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Characteristics of the Electric Eel Na,K-ATPase Phosphoprotein
CURRENT TOPICS IN MEMBRANES AND TRANSWRT. VOLUME 19
Characteristics of the Elecaic Eel Na,K-ATPase Phosphoprotein ATSUNORU YODA AND SHIZUKO YODA Depanmenr of Pharmacology University of WisconsinMedical School Madison, Wisconsin
I.
INTRODUCTION
The c l e a v a g e o f ATP by t h e N a , K - A T P a s e p r o c e e d s t h r o u g h p h o s p h o r y l a t e d i n t e r m e d i a t e s , t h e ADP- and K + - s e n s i t i v e phosphoenzymes (E1P and E 2 P ) . In a l l Na,K-ATPase p r e p a r a t i o n s known, E 2 P i s t h e m a j o r conpon e n t o f t h e p h o s p h o r y l a t e d form, whereas E 1 P i s t h e m a j o r component o n l y i n t h e p r e s e n c e of v e r y h i g h concent r a t i o n s of N a + o r i n a n enzyme p a r t i a l l y i n h i b i t e d by NEM. W e o b s e r v e d t h a t t h e e l e c t r i c e e l N a , K - A T P a s e p r e p a r e d w i t h o u t any d e t e r g e n t (Yoda and Yoda, 1 9 8 1 ) produced m o s t l y E 1 P i n t h e p r e s e n c e of 1 0 0 mM Na+, b u t t h e microsomes o f s h a r k r e c t a l g l a n d , a f a i r l y a c t i v e form of N a , K - A T P a s e when p r e p a r e d w i t h o u t d e t e r g e n t (Hokin e t a l . , 19731, produced m a i n l y E 2 P .
343
Copyright 0 1983 by Academic Press. Inc. All rights ofreproduction m any form reserved. ISBN 0-12-153319-0
ATSUNOBU YODA AND SHIZUKO YODA
344 11.
MATERIALS AND METHODS
I n t h i s s t u d y , t h e enzyme p h o s p h o r y l a t i o n w a s terminated a f t e r t h e p h o s p h o r y l a t i o n o f [ Y - ~ ~ATP P ] reached e q u i l i b r i u m i n t h e p r e s e n c e of Na+ and Mg2+ by t h e add i t i o n of 50 mM CDTA w i t h or w i t h o u t 5 0 vg/ml oligomyc i n , w i t h 1 mM ADP o r 2 mM K + added 0 . 5 sec l a t e r . The E1P and E 2 P p e r c e n t a g e s w e r e r o u g h l y c a l c u l a t e d from t h e r a t i o of t h e value of t h e dephosphorylated p r o t e i n 1 sec a f t e r t h e a d d i t i o n of ADP o r K+ t o t h e v a l u e of t h e p h o s p h o r y l a t e d p r o t e i n 1 sec a f t e r a d d i t i o n of water i n t h e absence o f ADP and K+. The p h o s p h o r y l a t i o n and dephosphorylation occurred i n t h e presence of a c o n s t a n t N a + c o n c e n t r a t i o n and a t a t e m p e r a t u r e of 4OC.
111.
