o l y s i s of t h e r e a g e n t o c c u r s i m u l t a n e o u s l y , so t h a t d e p l e t i o n of FSBA i n t h e r e a c t i o n m i x t u r e a f t e r a s i n g l e a d d i t i o n l i m i t s t h e e x t e n t o f r e a c t i o n w i t h t h e enzyme. Repeated a d d i t i o n s of FSBA c a n p r o d u c e e s s e n t i a l l y c o m p l e t e i n h i b i t i o n of enzyme a c t i v i t y . 367
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction inany form reserved.
ISBN 0-12-153319-0
JAMES 6. COOPER et el.
368
11.
RESULTS AND DISCUSSION
R e a c t i o n o f t h e enzyme i n 0 . 1 M K C 1 w i t h FSBA i s s l i g h t l y more r a p i d t h a n i n 0 . 1 M N a C 1 . Inactivation of N a , K - A T P a s e a c t i v i t y by FSBA c a n be p r e v e n t e d by i n c l u s i o n of ATP i n t h e i n c u b a t i o n medium. When 0 . 1 M N a C l i s p r e s e n t , 2.5 pM ATP p r o t e c t s half-maximally a g a i n s t i n a c t i v a t i o n by FSBA. But c a r r y i n g o u t t h e i n c u b a t i o n i n 0 . 1 M K C 1 medium l e a d s t o much l a r g e r ATP r e q u i r e m e n t f o r half-maximal p r o t e c t i o n ( a b o u t 2 0 0 U M ) The a p p a r e n t d i s s o c i a t i o n c o n s t a n t s f o r ATP p r o t e c t i o n a g a i n s t FSBA i n h i b i t i o n t h e r e f o r e a r e t h o s e e x p e c t e d f o r t h e high- and l o w - a f f i n i t y forms of t h e ATP-binding s i t e . A t 1 0 0 pM c o n c e n t r a t i o n s i n 0 . 1 M N a C 1 , b o t h ATP and ADP are e f f e c t i v e p r o t e c t o r s , whereas AMP i s n o t , a f i n d i n g c o n s i s t e n t w i t h t h e known s p e c i f i c i t y of t h e Na,K-ATPase. I n h i b i t i o n o f t h e enzyme by FSBA i s i r r e v e r s i b l e . Addition of p r o t e c t i n g c o n c e n t r a t i o n s of n u c l e o t i d e a f t e r r e a c t i o n w i t h FSBA h a s a l r e a d y o c c u r r e d d o e s n o t r e s t o r e a c t i v i t y but simply prevents f u r t h e r i n a c t i v a t i o n . Study of t h e k i n e t i c s of r e a c t i o n i s c o m p l i c a t e d by s o l v o l y s i s of t h e r e a g e n t . However, measurement o f f l u o r i d e release from FSBA w i t h t h e a i f of a f l u o r i d e s p e c i f i c e l e c t r o d e p e r m i t s m o n i t o r i n g of t h e l o s s of r e a g e n t . Repeated small a d d i t i o n s of FSBA t o m a i n t a i n i t s c o n c e n t r a t i o n c o n s t a n t t o w i t h i n 215% a l l o w s d e t e r m i n a t i o n of t h e c o n c e n t r a t i o n dependence of FSBA i n h i b i t i o n . I n a c t i v a t i o n i s e s s e n t i a l l y l i n e a r w i t h FSBA c o n c e n t r a t i o n t h r o u g h 1 mM, t h e approximate s o l u b i l i t y l i m i t of t h e r e a g e n t . This finding i s c o n s i s t e n t with t h e f a c t t h a t FSBA f a i l s t o compete w i t h ATP f o r hydrol y s i s on t h e h i g h - a f f i n i t y ATP s i t e u s i n g Na-ATP ase a s s a y c o n d i t i o n s . T h i s r e s u l t i n d i c a t e s t h a t t h e appar e n t d i s s o c i a t i o n c o n s t a n t f o r FSBA must exceed 5 mM. To e s t a l i s h t h a t FSBA re a c ts c o v a l e n t l y a t t h e ATPb i n d i n g s i t e , t h e a b i l i t y o f [3H]ADP t o b i n d t o t h a t s i t e a f t e r t r e a t m e n t o f t h e enzyme w i t h FSBA w a s determined. A l o s s of ADP-binding c a p a c i t y i n d i r e c t proport i o n t o loss of N a , K - A T P a s e a c t i v i t y was o b t a i n e d . T h i s f i n d i n g i n d i c a t e s t h a t o c c u p a t i o n o f t h e ATP-binding s i t e by FSBA i r r e v e r s i b l y p r e v e n t s s u b s e q u e n t [ 3H] ADP b i n d i n g . I n a d d i t i o n , t h e r e l a t i v e s t o i c h i o m e t r y of s p e c i f i c (ATP-protectable) [ 3H] FSBA a t t a c h m e n t t o t h e enzyme w a s found t o b e 0.94 k 0 . 2 4 moles bound p e r mole of o u a b a i n bound. An a p p r o x i m a t e l y e q u i v a l e n t amount of n o n s p e c i f i c l a b e l i n g w a s a l s o found, b u t t h i s a t t a c h m e n t of FSBA t o t h e enzyme h a s no e f f e c t on Na,K-ATPase act i v i t y . Most of t h e n o n s p e c i f i c l a b e l i n g and a l l of t h e s p e c i f i c l a b e l i n g i s found i n t h e c a t a l y t i c ( a ) s u b u n i t .
