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ADP-ATP Exchange in Internally Dialyzed Squid Giant Axons
CURRENT TOPICS M MEMBRANES AND TRANSPORT, VOLUME 19
ADP-ATP Exchange in Internally Dialyzed Squid Giant Axons PAUL DE WEER, GERDA E. BREIWIESER, AND H. GILBERT SMITH Department of Physiology and Biophysics Washington Universio School of Medicine St. Louis, Missouri
BRIAN G. KENNEDY' Department of Physiology Yale University Scltwl of Medicine New Haven, Connecticut
I.
MATERIALS AND METHODS
E x p e r i m e n t s were d e s i g n e d t o t e s t w h e t h e r t h e sodium pump o r N a , K - A T P a s e o f i n t e r n a l l y d i a l y z e d s q u i d g i a n t a x o n s c a n c a t a l y z e ADP-ATP exchange u n d e r c o n d i t i o n s where o u a b a i n - s e n s i t i v e N a - N a exchange i s known t o take place. Sodium-sodium exchange i s e l e c t r o n e u t r a l ( G a r r a h a n and Glynn, 1 9 6 7 a ; Abercrombie and D e Weer, 19781, r e q u i r e s ADP ( D e Weer, 1 9 7 0 ; Glynn and Hoffman, 1971) a s w e l l as ATP ( C a v i e r e s and Glynn; 1 9 7 9 1 , b u t d o e s n o t h y d r o l y z e t h e l a t t e r ( G a r r a h a n and Glynn, 1 9 6 7 b ) . S i n c e one of t h e r e a c t i o n s c a t a l y z e d by membrane-bound Na,K-ATPase i s a sodium-dependent ADP-ATP exchange (Skou, 1 9 6 0 1 , i t h a s been p r o p o s e d ( D e Weer, 1970; Glynn and Hoffman, 1 9 7 1 ) t h a t N a - N a exchange and ADP-ATP exchange a r e m a n i f e s t a t i o n s o f a s i n g l e molecular operation. The i n t e r n a l d i a l y s i s t e c h n i q u e of B r i n l e y and M u l l i n s (1967) w a s employed. G i a n t a x o n s of L o l i g o p e a l e i ( d i a m e t e r 1: 5 0 0 p m ) were b a t h e d i n p o t a s s i u m ' P r e s e n t a d d r e s s : Department of B i o c h e m i s t r y and M o l e c u l a r Biology, Lhiversity of T e x a s M e d i c a l S c h o o l , H o u s t o n , T e x a s . 665
Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved.
ISBN &IZ-l533194
666
PAUL DE WEER et el.
f r e e a r t i f i c i a l seawater and p e r f u s e d i n t e r n a l l y v i a a c e l l u l o s e a c e t a t e c a p i l l a r y t u b e ( d i a m e t e r = 100-150 p m ) running down t h e c e n t e r of t h e axon. The c e l l u l o s e acet a t e c a p i l l a r y had been r e n d e r e d porous t o i o n s and molecules of up t o 1 0 0 0 d a l t o n s i n i t s middle 1 5 mm reg i o n by exposure t o a l k a l i . The i n t e r n a l e r f u s a t e cont a i n e d , b e s i d e s g l y c i n e , g l u t a m a t e , C1-, K , and HEPES b u f f e r : 1 0 0 m Na+, 5 mM ATP, 5 m M ADP, and 1 5 mM Mg; L-arginine was a b s e n t . The flow r a t e of t h e i n t e r n a l p e r f u s a t e was a b o u t 1 . 