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Na+-dependent phosphorylation of the rat brain (Na+ + K+)-ATPase Possible non-equivalent activation sites for Na+
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Biochimica et Biophysica Acta, 429 ( 1 9 7 6 ) 2 5 8 - - 2 7 3 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 67713
Na÷-DEPENDENT P H O S P H O R Y L A T I O N OF T H E RAT BRAIN (Na + + K+)-ATPase POSSIBLE NON-EQUIVALENT ACTIVATION SITES FO R Na ÷
DONALD FOSTER and KHALIL AHMED
Toxicology Research Laboratory, Minneapolis Veterans Hospital, Minneapolis, Minn. 55417 *, and the Department o f Laboratory Medicine and Pathology, University o f Minnesota 55455 (U.S.A.) (Received July 28th, 1975)
S u m mar y 1. The steady state levels of Na÷-dependent p h o s p h o e n z y m e (E-P) in the (Na + + K÷)-ATPase (EC 3.6.1.3) of rat brain, obtained from a time course study of p h o s p h o e n z y m e f or m a t i on at 4 ° C, were d e p e n d e n t on the concent rat i on of Na ÷ in the reaction and were maximal in the presence of 64 mM Na ÷. The plot of p h o s p h o e n z y m e vs. Na* c onc e nt r at i on gave a curve which on conversion to a double reciprocal plot {1/E-P vs. 1/Na +) gave a line with two breaks, yielding apparently three linear segments. This may be taken to indicate the presence of multiple Na ÷ sites for the formation of the p h o s p h o e n z y m e . To test this hypothesis further, the following approach was taken. By making the assumption t h a t the p h o s p h o e n z y m e may represent bound Na ÷, it was possible to subject the data to rigorous multiple-site analysis by utilizing steady-state binding equations described by Klotz and Hunston (1971) (Biochemistry 10, 3065--3069), and by Scatchard (1949) (Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 ) . The analysis of the data by these methods suggests that there may be three non-equivalent Na ÷ activation sites for the f or m a t i on of Na÷-dependent p h o s p h o e n z y m e in the (Na ÷ + K÷)-ATPase. The estimated intrinsic association constants (K a) for activation by Na ÷ at each o f the three sites were 3.4, 0.295, and 0.025 mM -1 , respectively. 2. The steady-state level of Na÷-dependent p h o s p h o e n z y m e was reduced by 2 H2 O (deuterated water) and Me2 SO {dimethylsulfoxide). This inhibition was reversed by increasing the c onc e nt r at i on of Na ÷ in the reaction but remained constant over a time course at any given Na + concentration. An analysis of the effect of : H2 O on Na÷-dependent p h o s p h o e n z y m e form at i on in the presence * Please address all communications to this address. Abbreviations: 2H 20, deuterated water; Me2SO. dimethyl sulfoxide; (Na + + K÷)-ATPase, (Na + + K+)-stimulated adenosine triphosphatase.
259 o f gradually increasing Na ÷ c o n c e n t r a t i o n also revealed the presence o f t h r e e n o n - e q u i v a l e n t sites for Na t. The intrinsic association c o n s t a n t s (K a) for the activation o f Na * at each o f the t h r e e sites in the p r e s e n c e o f 2 H2 O were changed to 1.4, 0.232, and 0 . 0 3 3 mM -1 , respectively, which suggests a differential e f f e c t o f 2 H~ O o n the t h r e e n o n - e q u i v a l e n t Na t sites. 3. On statistical g r o u n d s (-+2 S.E. o f m e a n ) a t w o n o n - e q u i v a l e n t site m o d e l also fits the data. In this case, the intrinsic association c o n s t a n t s ( g a ) were 2.44 and 0.041 mM -~ in H~ O m e d i u m , and 1.062 and 0 . 0 4 8 m M - ' , in 2 H~ O med i u m , respectively, showing a differential e f f e c t o f 2 H~ O on the t w o none q u i v a l e n t sites. 4. The i n h i b i t o r y e f f e c t o f ~ H~ O and Me~ SO on the f o r m a t i o n o f Na ÷d e p e n d e n t p h o s p h o e n z y m e was m a x i m a l w h e n the e n z y m e was allowed a cont a c t with these agents p r i o r to the a d d i t i o n o f Na t in the reaction. On the o t h e r h a n d , p r i o r c o n t a c t o f the e n z y m e with Na ÷ r e d u c e d or abolished the i n h i b i t o r y e f f e c t o f 2 H~ O or Me~ SO. Prior c o n t a c t o f the e n z y m e with ATP also abolished the i n h i b i t i o n o f Me~ SO. These results s u p p o r t the view t h a t H~ O plays a r e g u l a t o r y role in the active c e n t e r o f the (Na ÷ + K÷)-ATPase so t h a t its presence t e n d s to favor the E2 (Kt-accepting) f o r m o f the e n z y m e whereas Na t binds to at least t w o o f its activation sites, in an a p p a r e n t l y c o m p e t i t i v e m a n n e r with r e s p e c t to H~ O, yielding the c o n f o r m a t i o n suitable f o r the p h o s p h o r y l a tion ( E l ) . At the l o w e s t affinity Na ÷ activation site for e i t h e r the 2 or 3 n o n - e q u i v a l e n t site case the p r e s e n c e o f H 2 0 m a y facilitate its binding. The results also suggest t h a t b o t h Na * or A T P can i n d e p e n d e n t l y shift the e n z y m e c o n f o r m a t i o n to E l .