RESULTS AND DISCUSSION
I n t h e p r e s e n c e of 1 0 0 mM N a + , a b o u t 7 0 % of t h e phosphoenzyme from t h e s h a r k enzyme w a s E2P ( i . e . , dep h o s p h o r y l a t e d by 2 mM K+ w i t h i n 1 sec) and a p p r o x i m a t e l y 3 0 % w a s ADP-sensitive ( i . e . , d e p h o s p h o r y l a t e d by 1 mM ADP w i t h i n 1 s e c ) , s i m i l a r t o t h e r e s u l t s r e p o r t e d f o r t h e kidney and b r a i n enzymes (Klodos and Ngkby, 1 9 7 9 ) . I n t h e e e l enzyme, however, a b o u t 8 0 % o f t h e In the phosphoenzyme was s e n s i t i v e t o b o t h ADP and.'K s h a r k and e e l enzymes, more t h a n 95% o f t h e phosphoenzymes were d e p h o s p h o r y l a t e d by t h e s i m u l t a n e o u s a d d i t i o n of ADP and K+. These d e p h o s p h o r y l a t i o n r e s u l t s f o r b o t h enzymes were n o t changed by t h e s u b s t i t u t i o n of unl a b e l e d ATP ( 2 mM) f o r 50 mM CDTA. The sum of t h e E 1 P and E2P p e r c e n t a g e s of t h e s h a r k enzyme was a b o u t l o o % , b u t t h a t of t h e eel enzyme w a s more t h a n 1 5 0 % , a s shown i n F i g . 1. Two d i f f e r e n t c o n c l u s i o n s c a n be drawn from t h e u n e x p e c t e d l y l a r g e sums of t h e e e l enzyme E1P and E2P p e r c e n t a g e s : (1) a s i g n i f i c a n t p e r c e n t a g e of t h e E 1 P may c o n v e r t t o E 2P o r v i c e v e r s a d u r i n g t h e dephosp h o r y l a t i o n p e r i o d (1 s e c ) ; o r ( 2 ) t h e e e l enzyme may produce a new t y p e of phosphoenzyme which i s b o t h K+and ADP-sensitive. To examine t h e s e p o s s i b i l i t i e s , t h e o l i g o m y c i n e f f e c t s on t h e d e p h o s p h o r y l a t i o n of t h e EP w e r e s t u d i e d , s i n c e s e v e r a l s t u d i e s have i n d i c a t e d t h a t oligomycin i s t h e i n h i b i t o r of t h e conversion of E1P t o E2P i n Na,KA T P a s e (Fahn e t a l . , 1 9 6 6 ) . I n t h e eel enzyme, 5 0 pg/ml oligomycin s u b s t a n t i a l l y reduced t h e K+ e f f e c t on t h e d e p h o s p h o r y l a t i o n r a t e , and reduced t h e sum of t h e E1P
ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN
345
F i g . 1 . Percentages of E I P and E2P i n the phosphorylated Na,K-ATPases i n various Nat concentrations. The experimental cond i t i o n s and the calculations are shown i n the t e x t , and the open bar or the half-shadowed bar represents the r e s u l t obtained with or without oligornycin, respectively.
and E2P p e r c e n t a g e s from 1 6 0 t o 1 2 4 % , r e s p e c t i v e l y , b u t no such oligomycin e f f e c t s were o b s e r v e d i n t h e s h a r k enzyme. From t h e o l i g o m y c i n e f f e c t s , w e concluded t h a t t h e e e l enzyme formed mainly E I P from ATP i n t h e p r e s e n c e of 1 0 0 m N a + , and t h a t t h i s E 1 P c o n v e r t e d t o E2P and w a s t h e n d e p h o s p h o r y l a t e d by K+. A s shown i n t h e o t h e r Na,K-ATPase p r e a r a t i o n s ( P o s t e t a l . , 19751, an i n c r e a s e i n t h e N a p c o n c e n t r a t i o n l e d t o a h i g h e r E J P p e r c e n t a g e i n t h e s h a r k enzyme a l s o . t h e oligomyUnder s u c h c o n d i t i o n s ( 5 0 0 o r 7 0 0 mM Na'), c i n - i n h i b i t a b l e c o n v e r s i o n o f E 1 P t o E2P was a l s o obs e r v e d , s i m i l a r t o t h e e e l enzyme i n t h e p r e s e n c e o f 1 0 0 mM N a + . On t h e o t h e r hand, t h e e e l enzyme formed mainly E2P i n t h e p r e s e n c e of 1 0 mM N a + and d i d n o t show t h e oligomycin e f f e c t a s i n t h e s h a r k enzyme i n t h e p r e s e n c e o f 1 0 0 m Na+. W e t h e r e f o r e c o n c l u d e t h a t t h e d i f f e r e n c e s between e e l and s h a r k enzymes r e s u l t from t h e d i f f e r e n t a f f i n i t i e s of t h e two enzymes t o N a + . The c o n v e r s i o n of E1P t o E2P i n t h e e e l enzyme w a s s t i m u l a t e d by o u a b a i n . When 2 m~ o u a b a i n w a s added a f t e r t h e p h o s p h o r y l a t i o n w a s t e r m i n a t e d by 50 mM CDTA, t h e p h o s p h o r y l a t e d e e l enzyme bound w i t h t h e o u a b a i n . T h i s b i n d i n g w a s d e t e c t e d by t h e i n h i b i t i o n of A T P a s e ( 5 0 % a t
ATSUNOBU YODA AND SHIZUKO YODA
346
I n h i b i t i o n by Ouabain-EP I n t e r a c t i o n and t h e E f f e c t s of ADP, and Oligomycina
TABLE I .