2 pl/min, r e s u l t i n g i n an ADP del i v e r y r a t e ( t o t h e c a p i l l a r y ) of a b o u t 50 pmoles/sec, and a l i n e a r flow r a t e of 2.5 mm/sec, g i v i n g a 6-sec dwell t i m e i n t h e porous r e g i o n . N u c l e o t i d e s and nuc l e o s i d e s i n t h e e f f l u e n t from t h e d i a l y s i s c a p i l l a r y were s e p a r a t e d by P E I - c e l l u l o s e t h i n - l a y e r chromatography. E s s e n t i a l l y a l l [14C]ADP o r ATP p r e s e n t e d t o t h e axon was r e c o v e r e d a s ATP, ADP, AMP, o r adenosine. The p e r m e a b i l i t y of t h e porous r e g i o n i n s i t u t o ATP and ADP was c a l c u l a t e d from t h e r a t e c o e f f i c i e n t of washout of [14C]ATP o r ADP from an axon e q u i l i b r a t e d with t h e s e l a b e l s . From t h i s i t was c a l c u l a t e d t h a t about 1 5 % of t h e n u c l e o t i d e d e l i v e r e d t o t h e c a p i l l a r y exchanges w i t h t h e axoplasm; t h e remainder e x i t s from t h e c a p i l l a r y (see F i g . 1 ) . The sodium pump of axons p e r f u s e d under t h e above c o n d i t i o n s engages i n Na-Na exchange ( D e Weer e t a l . , 1 9 7 9 ) . S i n c e b o t h NEM and oligomycin enhance ADP-ATP exchange (Fahn et a l . , 1966; B l o s t e i n , 1 9 7 0 1 , t h e i r e f f e c t on t h e s q u i d axon sodium pump was i n v e s t i g a t e d . Oligomycin, which b l o c k s Na-Na exchange i n e r y t h r o c y t e s (Garrahan and Glynn, 1 9 6 7 b ) , had no such e f f e c t when NEM a p p l i e d i n t r a a p p l i e d i n t r a c e l l u l a r l y a t 10-5 M . c e l l u l a r l y a t 5 m~ i n h i b i t e d o u a b a i n - s e n s i t i v e Na-K and Na-Na exchange, and r e n d e r e d t h e membrane l e a k y t o sodium i o n s . When axons were p e r f u s e d w i t h s o l u t i o n s c o n t a i n i n g [14c]ADP, up t o 8 % of t h e l a b e l w a s recovered a s *ATPI s u g g e s t i n g t h a t a t l e a s t h a l f of t h e *ADP t h a t r e a c h e s t h e axoplasm i s c o n v e r t e d t o ATP b e f o r e it r e e n t e r s t h e porous c a p i l l a r y . A d d i t i o n s of cyanide ( 2 m M ) , iodoa c e t a t e ( 2 m M ) , and a t r a c t y l o s i d e (50 U M ) , which s h o u l d block m e t a b o l i c r e c o n v e r s i o n of ADP t o ATP, and diadenos i n e pentaphosphate (0.5 m M ) , which b l o c k s a d e n y l a t e k i n a s e , brought t h e r e c o v e r y of l a b e l a s *ATP down t o 1% o r less. T h i s d e m o n s t r a t e s t h a t o u r p r o c e d u r e d e t e c t s p r e d i c t a b l e changes i n [ 14C]ATP f o r m a t i o n , and t h a t , w i t h t h e l i s t e d i n h i b i t o r s p r e s e n t , a b o u t 1/15 of t h e "ADP r e a c h i n g t h e axoplasm o r 1 pmole/sec, is recapt u r e d by t h e c a p i l l a r y a s 'ATP. T h i s c o u l d , of c o u r s e , mean t h a t ATP , i n c l u d i n g f r e s h l y produced [ 14C]ATP , i s
?
ADP - ATP EXCHANGE IN SQUID GIANT AXONS
copi llory
667
Axoplasm
nucleotide flow 100 pmol /sec
F i g . 1 . S c h e m a t i c summary of e x c h a n g e s b e t w e e n i n t e r n a l p e r f u s i o n c a p i l l a r y and a x o p l a s m , both c o n t a i n i n g 1 5 mM e a c h of ATP and ADP i n the s t e a d y s t a t e . F u r t h e r d e s c r i p t i o n i s cont a i n e d i n the t e x t .