Introduction (Na ÷ + K÷)-stimulated t r a n s p o r t ATPase (EC 3.6.1.3) p r e s e n t in m o s t m a m malian p l a s m a cell m e m b r a n e s , is c o n s i d e r e d t o r e p r e s e n t the e n z y m i c basis for the m o v e m e n t o f cations across the cell m e m b r a n e [ 1 ] . Evidence f r o m several l a b o r a t o r i e s has suggested t h a t the o p e r a t i o n o f this e n z y m e p r o c e e d s t h r o u g h a series o f i n t e r m e d i a r y steps involving c o n f o r m a t i o n a l changes related t o Na ÷d e p e n d e n t p h o s p h o r y l a t i o n o f the e n z y m e and the K÷-dependent b r e a k d o w n o f the p h o s p h o e n z y m e . In this c o n t e x t , the use o f modifiers or inhibitors o f the partial r e a c t i o n s o f this e n z y m e s y s t e m has p r o v i d e d a considerable a m o u n t o f i n f o r m a t i o n o n the ionic i n t e r a c t i o n s in the ATPase, and possible m e c h a n i s m ( s ) o f its o p e r a t i o n (for r e c e n t reviews see e.g. refs. 1--3). In previous work, we d e m o n s t r a t e d t h a t s u b s t i t u t i o n o f 2 H 2 0 ( d e u t e r a t e d w a t e r ) f o r H 2 0 in the ATPase r e a c t i o n r e s u l t e d in an i n h i b i t i o n o f the Na ÷ activation o f the ATPase in an a p p a r e n t l y c o m p e t i t i v e m a n n e r , while the associated K ÷ - d e p e n d e n t p - n i t r o p h e n y l p h o s p h a t a s e was s t i m u l a t e d b y 2 H: O [ 4 ] . These observations based o n a s t u d y o f the kinetics o f (Na ÷ + Kt)-ATPase led us to p o s t u l a t e t h a t the binding o f Na ÷ t o t h e (Na t + K÷)-ATPase p r o d u c e d a c o n f o r m a t i o n which was r e q u i r e d f o r the initiation o f the p h o s p h o e n z y m e f o r m a t i o n in the p r e s e n c e o f ATP, and f u r t h e r , t h a t H 2 0 p l a y e d a role in this i n t e r a c t i o n b e t w e e n Na t and t h e e n z y m e at this site. Several studies o n t h e effects o f a variety o f solvents o n
260 the (Na t + K~)-ATPase have been r e por t ed recently | 5 - - 9 ] . Among the various solvents examined, dimethylsulfoxide (Me2 SO) appears to have some features in c o m m o n with 2 H~ O, in that it inhibits the Na%dependent activation of the e n z y m e while c o n c o m i t a n t l y stimulating the K%dependent p - n i t r o p h e n y l phosphatase [6,7]. However, the results reported on the effect of Me~ SO on the Na%dependent p h o s p h o e n z y m e formation in the (Na ~ + K+)-ATPase are at variance [6,8]. We have e x t e n d e d these studies and have examined in detail the action of ~H~O and Me2 SO on the formation of Na%dependent phosphoe n z y m e in the (Na t + K÷)-ATPase complex. These experiments offer some clues to the possible nature of the interaction of Na ÷ and H~ O at the phosphorylation sites in the (Na t + Kt)-ATPase. Further, we have measured the steady-state levels of p h o s p h o e n z y m e formed when the concentrations of Na t are gradually increased (in K%free reaction media), and have devised an approach to analyze and relate these data in terms of the possible activation sites for Na ÷ for the formation of Na%dependent p h o s p h o e n z y m e in the ATPase complex. On the basis of these studies, we suggest the presence of three non-equivalent sites for Na t interaction with the (Na ÷ + Kt)-ATPase. A preliminary account of this work has been given [ 1 0 ] . Experimental procedure
Materials 2 H 2 0 (99.5%, lot TTS) was purchased from Mallinckrodt Chemical works, and was distilled twice in an all-glass apparatus before use. Me~ SO of the analytical grade was purchased from Baker Chemicals. Sucrose and Tris were of the ultrapure grade from Schwartz-Mann, New York; NaC1, KC1, and MgC12 were spectroscopically pure. Solutions of ATP, including those containing [~,~ ~ P] ATP, were passed through large columns of Tris form of AG-50 × 8 cation exchange resin, to eliminate any contamination from Na t, K + or NH~. EDTA was dissolved in Tris base, and treated the same way as ATP, described above. All other details concerning the materials used have been given previously [4,11--13]. Methods Preparatio~ of (Na ~ ÷ K÷)-A TPase. The details concerning the preparation of rat brain membrane (Na t + Kt)-ATPase and its properties are the same as described in previous work [4,14]. The e n z y m e preparation was suspended in a medium consisting of 0.25 M sucrose, 10 mM imidazole-HC1 and 1 mM EDTA, pH 7.4, following two additional washes in this medium. It was stored frozen in small aliquots and was stable over several weeks. The specific activity of the (Na t + K* )-stimulated ATPase on the average was 125 pm ol / m g of protein per h while the basic Mg2÷-stimulated c o m p o n e n t was generally between 5--10% of the total (Mg 2÷ + Na ÷ + K÷)-dependent ATPase. Preparation of phosphoenzyrne. The standard reaction medium, in a final volume of 2 ml, maintained at 4 ° C, consisted of 30 mM Tris • HC1, pH 7.45 (at 4°C), 0.3 mM MgC12,8 mM NaC1, 0.05 mM [~/.32 p] ATP {6 • 104 d p m / n m o l of ATP) and between 350 to 500 pg of rat brain membrane (Na t + K÷)-ATPase preparation. In general, the reaction time was 4 s which gave the steady-state