K+,
C o n d i t i o n of p h o s p h o r y l a t i o n Na
Temp. ("C)
+
ATP
Mg
2+
--
I n h i b i t i o n o f Na,K-ATPase + 2 mM + 1 mM + 50 ug/ml Kf ADP oligomycin
(mM)
(mM)
(mM)
(%I
4 4 4
10 100 1000
0.02 0.02 0.10
2 2 10
49 64 12
0.3 0.1 2.0
45 42 lo
--
25 25 25
10 100 1000
0.02 0.02 0.10
2 2 10
54 71 66
1.0 7.5 54
45 31 14
54 50 5
a
(0)
--
--
T h e e x p e r i m e n t a l p r o c e d u r e i s s h o w n i n the t e x t .
4 O C , 7 1 % a t 25OC) a f t e r t h e removal of t h e unbound ouab a i n by a Sephadex G-50 column. A s shown i n T a b l e I , t h i s ouabain b i n d i n g was reduced w i t h K+, n o t w i t h ADP a t 4 O C , b u t a t 25OC, it was a l s o reduced w i t h ADP. Oligomycin a l s o p r e v e n t e d t h i s o u a b a i n b i n d i n g i n t h e These r e s u l t s p r e s e n c e of h i g h c o n c e n t r a t i o n s of N a + . a l s o s u g g e s t t h a t E2P c a n b i n d w i t h o u a b a i n a t 4OC, unl i k e E l P , and a t 25OC, b u t t h e r a p i d change o f E 1 P t o E 2 P o c c u r s a t 25OC w i t h o u a b a i n . From t h e s e r e s u l t s , w e concluded t h a t t h e e e l enzyme c h a r a c t e r i s t i c a l l y forms E1P-rich phosphoenzyme i n t h e p r e s e n c e of 1 0 0 mM N a + and p r o b a b l y h a s a s p e c i f i c a l l y h i g h e r a f f i n i t y f o r N a + t h a n o t h e r Na,K-ATPase p r e p a r a t i o n s . The c o n v e r s i o n of E 1 P t o E 2 P by K+ o r ouabain, r e s u l t i n g i n t h e dephosphorylation o r t h e bindof o u a b a i n , was a l s o o b s e r v e d i n t h e a b s e n c e of f r e e M9
ins+-
REFERENCES Fahn, S., Kova-, G. J., and A bers, R. W. ( 3 6 ) . Sodium-potassiuma c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e o f E l ec trophorus e l e c t r i c organ. J . B i o l . Chem. 2 4 1 , 1882-1889. Hokin, L. E . , Dahl, J. L., Deupree, J. D . , Dixon, J. F., Hackney, J. F . , and Perdue, J. F. (1973). S t u d i e s on t h e c h a r a c t e r i z a t i o n o f t h e sodium-potassium t r a n s p o r t a d e n o s i n e t r i p h o s J . B i o l C h e m . 248 , 2593-2605. phatase
.
.
347
ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN
+
Klodos, I . , and Ndrby, J . G. ( 1 9 7 9 ) . E f f e c t of K+ and L i on i n t e r m e d i a r y s t e p s i n t h e N a ,K-ATPase r e a c t i o n . In " N a , K ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou a n d J. G. Ngkby, eds.) , p p . 331-342. Academic P r e s s , N e w York. P o s t , R. L . , Toda, G . , K u m e , S . , and T a n i g u c h i , K. ( 1 9 7 5 ) . Synthesis of a d e n o s i n e t r i p h o s p h a t e by N a , K-ATPase. J. Supraml. Struct. 3, 479-497. Yoda, A . , a n d Yoda, S. (1981). A new s i m p l e p r e p a r a t i o n method f o r Na,K-ATPase-rich membrane f r a g m e n t s . A n a l . Biochern. 110, 82-88.