(relconverted to ADP at a very high rate. We checked this possibility by perfusing axons with [14C]ATP as the label, and found that ATP + ADP conversion in a 15-mm stretch of axon amounted to less than 8 pmoles/ sec (see Fig. 1). If the Na-Na exchange say 2.25 pmoles/cm2 sec were concomitant with ADP-ATP exchange (3:l stoichiometry), then the sodium pumps present on an axon 15 mm long and 0.5 mm across would produce 2 pmoles *ATP from *ADP every second. About 1/3 to 1/2 would be reconverted to *ADP and the remainder would be captured by the porous capillary (Fig. 1). That is, we should expect to detect ouabain-sensitive production of *ATP at a rate of 1.0-1.3 pmoles/sec in our system. In fact, we never found as much as 2.0.5 pmole/sec, the limit of our sensitivity.
-
PAUL DE WEER eta/.
668
11.
RESULTS AND DISCUSSION
W e i n t e r p r e t t h i s f i n d i n g t o mean t h a t , under o u r e x p e r i m e n t a l c o n d i t i o n s , t h e sodium-carrying enzyme can skpttle Na+ s e v e r a l times a c r o s s t h e membrane bef o r e ATP i s r e l e a s e d . T h i s r e q u i r e s t h a t , i n t h e s q u i d axon enzyme, a d d i t i o n of ATP and Nai be o r d e r e d , n o t random: ADP
ATP -E%P*ADP(Na)
NaO
t
P-NaoG=E%P
Support f o r o r d e r e d r e l e a s e of ADP and Nao i n t h e r e d blood c e l l enzyme has been p r e s e n t e d by Glynn and Hoffman ( 1 9 7 1 ) . I t i s h i g h l y u n l i k e l y t h a t t h e f a i l u r e t o r e l e a s e *ATP i s due t o t h e h i g h [Na]i i n o u r e x p e r i ments s i n c e , i n f a c t , h i g h l e v e l s of sodium are known t o s t i m u l a t e ADP-ATP exchange i n kidney membranes (Beau94 and Glynn, 1 9 7 9 ) . R a t h e r , it a p p e a r s t h a t t h e a d d i t i o n of Nai must be preceded by t h e o r d e r e d a d d i t i o n of f i r s t ATP, t h e n Mg: ATP
M9
Nai E*ATPMg*Na,-.
2
--
High l e v e l s of M g would a l l o w Na-Na exchange t o t a k e p l a c e , y e t i n h i b i t t h e r e l e a s e of "ATP formed from "ADP. I n h i b i t i o n of ADP-ATP exchange by h i g h [Mg] i s w e l l documented (Fahn et a l . , 1 9 6 6 ; Robinson, 1 9 7 6 ; Beauge and Glynn, 1 9 7 9 ) .
ACKNOWLEDGMENT
Supported by NIH Grant NS 11223.
ADP - ATP EXCHANGE IN SQUID GIANT AXONS
669
REFERENCES Abercrombie, R. F., and De Weer, P. (1978). Electric current generated by squid giant axon sodium pump: External K and internal ADP effects. Am. J. Physiol. 235, C63-C68, Beaugd, L. A., and Glynn, I. M. (1979). Sodium ions, acting at high-affinity extracellular sites, inhibit sodium-ATPase activity of the sodiun pump by slowing dephosphorylation. J . Physiol. (London) 289, 17-31. Blostein, R. (1970). Sodium activated adenosine triphosphatase activity of the erythrocyte membrane. J. Biol. Chem. 245, 270-275. Brinley, F. J., Jr., and Mullins, L. J. (1967). Sodium extrusion by internally dialyzed squid axons. J . Gen. Physiol. 50, 2303-2331. Cavieres, J. D., and Glynn, I. M. (1979). Sodium-sodium exchange through the sodium pump: The roles of ATP and ADP. J . Physiol . (London) 297, 637-645. De Weer, P. (1970). Effects of intracellular adenosine-5'-diphosphate and orthophosphate on the sensitivity of sodium efflux from squid axon to external sodium and potassium. J. Gen. Physiol. 