261 level p h o s p h o r y l a t i o n , and unless o t h e r w i s e stated, the r e a c t i o n was initiated by the a d d i t i o n o f [7 -3 a P] ATP and was t e r m i n a t e d by adding 25 ml o f ice cold 5% (w/v) t r i c h l o r o a c e t i c acid c o n t a i n i n g 15 mM NaH~ PO4 and 0.6 mM ATP. The r a d i o a c t i v i t y d u e to 3 ~p i n c o r p o r a t e d in the p r o t e i n was m e a s u r e d as described before [ 11]. T o d e t e r m i n e the a c c u r a c y o f the p h o s p h o e n z y m e activity m e a s u r e d at low a d d e d Na * c o n c e n t r a t i o n s (e.g. at 0.5 mM Na*), c o n t r o l e x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e the c o n t r i b u t i o n o f the e n d o g e n o u s Na ÷ in the ATPase. F o r this, p h o s p h o e n z y m e f o r m e d in the presence o f 0.3 mM Mg ~÷ alone, and 0.3 mM Mg 2÷ + 16 mM K ÷ was measured. T h e d i f f e r e n c e b e t w e e n these t w o values should r e p r e s e n t t h e a m o u n t o f p h o s p h o e n z y m e activity due to the e n d o g e n o u s c o n t a m i n a t i o n o f the e n z y m e b y Na÷; in several e x p e r i m e n t s the m e a n values o f p h o s p h o e n z y m e activity were precisely the same in b o t h cases. F u r t h e r , b y a t o m i c a b s o r p t i o n s p e c t r o s c o p y , the e n z y m e p r e p a r a t i o n was f o u n d to c o n t a i n n o d e t e c t a b l e NH~ and K *, while the a m o u n t o f Na ÷ p r e s e n t was 60 n m o l / m g p r o t e i n , which would c o n t r i b u t e negligible a m o u n t s of Na ÷ in t h e reaction. T h e value o f the p h o s p h o e n z y m e o b t a i n e d in the presence o f (Mg ~÷ + K ÷) was s u b t r a c t e d f r o m t h a t in the presence o f (Mg 2÷ + Na÷), the diff e r e n c e being the Na÷-dependent p h o s p h o e n z y m e f o r m e d in the (Na÷+ K÷) ATPase system. T h e value o f p h o s p h o e n z y m e in the presence o f (Mg ~÷ + K ÷) was no m o r e t h a n 6% o f t h a t o b t a i n e d in the presence o f (Mg 2÷ + 64 mM Na÷). All d a t a are calculated as p m o l 3 ~p per mg o f p r o t e i n or as p e r c e n t p h o s p h o r y l a t i o n ( c o m p a r e d with m a x i m a l p h o s p h o e n z y m e f o r m e d in the presence o f 64 mM Na ÷) w h e n d i f f e r e n t e n z y m e p r e p a r a t i o n s were used. Other procedures. M e t h o d s for the e s t i m a t i o n o f p r o t e i n , and p r e p a r a t i o n o f [7 -3~ P] A T P were the same as given b e f o r e [ 1 1 , 1 2 ] . P r e p a r a t i o n o f the solutions in 2 H~ O was the same as r e p o r t e d previously [ 4 , 1 1 ] . Results
Effect o f 2 H2 0 and Me2 SO on Na%dependent p h o s p h o e n z y m e formation. It has been r e p o r t e d t h a t the c o n c e n t r a t i o n s at which 2 H 2 0 [4] and Me~ SO [6,7] p r o d u c e maximal, reversible, i n h i b i t o r y effects on the (Na + + K÷)-ATPase are 80---90 and 30%, respectively. T h e m a x i m a l inhibition o f Na÷-dependent p h o s p h o e n z y m e f o r m a t i o n by 2 H~ O was also observed at c o n c e n t r a t i o n s o f 8 0 - - 9 0 % [ 1 1 ] . In the p r e s e n c e o f 80% 2 H~ O or 30% Me~ SO, the i n h i b i t i o n o f the f o r m a t i o n o f Na÷-dependent p h o s p h o e n z y m e was m o d i f i e d by the level o f Na ÷ in the r e a c t i o n . The i n h i b i t i o n at 0.5, 1.0, 8.0, and 64 mM Na ÷ in the p r e s e n c e o f 2 H 2 0 was 50, 28, 23, and 7%, respectively. H o w e v e r , even at 128 mM Na ÷, a small b u t r e p r o d u c i b l e inhibition ( a b o u t 7%) by ~ H 2 0 was still apparent. Similarly, i n h i b i t i o n b y Me2 SO was r e d u c e d f r o m 69% at 8 mM Na ÷ t o 20% at 64 m M Na ÷. It m a y be m e n t i o n e d t h a t in previous work, it was established t h a t the i n h i b i t i o n o f (Na ÷ + K÷)-ATPase [4] or N a % d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n [11] b y ~ H 2 0 was n o t due t o alterations in the p H o p t i m a f o r these activies in the p r e s e n c e o f ~ H~ O. Time course o f the effect o f 2 H~ 0 on the formation o f p h o s p h o e n z y m e . T h e d a t a given in Fig. 1 d e p i c t the variations o f steady-state p h o s p h o e n z y m e with varying c o n c e n t r a t i o n s o f Na ÷. H o w e v e r , at any given c o n c e n t r a t i o n o f
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No* ( m ~ ) Fig. 2. E f f e c t of v a r y i n g c o n c e n t r a t i o n o f N a + o n t h e s t e a d y - s t a t e level of p h o s p h o e n z y m e in t h e p r e s e n c e and a b s e n c e o f 2 H 2 0 . Na + c o n c e n t r a t i o n was v a r i e d as s h o w n . P h o s p h o e n z y m e is p l o t t e d as the p e r c e n t of t h e m a x i m a l p h o s p h o e n z y m e f o r m e d (i.e. in t h e p r e s e n c e of 6 4 m M Na+). T h e a c t u a l a m o u n t of s t e a d y state p h o s p h o e n z y m e f o r m e d in t h e p r e s e n c e o f 64 m M Na + was 2 6 4 . 5 p m o l / m g of p r o t e i n in the H 2 0 s y s t e m , a n d 2 4 6 . 1 p m o l / m g of p r o t e i n in t h e 2 H 2 0 s y s t e m . All lines in p a n e l s B1, C2 a n d D 3 w e r e f i t t e d b y t h e m e t h o d o f l e a s t squares. All o t h e r e x p e r i m e n t a l details w e r e as d e s c r i b e d u n d e r M e t h o d s . ©, H 2 0 c o n t r o l s ; e, 80% 2 H 2 0 . Insets, p a n e l A s h o w s the d o u b l e r e c i p r o c a l p l o t (1/% E-P vs. l / N a +) in t h e H 2 0 s y s t e m ; the e r r o r b a r s d e n o t e S.E. d e r i v e d a c c o r d i n g t o t h e m e t h o d of J o h a n s e n a n d L u m r y [ 4 4 ] ; p a n e l s B1, C2 a n d D3 r e p r e s e n t t h e t h r e e s e g m e n t s m a r k e d at 1, 2 and 3 in p a n e l A in t h e p r e s e n c e of H 2 0 (o) or 80% 2 H 2 0 ( e ) r e a c t i o n m e d i a .