Characteristics of the Elecaic Eel Na,K-ATPase Phosphoprotein ATSUNORU YODA AND SHIZUKO YODA Depanmenr of Pharmacology University of WisconsinMedical School Madison, Wisconsin
I.
INTRODUCTION
The c l e a v a g e o f ATP by t h e N a , K - A T P a s e p r o c e e d s t h r o u g h p h o s p h o r y l a t e d i n t e r m e d i a t e s , t h e ADP- and K + - s e n s i t i v e phosphoenzymes (E1P and E 2 P ) . In a l l Na,K-ATPase p r e p a r a t i o n s known, E 2 P i s t h e m a j o r conpon e n t o f t h e p h o s p h o r y l a t e d form, whereas E 1 P i s t h e m a j o r component o n l y i n t h e p r e s e n c e of v e r y h i g h concent r a t i o n s of N a + o r i n a n enzyme p a r t i a l l y i n h i b i t e d by NEM. W e o b s e r v e d t h a t t h e e l e c t r i c e e l N a , K - A T P a s e p r e p a r e d w i t h o u t any d e t e r g e n t (Yoda and Yoda, 1 9 8 1 ) produced m o s t l y E 1 P i n t h e p r e s e n c e of 1 0 0 mM Na+, b u t t h e microsomes o f s h a r k r e c t a l g l a n d , a f a i r l y a c t i v e form of N a , K - A T P a s e when p r e p a r e d w i t h o u t d e t e r g e n t (Hokin e t a l . , 19731, produced m a i n l y E 2 P .
343
Copyright 0 1983 by Academic Press. Inc. All rights ofreproduction m any form reserved. ISBN 0-12-153319-0
ATSUNOBU YODA AND SHIZUKO YODA
344 11.
MATERIALS AND METHODS
I n t h i s s t u d y , t h e enzyme p h o s p h o r y l a t i o n w a s terminated a f t e r t h e p h o s p h o r y l a t i o n o f [ Y - ~ ~ATP P ] reached e q u i l i b r i u m i n t h e p r e s e n c e of Na+ and Mg2+ by t h e add i t i o n of 50 mM CDTA w i t h or w i t h o u t 5 0 vg/ml oligomyc i n , w i t h 1 mM ADP o r 2 mM K + added 0 . 5 sec l a t e r . The E1P and E 2 P p e r c e n t a g e s w e r e r o u g h l y c a l c u l a t e d from t h e r a t i o of t h e value of t h e dephosphorylated p r o t e i n 1 sec a f t e r t h e a d d i t i o n of ADP o r K+ t o t h e v a l u e of t h e p h o s p h o r y l a t e d p r o t e i n 1 sec a f t e r a d d i t i o n of water i n t h e absence o f ADP and K+. The p h o s p h o r y l a t i o n and dephosphorylation occurred i n t h e presence of a c o n s t a n t N a + c o n c e n t r a t i o n and a t a t e m p e r a t u r e of 4OC.
111.
