Sidedness of Sodium Interactions with the Sodium Pump in the Absence of K+

Sidedness of Sodium Interactions with the Sodium Pump in the Absence of K+

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19 Sidedness of Sodium Interactions with the Sodium Pump in the Absence of K+ RHONA BLOSTEIN' Depar...

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 19

Sidedness of Sodium Interactions with the Sodium Pump in the Absence of K+ RHONA BLOSTEIN' Departments of Biochemisry and Erperimental Medicine McGiN University, and the Division of Hematology Royal Victoria Hospital Montreal, Quebec, Canada

I.

INTRODUCTION

T h i s d i s c u s s i o n i s concerned w i t h t h e e f f e c t s of r e a c t i o n sequence i n r e l a t i o n t o t h e mechanism of o u a b a i n - s e n s i t i v e N a + t r a n s p o r t . I n p r e v i o u s s t u d i e s u s i n g i n s i d e - o u t v e s i c l e s o f human red c e l l membranes, w e have shown t h a t N a + - s t i m u l a t e d p h o s p h o r y l a t i o n of t h e enzyme o c c u r s w i t h less t h a n micromolar amounts of ATP and r e q u i r e s N a + a t o n l y t h e c y t o p l a s m i c s u r f a c e . Although e x t r a v e s i c u l a r N a + (Nacyt, N a + a t t h e c y t o p l a s m i c s u r f a c e ) a t v e r y low c o n c e n t r a t i o n (10.2 m M ) s t i m u l a t e s ATP h y d r o l y s i s , h i g h e r amounts a r e needed f o r N a + t r a n s l o c a t i o n from t h e c y t o p l a s m i c t o t h e e x t r a c e l l u l a r s i d e of t h e membrane (Blostein, 1979). I t w a s suggested t h a t a transl o c a t i o n s t e p , e . g . , t r a n s i t i o n from o n e form o f phosphoenzyme t o a n o t h e r , El-P -+ E 2 - P , r e q u i r e s a l l ( t h r e e ? ) Na+ b i n d i n g s i t e s t o be s a t u r a t e d . N a + on t h e N a , K - A T P a s e

'Present a d d r e s s : Research I n s t i t u t e , Montreal Hospital , Montreal, Canada. 649

General

Copyright 0 1983 by Academic press. Inc. All rights of reproduction in any form reserved. ISBN 012-153319-0

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S e v e r a l o t h e r p r o p e r t i e s of Na+-stimulated A T P a s e (Na-ATPase) s u b s t a n t i a t e t h e c o n c l u s i o n t h a t t h i s aci s t h e enzymic b a s i s f o r ATP-dependent t r a n s p o r t i n t h e absence of K+. Thus, a t low c o n c e n t r a t i o n s of ATP, L i + and Rb+ ( o r K + ) a f f e c t Na+ t r a n s p o r t and Na+-ATPase s i m i l a r l y : i n t r a v e s i c u l a r L i + ( L i e x t r Li+ a t t h e normal e x t r a c e l l u l a r s u r f a c e ) s t i m u l a t e s , whereas i n t r a v e s i c u l a r Rb+ (Rbext) o r K+ i n h i b i t s b o t h a c t i v i t i e s . These e f f e c t s are c o n s i s t e n t w i t h t h e i r K+-like e f f e c t s on t h e sequence E2-P f K+ + K * E 2 +K.El-+-El + K+ whereby r e l e a s e of Rb+ ( o r K+) , b u t n o t L i + , from t h e enzyme i s r a t e - l i m i t i n g and i s acc e l e r a t e d only w i t h ATP bound t o a l o w - a f f i n i t y s i t e . I n a d d i t i o n , i n t r a v e s i c u l a r Na+ (NaeVt, Na+ a t t h e e x t r a c e l l u l a r s u r f a c e ) modulates b o t h a c t i v i t i e s i n a s i m i l a r , b i p h a s i c manner as f o l l o w s : u s i n g r e d c e l l g h o s t s , Glynn and K a r l i s h ( 1 9 7 6 ) showed t h a t e x t r a c e l l u l a r Na+, a t l o w c o n c e n t r a t i o n , i n h i b i t s Na+-ATPase a s w e l l as t h e a s s o c i a t e d ATP h y d r o l y s i s , Using i n s i d e o u t v e s i c l e s , w e showed t h a t t h e i n h i b i t o r y e f f e c t of low c o n c e n t r a t i o n s of Naext i s a s s o c i a t e d w i t h a dec r e a s e i n a p p a r e n t t u r n o v e r of phosphoenzyme (E-P) ( B l o s t e i n e t al., 1 9 7 9 ) ; t h i s i s i n c o n t r a s t t o e f f e c t s of Kext, Rbext, L i e x t , o r h i g h e r l e v e l s of N a e x t r a l l of which i n c r e a s e t h e a p p a r e n t t u r n o v e r of phosphoenzyme, whether o r n o t t h e y a c t i v a t e ( L i e x t , 2 25 mM Naext) o r i n h i b i t o v e r a l l pump and A T P a s e a c t i v i t y (Rbextr Kextr I 5 m~ Naext)

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11.