263 Na ÷, the level of phosphoenzyme formed remains steady over a time course of 30 s as shown in the figure; in other control experiments the steady-state level of phosphoenzyme formed in the presence of 8 mM Na ÷ did not change significantly ovel a period of 80 s (data not shown). The inhibition produced by 2 H2 O in the presence of 8 mM Na ÷ was also constant over a reaction period of 30 s. Formation of p h o s p h o e n z y m e in the presence of varying Na ÷. The effect of gradually increasing concentrations of Na ÷ on the formation of steady-state Na÷-dependent phosphoenzyme in the H~ O reaction media is shown in Fig. 2 (open circles). Maximal steady-state levels of Na÷-dependent phosphoenzyme were obtained at some 64 mM Na ÷. As is evident from Fig. 2 (inset A), a double reciprocal plot of the data showed a break at two points, yielding three linear segments designated in Fig. 2 inset A, as 1, 2 and 3. These segments are depicted as the expanded plots in the insets B1, C2, and D3 to illustrate that the slopes and the points of intersection on the ordinate are different for each of the three segments. The non-linearity of the plot in Fig. 2, inset A, may possibly be interpreted as empirical evidence for the presence of multiple nonequivalent Na ÷ sites for the formation of phosphoenzyme in the (Na ÷ + K÷)ATPase. However, this plot alone is considered inadequate to determine the number of sites and their constants [ 1 5 - - 1 7 ] . Therefore, the procedures described by Klotz and Hunston [15] and by Scatchard [18] to determine the number of binding sites and their kinetic constants, were considered. Since the steadystate phosphoenzyme varies with Na ÷ concentration (Fig. 1), we let the phosphoenzyme represent Na ÷ binding for its formation (see also the Discussion).
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Fig. 3. S c a t c h a r d p l o t a n a l y s i s o f N a + a c t i v a t i o n sites. Panel A , t h e v a l u e s f o r [ E - P ] a n d N a + are t h e s a m e as in T a b l e I. E, as d e f i n e d in t h e A p p e n d i x , is t h e t o t a l c o n c e n t r a t i o n o f e n z y m e in t h e r e a c t i o n ( 3 0 4 p m o l / m g o f p r o t e i n ) . T h e t h e o r e t i c a l v a l u e s ( × ) w e r e c a l c u l a t e d as d e s c r i b e d u n d e r t h e A p p e n d i x ; o, experimental values, where [E-P]/[El represents the moles of Na + bound per unit of the enzyme which h a s o n e c o v a l e n t l y l i n k e d p h o s p h a t e . P a n e l B, a t e s t f o r t w o n o n - e q u i v a l e n t s i t e s is s h o w n , o, e x p e r i m e n t a l v a l u e s ; + o r - - , t w o p o s s i b l e t h e o r e t i c a l fits w h i c h p r e s u m a b l y b r a c k e t t h e b e s t fit for this m o d e l . Panel C, t h r e e n o n - e q u i v a l e n t site m o d e l a p p l i e d t o N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n in t h e p r e s e n c e o f 2 H 2 0 . T h e v a l u e s o f t h e N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m e d in t h e p r e s e n c e o f 2 H 2 0 are as s h o w n in Fig. 2; t h e a m o u n t o f p h o s p h o e n z y m e at 6 4 m M N a + in t h e p r e s e n c e o f 8 0 % 2 H 2 0 w a s 2 4 6 . 1 p m o l / m g o f p r o t e i n , o, e x p e r i m e n t a l p o i n t s ; X , c a l c u l a t e d p o i n t s o n t h e basis o f t h r e e n o n - e q u i v a l e n t sites f o r N a +. All l i n e s are d r a w n t o fit t h e e x p e r i m e n t a l p o i n t ~ .
264
By doing so it is possible to plot the data in terms of a Scatchard plot as shown in Fig. 3 (panel A) to describe Na ÷ binding. The actual values of phosphoenzyme activity and standard errors are given in Table I. The three intercepts on the abscissa may suggest three classes of binding sites while the uppermost intercept on the ordinate gives a stoichiometric binding constant designated in the Appendix as/~. Based on the general equation (2) given in the Appendix, models for two and three equivalent and non-equivalent binding sites were tested by plotting r t = t[E-P]/[E] vs. [Na t] , as shown in Fig. 4, where [E-P] represents the phosphoenzyme concentration. The theoretical values for data points in Fig. 4, panels A and B, for the three and two non-equivalent site models were also plotted in Fig. 3, for comparison of experimental values with the theoretical values for three non-equivalent site (Fig. 3, panel A) or two non-equivalent site models (Fig. 3, panel B). As shown in Figs. 3 and 4, the two and three equivalent site models showed considerable deviation between the theoretical and experimental points; the result with a one-site model was so deviant as not to merit inclusion in the figure. The model with two non-equivalent sites also appeared to be incapable of fitting the experimental points within -+1 S.E. on both ends of the curve in Fig. 4. The deviation was minimal {within + 1 S.E.) when the data were fitted to a three non-equivalent site model, and a good agreement was noted between the calculated and the experimental points (Fig. 3, panel A and Fig. 4, panel A). The intrinsic association constants (K a) for the activation by Na ÷ were estimated from the three non-equivalent site model shown in Fig. 4, panel A, as described under the Appendix; the values of these constants in the H 2 0 system were 3.4, 0.295, and 0.025 mM -~ Other theoretical details for the above plots are given in the Appendix. Effect of 2H2 0 on the formation of phosphoenzyme in the presence o f varying Na ÷. The data given in Fig. 2 also show the effect of gradually increas-
TABLE I VALUES
O F D A T A P O I N T S I N F I G S . 2, 3 A N D 4
E-P r e f e r s t o t h e s t e a d y - s t a t e N a + - d e p e n d e n t p h o s p h o e n z y m e as p m o l / m g o f p r o t e i n , i n t h e p r e s e n c e o f t h e v a r i o u s c o n c e n t r a t i o n s o f N a + s h o w n . E r e f e r s t o t h e t o t a l c o n c e n t r a t i o n o f e n z y m e i n t h e r e a c t i o n as d e f i n e d i n t h e A p p e n d i x , a n d was 3 0 4 p m o l / m g o f p r o t e i n . T h e c a l c u l a t i o n s o f t h e s t a n d a r d e r r o r ( S . E . ) i n t h e v a r i o u s t r a n s f o r m a t i o n s o f E-P w e r e b a s e d o n t h e p r o c e d u r e o f J o h a n s e n a n d L u m r y [ 1 4 ] as d e scribed in the Appendix. No. of deterrninations
Na+ (raM)
[E-P] -+ S.E.