RESULTS AND DISCUSSION
I n t h e p r e s e n c e of 1 0 0 mM N a + , a b o u t 7 0 % of t h e phosphoenzyme from t h e s h a r k enzyme w a s E2P ( i . e . , dep h o s p h o r y l a t e d by 2 mM K+ w i t h i n 1 sec) and a p p r o x i m a t e l y 3 0 % w a s ADP-sensitive ( i . e . , d e p h o s p h o r y l a t e d by 1 mM ADP w i t h i n 1 s e c ) , s i m i l a r t o t h e r e s u l t s r e p o r t e d f o r t h e kidney and b r a i n enzymes (Klodos and Ngkby, 1 9 7 9 ) . I n t h e e e l enzyme, however, a b o u t 8 0 % o f t h e In the phosphoenzyme was s e n s i t i v e t o b o t h ADP and.'K s h a r k and e e l enzymes, more t h a n 95% o f t h e phosphoenzymes were d e p h o s p h o r y l a t e d by t h e s i m u l t a n e o u s a d d i t i o n of ADP and K+. These d e p h o s p h o r y l a t i o n r e s u l t s f o r b o t h enzymes were n o t changed by t h e s u b s t i t u t i o n of unl a b e l e d ATP ( 2 mM) f o r 50 mM CDTA. The sum of t h e E 1 P and E2P p e r c e n t a g e s of t h e s h a r k enzyme was a b o u t l o o % , b u t t h a t of t h e eel enzyme w a s more t h a n 1 5 0 % , a s shown i n F i g . 1. Two d i f f e r e n t c o n c l u s i o n s c a n be drawn from t h e u n e x p e c t e d l y l a r g e sums of t h e e e l enzyme E1P and E2P p e r c e n t a g e s : (1) a s i g n i f i c a n t p e r c e n t a g e of t h e E 1 P may c o n v e r t t o E 2P o r v i c e v e r s a d u r i n g t h e dephosp h o r y l a t i o n p e r i o d (1 s e c ) ; o r ( 2 ) t h e e e l enzyme may produce a new t y p e of phosphoenzyme which i s b o t h K+and ADP-sensitive. To examine t h e s e p o s s i b i l i t i e s , t h e o l i g o m y c i n e f f e c t s on t h e d e p h o s p h o r y l a t i o n of t h e EP w e r e s t u d i e d , s i n c e s e v e r a l s t u d i e s have i n d i c a t e d t h a t oligomycin i s t h e i n h i b i t o r of t h e conversion of E1P t o E2P i n Na,KA T P a s e (Fahn e t a l . , 1 9 6 6 ) . I n t h e eel enzyme, 5 0 pg/ml oligomycin s u b s t a n t i a l l y reduced t h e K+ e f f e c t on t h e d e p h o s p h o r y l a t i o n r a t e , and reduced t h e sum of t h e E1P
ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN
345
F i g . 1 . Percentages of E I P and E2P i n the phosphorylated Na,K-ATPases i n various Nat concentrations. The experimental cond i t i o n s and the calculations are shown i n the t e x t , and the open bar or the half-shadowed bar represents the r e s u l t obtained with or without oligornycin, respectively.
and E2P p e r c e n t a g e s from 1 6 0 t o 1 2 4 % , r e s p e c t i v e l y , b u t no such oligomycin e f f e c t s were o b s e r v e d i n t h e s h a r k enzyme. From t h e o l i g o m y c i n e f f e c t s , w e concluded t h a t t h e e e l enzyme formed mainly E I P from ATP i n t h e p r e s e n c e of 1 0 0 m N a + , and t h a t t h i s E 1 P c o n v e r t e d t o E2P and w a s t h e n d e p h o s p h o r y l a t e d by K+. A s shown i n t h e o t h e r Na,K-ATPase p r e a r a t i o n s ( P o s t e t a l . , 19751, an i n c r e a s e i n t h e N a p c o n c e n t r a t i o n l e d t o a h i g h e r E J P p e r c e n t a g e i n t h e s h a r k enzyme a l s o . t h e oligomyUnder s u c h c o n d i t i o n s ( 5 0 0 o r 7 0 0 mM Na'), c i n - i n h i b i t a b l e c o n v e r s i o n o f E 1 P t o E2P was a l s o obs e r v e d , s i m i l a r t o t h e e e l enzyme i n t h e p r e s e n c e o f 1 0 0 mM N a + . On t h e o t h e r hand, t h e e e l enzyme formed mainly E2P i n t h e p r e s e n c e of 1 0 mM N a + and d i d n o t show t h e oligomycin e f f e c t a s i n t h e s h a r k enzyme i n t h e p r e s e n c e o f 1 0 0 m Na+. W e t h e r e f o r e c o n c l u d e t h a t t h e d i f f e r e n c e s between e e l and s h a r k enzymes r e s u l t from t h e d i f f e r e n t a f f i n i t i e s of t h e two enzymes t o N a + . The c o n v e r s i o n of E1P t o E2P i n t h e e e l enzyme w a s s t i m u l a t e d by o u a b a i n . When 2 m~ o u a b a i n w a s added a f t e r t h e p h o s p h o r y l a t i o n w a s t e r m i n a t e d by 50 mM CDTA, t h e p h o s p h o r y l a t e d e e l enzyme bound w i t h t h e o u a b a i n . T h i s b i n d i n g w a s d e t e c t e d by t h e i n h i b i t i o n of A T P a s e ( 5 0 % a t
ATSUNOBU YODA AND SHIZUKO YODA
346
I n h i b i t i o n by Ouabain-EP I n t e r a c t i o n and t h e E f f e c t s of ADP, and Oligomycina
TABLE I .