METHODS AND RESULTS

To g a i n i n s i g h t i n t o t h e q u e s t i o n of whether t h e r e s t o r a t i o n of ATP h y d r o l y s i s by r e l a t i v e l y h i g h l e v e l s of Naext was a s s o c i a t e d w i t h Na t r a n s p o r t , w e measured Na+ e f f l u x (normal i n f l u x ) a s w e l l as i n f l u x (normal e f f l u x ) u s i n g i n s i d e - o u t membrane v e s i c l e s . I t w a s observed t h a t t h e sodium pump can o p e r a t e i n a mode i n which i n f l u x and e f f l u x a r e a s s o c i a t e d w i t h ATP h y d r o l y s i s ( L e e and B l o s t e i n , 1 9 8 0 ) . D e s p i t e v a r i a t i o n i n a b s o l u t e a c t i v i t i e s of d i f f e r e n t p r e p a r a t i o n s , i n each c a s e , v a l u e s f o r b o t h f l u x e s a r e of s i m i l a r magn i t u d e when b o t h a r e measured i n t h e same way (22Na retained i n Millipor e- f ilter ed v e s i c l e s ) . This r e s u l t i s c o n s i s t e n t w i t h roughly one-for-one exchange. Moreover, w e have a s c e r t a i n e d t h a t 22Na e f f l u x i s n o t due t o ADP formed d u r i n g t h e c o u r s e of t h e experiment.

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Na-Na exchange i n t h e absence of ADP c a n be d i s t i n g u i s h e d from ADP-sensitive exchange i n t h e f o l l o w i n g way. Vanadate i n h i b i t s ATP-dependent Na+ i n f l u x i n both t h e p r e s e n c e and absence of Kext (normal N a - K exc h a n g e ) , a l t h o u g h t o a lesser e x t e n t w i t h r e l a t i v e l y high l e v e l s of Naext ( c f . Beauge et al., 1 9 8 0 ) . Howe v e r , w i t h 50 m~ Naext, it i s observed t h a t ADP-sensit i v e Na-Na exchange, measured a s ADP-sensitive 22Na i n f l u x , i s v i r t u a l l y i n s e n s i t i v e t o vanadate c o n c e n t r a t i o n s which p a r t i a l l y i n h i b i t Na-Na exchange i n t h e abs e n c e o f ADP. Na-Na exchange i n t h e absence of K+ and ADP may r e f l e c t exchange of Naext f o r Nac t i n a manner a n a l o gous t o t h e exchange of Kext f o r 8 a c y t , whereby NaeXt a c t s a t Kext s i t e s . However, t h e p o s s i b i l i t y remains sites, i n t h a t Naext a c t s a t s i t e s d i f f e r e n t from Ke,t p a r t i c u l a r , t h e Na+ d i s c h a r g e s i t e s ; N a e x t would t h e n s t i m u l a t e r e v e r s a l of E l - P + E 2 - P (see scheme below) i n f a v o r of E l - P which, i n t h e absence of ADP, i s hydrolyzed. ATP

I n f a v o r of t h e l a t t e r i n t e r p r e t a t i o n i s t h e remarkably s i m i l a r r e s p o n s e of ADP-ATP exchange, r e p o r t e d by Kaplan and H o l l i s ( 1 9 8 0 1 , and Na+-ATPase, a s d e s c r i b e d by Glynn and K a r l i s h ( 1 9 7 6 1 , t o changes i n Naext c o n c e n t r a t i o n . On t h e o t h e r hand, i f Naext a t h i g h l e v e l s s t i m u l a t e s h y d r o l y s i s v i a E l - P , i t may be argued t h a t t h e r a t e of t h e o v e r a l l ATPase could t h e n proceed a t a r a t e a t l e a s t as g r e a t a s t h a t v i a t h e "normal" r o u t e through E2-P. To t h e c o n t r a r y , w e o b s e r v e t h a t , under t h e p r e s e n t cond i t i o n s f o r o b s e r v i n g Na e f f l u x coupled t o Na-ATPase (5 m M N a c y t ) , oligomycin, which s t i m u l a t e s Na+-depend e n t ADP-ATP exchange and i s b e l i e v e d t o i n h i b i t t h e c o n v e r s i o n of E l - P t o E z - P , r e d u c e s ATP h y d r o l y s i s t o a r a t e less t h a n t h a t observed when h i g h l e v e l s of Naext are present.

ACKNOWLEDGMENT

This work was supported by The Medical Research Council of Canada.

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