11 2 5 4 6 4 6 6 2 2 12 2
0.5 0.8 1.0 1.2 2.0 4.0 8.0 16.0 32.0 48.0 64.0 96.0
79.8 92.0 106.0 109.3 129.0 175.0 198.2 209.3 236.5 251.8 264.5 242.8
[E-PI/[El + S.E.
-+ 3 . 8 3 ± 5.67 + 8.20 -+ 8 . 4 0 -+ 7 . 3 3 -+ 7 . 9 7 -+ 7 . 1 4
-+ 6 . 0 8
0 . 2 6 2 3 -+ 0 . 0 1 4 0.3026 0.3486 + 0.022 0 . 3 5 9 5 -+ 0 . 0 3 1 0 . 4 2 4 3 -+ 0 . 0 3 1 0 . 5 7 5 6 -+ 0 . 0 3 4 0.6519 ± 0.034 0 . 6 8 8 4 -+ 0 . 0 3 3 0.7779 0.8282 0.870 + 0.028
265 3D
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)
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,5~ 72
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Fig. 4. Multiple site analysis of Na ÷ a c t i v a t i o n o f p h o s P h o e n z y m e f o r m a t i o n . Panel A, t h e c o n c e n t r a t i o n of N a +, v a l u e s o f [ E ] , [ E - P ] , a n d c a l c u l a t e d [ E - P ] / [ E ] are t h o s e given in T a b l e I a n d Fig. 3. T h e e q u a t i o n rt = >?i=lm N i K i [ N a +] ]1 + K i [ N a +], ( m = 3), w a s s o l v e d for t h r e e e q u i v a l e n t (~) or n o n - e q u i v a l e n t ( × ) a s s o c i a t i o n c o n s t a n t s f o r Na + (see the A p p e n d i x for details); ©, e x p e r i m e n t a l p o i n t s for t × [E-P] / [ E ] . Panel B, t h e s a m e e q u a t i o n as in p a n e l A e x c e p t t h a t m = 2 was solved for the t w o e q u i v a l e n t (A) or n o n - e q u i v a l e n t (+ or - - ) a s s o c i a t i o n c o n s t a n t s for Na+; o, e x p e r i m e n t a l points. All lines are d r a w n to c o n n e c t t h e t h e o r e t i c a l p o i n t s . T h e a p p a r e n t intrinsic a s s o c i a t i o n c o n s t a n t s ( K a ) f o r t h e a c t i v a t i o n b y Na ÷ at e a c h o f the t h r e e n o n - e q u i v a l e n t sites w e r e 3.4, 0 . 2 9 5 , a n d 0 . 0 2 5 m M -1, r e s p e c t i v e l y , in t h e p r e s e n c e of H 2 0 , a n d w e r e 1.4, 0 . 2 3 2 , a n d 0 , 0 3 3 m M -l, r e s p e c t i v e l y , in t h e p r e s e n c e o f 80% 2 H 2 0 .
ing co n cen tr atio ns of Na ÷ on the f o r m a t i o n of steady-state Na÷-dependent p h o s p h o e n z y m e in the reaction media containing 80% 2 H~ O. A double reciprocal p lo t o f the data (i.e. 1/E-P vs. 1/Na ÷) gave three linear segments as in the case o f the p l o t obtained in the presence of H~ O (not shown in the inset A, but shown in the e x p a n d e d scale segments B1, C2, and D3 of Fig. 2). Further, it appeared th at the segments designated as no. 1 and as no. 2 in bot h H~ O and 2 H~ O in ter cep te d the ordinate at the same point in each case, whereas the segments designated as no. 3 did n o t m e e t at the same p o i n t on the ordinate in the presence of H~ O c o m p a r e d with ~ H~ O. The result suggests that over the range o f c o n c e n t r a t i o n of Na ÷ given in the insets B1 and C2, the maximal p h o s p h o e n z y m e activity attainable is essentially the same in the presence or absence o f ~ H2 O, and t ha t the r educt i on in the p h o s p h o e n z y m e levels in the presence o f 2 H~ O at these concentrations of Na ÷ may be due to a reduced affinity o f Na ÷. We f u r t h e r subjected the Na÷-dependent p h o s p h o e n z y m e data obtained in the presence of ~ H~ O to multiple-site analysis as in the case of the data obtained in the presence o f H~ O described above (Fig. 3, panel C), which also indicated three Na ÷ activation sites. Again, using the intercept on the ordinate o f Fig. 3, panel C, and assuming different ratios of Na ÷ binding per p h o s p h o e n z y m e molecule, tests o f the n u m b e r of Na ÷ sites and their equiva-