K+,
C o n d i t i o n of p h o s p h o r y l a t i o n Na
Temp. ("C)
+
ATP
Mg
2+
--
I n h i b i t i o n o f Na,K-ATPase + 2 mM + 1 mM + 50 ug/ml Kf ADP oligomycin
(mM)
(mM)
(mM)
(%I
4 4 4
10 100 1000
0.02 0.02 0.10
2 2 10
49 64 12
0.3 0.1 2.0
45 42 lo
--
25 25 25
10 100 1000
0.02 0.02 0.10
2 2 10
54 71 66
1.0 7.5 54
45 31 14
54 50 5
a
(0)
--
--
T h e e x p e r i m e n t a l p r o c e d u r e i s s h o w n i n the t e x t .
4 O C , 7 1 % a t 25OC) a f t e r t h e removal of t h e unbound ouab a i n by a Sephadex G-50 column. A s shown i n T a b l e I , t h i s ouabain b i n d i n g was reduced w i t h K+, n o t w i t h ADP a t 4 O C , b u t a t 25OC, it was a l s o reduced w i t h ADP. Oligomycin a l s o p r e v e n t e d t h i s o u a b a i n b i n d i n g i n t h e These r e s u l t s p r e s e n c e of h i g h c o n c e n t r a t i o n s of N a + . a l s o s u g g e s t t h a t E2P c a n b i n d w i t h o u a b a i n a t 4OC, unl i k e E l P , and a t 25OC, b u t t h e r a p i d change o f E 1 P t o E 2 P o c c u r s a t 25OC w i t h o u a b a i n . From t h e s e r e s u l t s , w e concluded t h a t t h e e e l enzyme c h a r a c t e r i s t i c a l l y forms E1P-rich phosphoenzyme i n t h e p r e s e n c e of 1 0 0 mM N a + and p r o b a b l y h a s a s p e c i f i c a l l y h i g h e r a f f i n i t y f o r N a + t h a n o t h e r Na,K-ATPase p r e p a r a t i o n s . The c o n v e r s i o n of E 1 P t o E 2 P by K+ o r ouabain, r e s u l t i n g i n t h e dephosphorylation o r t h e bindof o u a b a i n , was a l s o o b s e r v e d i n t h e a b s e n c e of f r e e M9
ins+-
REFERENCES Fahn, S., Kova-, G. J., and A bers, R. W. ( 3 6 ) . Sodium-potassiuma c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e o f E l ec trophorus e l e c t r i c organ. J . B i o l . Chem. 2 4 1 , 1882-1889. Hokin, L. E . , Dahl, J. L., Deupree, J. D . , Dixon, J. F., Hackney, J. F . , and Perdue, J. F. (1973). S t u d i e s on t h e c h a r a c t e r i z a t i o n o f t h e sodium-potassium t r a n s p o r t a d e n o s i n e t r i p h o s J . B i o l C h e m . 248 , 2593-2605. phatase
.
.
347
ELECTRIC EEL Na,K-ATPase PHOSPHOPROTEIN
+
Klodos, I . , and Ndrby, J . G. ( 1 9 7 9 ) . E f f e c t of K+ and L i on i n t e r m e d i a r y s t e p s i n t h e N a ,K-ATPase r e a c t i o n . In " N a , K ATPase: S t r u c t u r e and K i n e t i c s " (J. C. Skou a n d J. G. Ngkby, eds.) , p p . 331-342. Academic P r e s s , N e w York. P o s t , R. L . , Toda, G . , K u m e , S . , and T a n i g u c h i , K. ( 1 9 7 5 ) . Synthesis of a d e n o s i n e t r i p h o s p h a t e by N a , K-ATPase. J. Supraml. Struct. 3, 479-497. Yoda, A . , a n d Yoda, S. (1981). A new s i m p l e p r e p a r a t i o n method f o r Na,K-ATPase-rich membrane f r a g m e n t s . A n a l . Biochern. 110, 82-88.