.
STUDIES OF THE ATP BINDING SITE OF CANINE KIDNEY
369
One c u r r e n t view o f N a , K - A T P a s e s t r u c t u r e r e l a t i n g t o i t s f u n c t i o n i s t h a t a s i n g l e a c t i v e s i t e on a + d i m e r i c a , B - u n i t c a n c a t a l y z e a l l a c t i v i t i e s of t h e Na pump w i t h o u t any r e q u i r e m e n t f o r a-a s u b u n i t i n t e r a c t i o n i n a n ~ 1 2 ~ 8t e2 t r a m e r i c u n i t (Smith e t a l . , 1980; Moczydlowski and F o r t e s , 1 9 8 1 ) . I f t h i s were t r u e , t h e n i n h i b i t i o n of N a , K - A T P a s e a c t i v i t y by FSBA s h o u l d b l o c k p - n i t r o p h e n y l p h o s p h a t a s e (pNPPase) a c t i v i t y i n e q u a l p r o p o r t i o n , s i n c e t h e l a t t e r a c t i v i t y i s c a t a l y z e d by t h e l o w - a f f i n i t y form o f t h e ATP-binding s i t e ( R o b i n s o n , 1 9 7 6 ) . An a , B - u n i t would need t o c y c l e between t h e s e two forms t o c a t a l y z e c o m p l e t e Na,K-ATPase a c t i v i t y . In a c t u a l i t y , l o s s of N a , K - A T P a s e a c t i v i t y (measured w i t h 5 mM ATP) a f t e r FSBA t r e a t m e n t i s r o u g h l y t w i c e a s ext e n s i v e a s l o s s of K+-phosphatase a c t i v i t y , w h e t h e r t h e t r e a t m e n t i s c a r r i e d o u t i n 0 . 1 M N a + or K+ medium. The same r e s u l t s are o b t a i n e d i f N a + - A T P a s e a c t i v i t y i s measured u s i n g 2 0 pM ATP. T h e r e i s no change i n appar e n t K~ for pNPP €or t h e s u r v i v i n g p h o s p h a t a s e a c t i v i t y . These r e s u l t s imply t h a t t h e f u n c t i o n a l a 2 8 2 pump u n i t s have two s i t e s f o r pNPP h y d r o l y s i s p e r e a c h f u n c t i o n a l ATP s i t e . B l o c k i n g t h e l a t t e r w i t h a n u c l e o t i d e a n a l o g s u c h a s FSBA i n h i b i t s Na-ATPase and Na,K-ATPase a c t i v i t y a l o n g w i t h one of t h e two pNPP s i t e s . The una f f e c t e d s i t e i s u n a b l e t o assume t h e c o n f o r m a t i o n req u i r e d t o p r o d u c e h i g h a f f i n i t y €or n u c l e o t i d e , e v e n i n These r e s u l t s a r e c o n s i s N a + medium c o n t a i n i n g no K + . t e n t w i t h a model o f Na,K-ATPase s t r u c t u r e h a v i n g e i t h e r (1) one pNPPase c a t a l y t i c s i t e on e a c h of t h e two d i f f e r e n t a-subunits i n t h e a282 tetramer, with r e s t r i c t i v e c o n f o r m a t i o n a l i n t e r a c t i o n s between them; or ( 2 ) two pNPPase s i t e s on a s i n g l e - s u b u n i t , w i t h t h e p o s s i b i l i t y t h a t these sites a l t e r n a t e as t h e ATPase c a t a l y t i c s i t e . I n t h e l a t t e r case, t h e o t h e r a - s u b u n i t of t h e ~ 1 2 B 2 t e t r a m e r would b e c a t a l y t i c a l l y i n a c t i v e .
ACKNOWLEDGMENT S u p p o r t e d i n p a r t by N I H g r a n t 5S07RR5350.
REFERENCES
Cooper, J. B . ,
165-174.
a n d W i n t e r , C. G.
(1980).
J . S u p r a n w l . Struct. 1 3 ,
370
JAMES 9. COOPER eta/.
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