266 lency s h o w e d t h a t an equivalent site m o d e l would n o t fit the data (plots n o t included in Fig. 4). The intrinsic association c o n s t a n t s for Na ÷ ( g a ) i n the presence o f 2 H2 O for a three n o n - e q u i v a l e n t site m o d e l were e s t i m a t e d as 1.4, 0.232 and 0.033 mM -1, respectively. These values, c o m p a r e d with the constants o b t a i n e d f r o m the e x p e r i m e n t s with H2 O r e a c t i o n media, indicate a differential e f f e c t o f 2 H~ O on the three sites. The decrease in the first t w o constants (K a ) by 2 H~ O was 59 and 21%, respectively, while for the third one there was an increase o f 32%. F u r t h e r , a c o m p a r i s o n o f the i n t e r c e p t s on the [ E - P ] / [ N a ÷] [E] axis for H~O (Fig. 3, panel A) and ~ H~O (Fig. 3, panel C) shows an overall r e d u c t i o n o f 47% in the affinity o f Na*. It m a y be recalled t h a t 80% 2 H2 O gives an average o f 40% inhibition o f the (Na + + K ÷)-ATPase [4]. Consideration o f two non-equivalent site models. We have p r o p o s e d above t h a t a t h r e e n o n - e q u i v a l e n t Na ÷ activation site m o d e l shows the best fit to o u r d a t a w h e n all points are c o n s i d e r e d within +1 S.E. o f each mean. However, on the basis o f +2 S.E. o f each mean, the t w o n o n - e q u i v a l e n t site m o d e l (Fig. 4, panel B, b r o k e n line) is n o t distinguishable f r o m the three n o n - e q u i v a l e n t site model. If this were the case the K a values (derived b y the same p r o c e d u r e s as described above) for Na ÷ activation o f the e n z y m e in the presence o f H~ O m e d i u m were e s t i m a t e d at 2.440 and 0.041 mM -~ , respectively. In the presence o f ~ H~ O, these values were changed to 1.062 and 0 . 0 4 8 mM -1, respectively. The relative i n h i b i t o r y e f f e c t o f 2 H2 O on the high affinity n o n - e q u i v a l e n t site w o u l d thus be 56% with an increase o f 17% o n the low affinity site.
Effect o f the order o f addition o f ligands on the inhibition by ~H~ 0 and Me, SO. R e c e n t l y , Albers and Koval [6] s h o w e d t h a t Me~ SO did n o t inhibit the Na÷-dependent p h o s p h o e n z y m e f o r m a t i o n , whereas Kaniike et al. [8] were
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20
Time (s) Fig. 5. T i m e c o u r s e o f t h e e f f e c t o f M e 2 S O o n N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n a n d t h e e f f e c t o f t h e o r d e r o f t h e a d d i t i o n o f v a r i o u s c o m p o n e n t s . T h e e n z y m e w a s a l l o w e d a c o n t a c t w i t h N a + o r K +, a n d Me 2 S O i n t h e f o l l o w i n g o r d e r p ~ i o r t o t h e s t a x t o f t h e r e a c t i o n : ©, e n z y m e + N a + i n H 2 0 s y s t e m ; ~, e n z y m e + N a +, f o l l o w e d b y M e 2 S O ; A, e n z y m e + K +, f o l l o w e d b y M e 2 S O ; t , e n z y m e + K + i n H 2 0 s y s t e m ; u, N a + + M e 2 S O , f o l l o w e d b y t h e e n z y m e ; m, K + + M e 2 S O , f o l l o w e d b y t h e e n z y m e . A l l o t h e r e x p e r i m e n t a l d e t a i l s w e r e t h e s a m e as f o r e x p e r i m e n t s n o . 1 - - 6 i n T a b l e II.
267 O ATP__ E2
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(~) E 1 • (No o r ATP)
--~IMgATP'Na~'~--
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@ ~o -H2~
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Fig. 6. Sites o f a c t i o n o f s o l v e n t s a n d H 2 0 in t h e o p e r a t i o n o f t h e ( N a + + K+)-ATPase. ( 1 ) N a + or A T P s h i f t t h e e q u i l i b r i u m w i t h r e s p e c t to t h e e n z y m e c o n f o r m a t i o n f r o m E 2 to E l , w h e r e a s H 2 0 , 2 H 2 0 , a n d Me 2 SO will r e v e r s e it; o r g a n i c s o l v e n t s s u c h as a c e t o n e , e t h e r , a l c o h o l s [ 9 ] a n t a g o n i z e t h e a c t i o n o f H 2 O, a n d m a y s h i f t t h e e q u i l i b r i u m to t h e right~ ( 2 ) E 1 N a or E 1 A T P r e a c t s w i t h s u b s t r a t e M g A T P a n d Na~ to f o r m t h e i n t e r m e d i a t e [E 1 • M g A T P - N a x ] w h i c h l e a d s to t h e f o r m a t i o n o f E2-P • N a x ; ( 3 ) t h e i n t e r a c t i o n o f K~ is f a c i l i t a t e d b y H 2 O, 2 H2 O a n d Me 2 SO, a n d a n t a g o n i z e d b y o r g a n i c s o l v e n t s ; ( 4 ) t h e i n t e r a c t i o n o f H 2 0 on t h e b r e a k d o w n o f K + - m e d i a t e d p h o s p h o e n z y m e is e v i d e n c e d b y s t i m u l a t o r y e f f e c t o f 2 H 2 0 or M e 2 S O , a n d i n h i b i t o r y e f f e c t o f o r g a n i c s o l v e n t s ; (5) t h e p r e s e n c e o f N a + or A T P leads t o t h e d i s s o c i a t i o n o f E 2 • Ky b e c a u s e o f t h e a c t i o n o f t h e s e a g e n t s at s t e p 1, a b o v e .