56, 583-620. De Weer, P., Kennedy, B. G., and Abercrombie, R. F. (1979). Relationship between the Na:K exchanging and Na:Na exchanging modes of operation of the sodium pump. In "Na,K-ATPase: Structure and Kinetics" (J. C. Skou and J. G. Ndrby, eds.), pp. 504-515. Academic Press, New York. Fahn, S., Hurley, M. R., Koval, G. J., and Albers, R. W. (1966). Sodium-potassium-activated adenosine triphosphatase of Electrophorus electric organ. 11. Effects of N-ethylmaleimide and other sulfhydryl reagents. J . Biol. Chem. 241, 1890-1895. Garrahan, P. J., and Glynn, I. M. (1967a). The behaviour of the sodium pump in red cells in the absence of external potassium. J. Physiol. (London) 192, 159-174. Garrahan, P. J., and Glynn, I. M. (1967b). The Stoicheiometry of the sodium pump. J . Physiol. (London) ,192, 217-235. Glynn, I. M., and Hoffman, J. F. (1971). Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red blood cells. J. Physiol. (London) 218, 239-256. Robinson, J. D. (1976). The (Na++K+)-dependent ATPase. Mode of inhibition of ADP/ATP exchange activity by MgC12. Biochim. Biophys. Acta 440, 711-722. ++ + Na+-actiSkou, J. C. (1960). Further investigation on a Mg vated adenosinetriphosphatase, possibly related to the active, linked transport of Na' and K+ across the nerve membrane. Biochim. Biophys. A c t a 42, 6-23.
ADP-ATP Exchange in Internally Dialyzed Squid Giant Axons PAUL DE WEER, GERDA E. BREIWIESER, AND H. GILBERT SMITH Department of Physiology and Biophysics Washington Universio School of Medicine St. Louis, Missouri
BRIAN G. KENNEDY' Department of Physiology Yale University Scltwl of Medicine New Haven, Connecticut
I.
MATERIALS AND METHODS
E x p e r i m e n t s were d e s i g n e d t o t e s t w h e t h e r t h e sodium pump o r N a , K - A T P a s e o f i n t e r n a l l y d i a l y z e d s q u i d g i a n t a x o n s c a n c a t a l y z e ADP-ATP exchange u n d e r c o n d i t i o n s where o u a b a i n - s e n s i t i v e N a - N a exchange i s known t o take place. Sodium-sodium exchange i s e l e c t r o n e u t r a l ( G a r r a h a n and Glynn, 1 9 6 7 a ; Abercrombie and D e Weer, 19781, r e q u i r e s ADP ( D e Weer, 1 9 7 0 ; Glynn and Hoffman, 1971) a s w e l l as ATP ( C a v i e r e s and Glynn; 1 9 7 9 1 , b u t d o e s n o t h y d r o l y z e t h e l a t t e r ( G a r r a h a n and Glynn, 1 9 6 7 b ) . S i n c e one of t h e r e a c t i o n s c a t a l y z e d by membrane-bound Na,K-ATPase i s a sodium-dependent ADP-ATP exchange (Skou, 1 9 6 0 1 , i t h a s been p r o p o s e d ( D e Weer, 1970; Glynn and Hoffman, 1 9 7 1 ) t h a t N a - N a exchange and ADP-ATP exchange a r e m a n i f e s t a t i o n s o f a s i n g l e molecular operation. The i n t e r n a l d i a l y s i s t e c h n i q u e of B r i n l e y and M u l l i n s (1967) w a s employed. G i a n t a x o n s of L o l i g o p e a l e i ( d i a m e t e r 1: 5 0 0 p m ) were b a t h e d i n p o t a s s i u m ' P r e s e n t a d d r e s s : Department of B i o c h e m i s t r y and M o l e c u l a r Biology, Lhiversity of T e x a s M e d i c a l S c h o o l , H o u s t o n , T e x a s . 665
Copyright 0 1983 by Academic Press, Inc. All rights of reproductionin any form reserved.