able to d e m o n s t r a t e an i n h i b i t i o n o f the p h o s p h o e n z y m e f o r m a t i o n b y Me2 SO. T h e results s u m m a r i z e d in Table II clearly illustrate t h a t the p r i o r c o n t a c t o f the e n z y m e with Na ÷ or A T P c o n s i d e r a b l y alters the i n h i b i t o r y e f f e c t o f Me2 SO or ~ H 2 0 on the steady-state level o f p h o s p h o e n z y m e . When Me: SO and Na +, or 2 H2 O and Na ÷, are a d d e d t o g e t h e r , a large i n h i b i t i o n is observed which is even greater w h e n Me~ SO or : H: O are in c o n t a c t with the e n z y m e
T A B L E II EFFECT
OF THE ORDER
O F A D D I T I O N O F L I G A N D S O N T H E I N H I B I T I O N BY 2 H 2 0 O R M e 2 S O
A d d i t i o n s in t h e s e q u e n c e l i s t e d w i t h e a c h e x p e r i m e n t w e r e m a d e to t h e s t a n d a r d r e a c t i o n m e d i u m (as g i v e n u n d e r M e t h o d s ) . F o r e x p e r i m e n t nos. 1 ~ 6 , w h e r e i n d i c a t e d , c a t i o n s a n d e n z y m e w e r e in c o n t a c t f o r 10 r a i n f o l l o w e d b y a 6 0 - r a i n c o n t a c t w i t h M e 2 S O o r 2 H 2 0 . W h e n c a t i o n s a n d M e 2 S O o r 2 H 2 0 w e r e a d d e d t o g e t h e r to t h e e n z y m e , t h e i n c u b a t i o n w a s c a r r i e d o u t f o r 60 rain. W h e n M e 2 S O a n d 2 H 2 0 w e r e a d d e d p r i o r to c a t i o n s , t h e c o n t a c t w i t h t h e e n z y m e w a s f o r 10 m i n , t h e n an a d d i t i o n a l 50 r a i n f o l l o w i n g c a t i o n s . F o l l o w i n g t h e s e t r e a t m e n t s , t h e r e a c t i o n w a s s t a r t e d by t h e a d d i t i o n o f [7-52P] A T P a n d w a s t e r m i n a t e d 4 s later. In e x p e r i m e n t n o . 7, t h e e n z y m e w a s in c o n t a c t w i t h A T P ( c o n t a i n i n g [ 7 - ~ 2 P ] A T P ) f o r 1 0 s f o l l o w e d b y 10 s w i t h M e 2 S O , a n d t h e n t h e r e a c t i o n w a s i n i t i a t e d b y t h e a d d i t i o n o f Na+ a n d w a s t e r m i n a t e d 10 s l a t e r . I n e x p e r i m e n t 8, M e 2 S O w a s i n c o n t a c t w i t h t h e e n z y m e f o r 10 s f o l l o w e d bY [ 7 - 3 2 P ] A T P a n d Na+ o r K"~ f o r an a d d i t i o n a l 10 s. T h e a m o u n t o f e n z y m e (E) w a s 50 # g , M e 2 S O , 2 H 2 0 , a n d N a+ w e r e p r e s e n t at a c o n c e n t r a t i o n o f 3 0 % , 80%, a n d 8 raM, r e s p e c t i v e l y . V a l u e s f b r N a + - i n d e p e n d e n t p h o s p h o e n z y m e (i.e. in t h e p r e s e n c e o f Mg 2+ + 16 m M K+) w e r e s u b t r a c t e d f x o m e x p e r i m e n t a l values, to o b t a i n N a + - d e p e n d e n t p h o s p h o e n z y m e , a n d o t h e r a p p r o p r i a t e c o n t r o l s w e r e i n c l u d e d t h r o u g h o u t , All o t h e r d e t a i l s w e r e t h e s a m e as g i v e n u n d e r M e t h o d s . Exp. no.
Sequence of additions
Percent inhibition of phosphoenzyme
1 2 3 4 5 6 7 8
2 H 2 0 + E + Na+ 2H20+Na ++ E Na++ E+2H20 M e 2 S O + E + Na + Me2SO + Na + + E Na + + E + M e 2 S O ATP + E + Me2SO Me2SO + E + ATP
44 36 24 77 69 2 1 34
268 p r i o r to the a d d i t i o n o f Na ÷. This suggests t h a t Me2 SO a n d 2 H 2 0 b l o c k the i n t e r a c t i o n of Na ÷ with its a c t i v a t i o n sites. F u r t h e r , if A T P is p r e s e n t on the e n z y m e , Na ÷ can also b i n d to its sites w i t h o u t i n t e r f e r e n c e f r o m Me2 SO. This e x p e r i m e n t gives clear evidence t h a t the r a n d o m m e c h a n i s m w o r k s in the (Na * + K*)-ATPase since either Na ~ or A T P m a y get on to their sites i n d e p e n d e n t l y and t h a t with b o t h p r e s e n t , p h o s p h o e n z y m e f o r m a t i o n will t a k e place. T h e f a c t t h a t p r i o r c o n t a c t of the e n z y m e with A T P leads to the f o r m a t i o n o f p h o s p h o e n z y m e in the p r e s e n c e o f Na * and Me2 SO suggests t h a t A T P b o u n d to the e n z y m e assists Na * to a t t a c h to its active sites. The d a t a in Fig. 5 s h o w t h a t the e f f e c t o f t h e o r d e r o f a d d i t i o n o f ligands is n o t m o d i f i e d over a t i m e course in the p r e s e n c e o f Me~ SO. Similar results were o b s e r v e d with ~ H~ O (Fig. 2, and o t h e r d a t a n o t shown).