ISBN &IZ-l533194
666
PAUL DE WEER et el.
f r e e a r t i f i c i a l seawater and p e r f u s e d i n t e r n a l l y v i a a c e l l u l o s e a c e t a t e c a p i l l a r y t u b e ( d i a m e t e r = 100-150 p m ) running down t h e c e n t e r of t h e axon. The c e l l u l o s e acet a t e c a p i l l a r y had been r e n d e r e d porous t o i o n s and molecules of up t o 1 0 0 0 d a l t o n s i n i t s middle 1 5 mm reg i o n by exposure t o a l k a l i . The i n t e r n a l e r f u s a t e cont a i n e d , b e s i d e s g l y c i n e , g l u t a m a t e , C1-, K , and HEPES b u f f e r : 1 0 0 m Na+, 5 mM ATP, 5 m M ADP, and 1 5 mM Mg; L-arginine was a b s e n t . The flow r a t e of t h e i n t e r n a l p e r f u s a t e was a b o u t 1 . 2 pl/min, r e s u l t i n g i n an ADP del i v e r y r a t e ( t o t h e c a p i l l a r y ) of a b o u t 50 pmoles/sec, and a l i n e a r flow r a t e of 2.5 mm/sec, g i v i n g a 6-sec dwell t i m e i n t h e porous r e g i o n . N u c l e o t i d e s and nuc l e o s i d e s i n t h e e f f l u e n t from t h e d i a l y s i s c a p i l l a r y were s e p a r a t e d by P E I - c e l l u l o s e t h i n - l a y e r chromatography. E s s e n t i a l l y a l l [14C]ADP o r ATP p r e s e n t e d t o t h e axon was r e c o v e r e d a s ATP, ADP, AMP, o r adenosine. The p e r m e a b i l i t y of t h e porous r e g i o n i n s i t u t o ATP and ADP was c a l c u l a t e d from t h e r a t e c o e f f i c i e n t of washout of [14C]ATP o r ADP from an axon e q u i l i b r a t e d with t h e s e l a b e l s . From t h i s i t was c a l c u l a t e d t h a t about 1 5 % of t h e n u c l e o t i d e d e l i v e r e d t o t h e c a p i l l a r y exchanges w i t h t h e axoplasm; t h e remainder e x i t s from t h e c a p i l l a r y (see F i g . 1 ) . The sodium pump of axons p e r f u s e d under t h e above c o n d i t i o n s engages i n Na-Na exchange ( D e Weer e t a l . , 1 9 7 9 ) . S i n c e b o t h NEM and oligomycin enhance ADP-ATP exchange (Fahn et a l . , 1966; B l o s t e i n , 1 9 7 0 1 , t h e i r e f f e c t on t h e s q u i d axon sodium pump was i n v e s t i g a t e d . Oligomycin, which b l o c k s Na-Na exchange i n e r y t h r o c y t e s (Garrahan and Glynn, 1 9 6 7 b ) , had no such e f f e c t when NEM a p p l i e d i n t r a a p p l i e d i n t r a c e l l u l a r l y a t 10-5 M . c e l l u l a r l y a t 5 m~ i n h i b i t e d o u a b a i n - s e n s i t i v e Na-K and Na-Na exchange, and r e n d e r e d t h e membrane l e a k y t o sodium i o n s . When axons were p e r f u s e d w i t h s o l u t i o n s c o n t a i n i n g [14c]ADP, up t o 8 % of t h e l a b e l w a s recovered a s *ATPI s u g g e s t i n g t h a t a t l e a s t h a l f of t h e *ADP t h a t r e a c h e s t h e axoplasm i s c o n v e r t e d t o ATP b e f o r e it r e e n t e r s t h e porous c a p i l l a r y . A d d i t i o n s of cyanide ( 2 m M ) , iodoa c e t a t e ( 2 m M ) , and a t r a c t y l o s i d e (50 U M ) , which s h o u l d block m e t a b o l i c r e c o n v e r s i o n of ADP t o ATP, and diadenos i n e pentaphosphate (0.5 m M ) , which b l o c k s a d e n y l a t e k i n a s e , brought t h e r e c o v e r y of l a b e l a s *ATP down t o 1% o r less. T h i s d e m o n s t r a t e s t h a t o u r p r o c e d u r e d e t e c t s p r e d i c t a b l e changes i n [ 14C]ATP f o r m a t i o n , and t h a t , w i t h t h e l i s t e d i n h i b i t o r s p r e s e n t , a b o u t 1/15 of t h e "ADP r e a c h i n g t h e axoplasm o r 1 pmole/sec, is recapt u r e d by t h e c a p i l l a r y a s 'ATP. T h i s c o u l d , of c o u r s e , mean t h a t ATP , i n c l u d i n g f r e s h l y produced [ 14C]ATP , i s
?
ADP - ATP EXCHANGE IN SQUID GIANT AXONS
copi llory
667
Axoplasm
nucleotide flow 100 pmol /sec
F i g . 1 . S c h e m a t i c summary of e x c h a n g e s b e t w e e n i n t e r n a l p e r f u s i o n c a p i l l a r y and a x o p l a s m , both c o n t a i n i n g 1 5 mM e a c h of ATP and ADP i n the s t e a d y s t a t e . F u r t h e r d e s c r i p t i o n i s cont a i n e d i n the t e x t .
(relconverted to ADP at a very high rate. We checked this possibility by perfusing axons with [14C]ATP as the label, and found that ATP + ADP conversion in a 15-mm stretch of axon amounted to less than 8 pmoles/ sec (see Fig. 1). If the Na-Na exchange say 2.25 pmoles/cm2 sec were concomitant with ADP-ATP exchange (3:l stoichiometry), then the sodium pumps present on an axon 15 mm long and 0.5 mm across would produce 2 pmoles *ATP from *ADP every second. About 1/3 to 1/2 would be reconverted to *ADP and the remainder would be captured by the porous capillary (Fig. 1). That is, we should expect to detect ouabain-sensitive production of *ATP at a rate of 1.0-1.3 pmoles/sec in our system. In fact, we never found as much as 2.0.5 pmole/sec, the limit of our sensitivity.
-
PAUL DE WEER eta/.
668
11.
RESULTS AND DISCUSSION
W e i n t e r p r e t t h i s f i n d i n g t o mean t h a t , under o u r e x p e r i m e n t a l c o n d i t i o n s , t h e sodium-carrying enzyme can skpttle Na+ s e v e r a l times a c r o s s t h e membrane bef o r e ATP i s r e l e a s e d . T h i s r e q u i r e s t h a t , i n t h e s q u i d axon enzyme, a d d i t i o n of ATP and Nai be o r d e r e d , n o t random: ADP
ATP -E%P*ADP(Na)
NaO
t
P-NaoG=E%P
Support f o r o r d e r e d r e l e a s e of ADP and Nao i n t h e r e d blood c e l l enzyme has been p r e s e n t e d by Glynn and Hoffman ( 1 9 7 1 ) . I t i s h i g h l y u n l i k e l y t h a t t h e f a i l u r e t o r e l e a s e *ATP i s due t o t h e h i g h [Na]i i n o u r e x p e r i ments s i n c e , i n f a c t , h i g h l e v e l s of sodium are known t o s t i m u l a t e ADP-ATP exchange i n kidney membranes (Beau94 and Glynn, 1 9 7 9 ) . R a t h e r , it a p p e a r s t h a t t h e a d d i t i o n of Nai must be preceded by t h e o r d e r e d a d d i t i o n of f i r s t ATP, t h e n Mg: ATP
M9
Nai E*ATPMg*Na,-.
2
--
High l e v e l s of M g would a l l o w Na-Na exchange t o t a k e p l a c e , y e t i n h i b i t t h e r e l e a s e of "ATP formed from "ADP. I n h i b i t i o n of ADP-ATP exchange by h i g h [Mg] i s w e l l documented (Fahn et a l . , 1 9 6 6 ; Robinson, 1 9 7 6 ; Beauge and Glynn, 1 9 7 9 ) .
ACKNOWLEDGMENT
Supported by NIH Grant NS 11223.
ADP - ATP EXCHANGE IN SQUID GIANT AXONS
669
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