Discussion Previous studies utilizing the analyses o f the kinetics o f a c t i v a t i o n of the (Na ÷ + K*)-ATPase b y varying c o n c e n t r a t i o n s o f Na ÷ in the p r e s e n c e of varied or fixed c o n c e n t r a t i o n s o f K ÷ [ 1 4 , 1 9 ] have i n d i c a t e d t w o or t h r e e e q u i v a l e n t Na ÷ a c t i v a t i o n sites. T h e s e m o d e l s do n o t clearly distinguish b e t w e e n the e q u i v a l e n t and n o n - e q u i v a l e n t a c t i v a t i o n sites [ 1 9 ] . This t y p e o f a p p r o a c h is also c o m p l i c a t e d b y c o m p l e x i n t e r a c t i o n o f cations at e x t r e m e s o f c o n c e n t r a t i o n ratios. A T P - d e p e n d e n t binding o f Na t to the A T P a s e has been investigated, b u t has n o t b e e n utilized for binding-site analysis [ 2 0 - - 2 2 ] . T h e e x p e r i m e n t a l design in the p r e s e n t w o r k relies on the f o r m a t i o n o f N a ÷ - d e p e n d e n t p h o s p h o e n z y m e (the b r e a k d o w n o f which is sensitive to K÷), w h e r e b y the c o m p l i c a t e d i n t e r a c t i o n s o f N a ÷ a n d K ÷ are avoided, since t h e r e a c t i o n s y s t e m c o n t a i n s o n l y Na *, and only the N a ÷ - d e p e n d e n t s t e a d y - s t a t e p h o s p h o e n z y m e f o r m e d is examined. If, as suggested b y a large b o d y o f evidence, the p h o s p h o e n z y m e is the i n t e r m e d i a t e in the o p e r a t i o n o f the (Na" + K ' ) - A T P a s e and m a y be r e g a r d e d as the Na÷-carrier in the p u m p cycle [ 2 , 3 , 2 3 - - 2 6 ] (see also 1), t h e n it m a y be possible to use the p h o s p h o e n z y m e as a m e a s u r e of Na t binding. In this case, the s t e a d y state e q u a t i o n s and graphical m e t h o d s for s t u d y i n g the binding of small m o l e c u l e s to m a c r o m o l e c u l e s m a y be applied to analyse Na ÷ b o u n d t o p h o s p h o e n z y m e (see e.g. 1 5 , 1 8 , 2 7 , and the A p p e n d i x ) . By assuming t h a t cation binding sites are i n d e p e n d a n t with fixed affinities and w i t h o u t defining the p h y s i c a l n a t u r e o f the m a c r o m o l e c u l a r state o f t h e carrier, the s t e a d y - s t a t e b i n d i n g e q u a t i o n s w o u l d a p p l y w h e t h e r the e n z y m e were p r e s e n t as d i f f e r e n t species each having a distinct a f f i n i t y c o n s t a n t or as a single species with all o f the d i s t i n c t binding sites on a single m a c r o m o l e c u l e . T h e p r e s e n t m o d e l a t t e m p t s to relate the s t o i c h i o m e t r y o f Na * binding p e r p h o s p h o e n z y m e m o l e c u l e b a s e d on the o b s e r v e d c o r r e s p o n d e n c e b e t w e e n Na ÷ c o n c e n t r a t i o n a n d s t e a d y - s t a t e level o f p h o s p h o e n z y m e . T h e binding o f Na ÷ to its sites w o u l d be a c o n s e q u e n c e o f an e q u i l i b r i u m involving r e a c t i o n 1 as s h o w n in Fig. 6. It w o u l d be i m p l i e d f r o m the law o f mass a c t i o n as discussed b y S c a t c h a r d [18] t h a t t h e p r o b a b i l i t y o f o c c u p a n c y o f m u l t i p l e Na ÷ binding sites w o u l d be d e p e n d e n t u p o n the Na ÷ c o n c e n t r a t i o n . T h e result f r o m experim e n t s 4 and 6 in T a b l e II suggests t h a t p h o s p h o r y l a t i o n w o u l d o c c u r o n l y
269 when the conditions of reaction 2 (Fig. 6) are met, including a sufficient a m o u n t o f b o u n d Na ÷ per potential p h o s p h o r y l a t i o n site. Accordingly, the steady-state level of p h o s p h o r y l a t i o n may be used as a measure of Na ÷ t h a t was b o u n d prior to p h os phor yl a t i on. This is in agreement with the c o n c e p t t hat the c o n cen tr atio n s of Na ÷ and K ÷ m a y shift the steady-state concentrations of carrier moieties to control the rates of cation transport [ 2 8 ] . Our suggestion of three non-equivalent Na ÷ activation sites for (Na ÷ + K÷}-ATPase is compatible with the data on the kinetics of transport in nerve where it was shown that three sites with different Na ÷ affinities were necessary to a c c o u n t for the Na + activation o f Na÷-Na ÷ exchange [ 2 9 ] . However, in red cells, three' sites of equal apparent affinity for Na ÷ are involved in Na+-Na + exchange [ 3 0 ] . It would be interesting to determine if the nature of Na ÷ activation sites (i.e. equivalent or non-equivalent) varies with tissues. The possibility of interaction between the multiple non-equivalent Na ÷ sites postulated above c a n n o t be ruled o u t at the present. Nor can we rule o u t t hat our results are due to the possible i n h o m o g e n e i t y of the e n z y m e system used. Further, based on strict statistical considerations, the two non-equivalent site model may also fit the data. The results in Table II have several implications. If b o u n d H2 O plays a role at the Na ÷ sites [ 4 , 1 1 ] , it would seem reasonable that the agents such as Me2 SO and 2 H 2 0 which form stronger h y d r o g e n (or deuterium) bonding by replacing H~ O [32,33] would tend to block the interaction of Na ÷ with its activation sites. We propose that H~ O shifts the c o n f o r m a t i o n of the e n z y m e f r o m E, to E~, while Na ÷ reverses this trend (Fig. 6). This would imply that Na ÷ must expel the b o u n d H~ O f r om its activation sites in order to bind to these sites, although this does n o t seem to be the case at one of the Na ÷ sites (i.e. the weak Na ÷ binding site with the lowest Ka). Removal of H~ O from the higher affinity Na ÷ sites could increase the h y d r o p h o b i c i t y in the region, thereby preventing the spontaneous br eakdow n o f the p h o s p h o e n z y m e [ 9 ] . However, H2 O and K ÷ will tend to move the [K-E~-P] com pl ex back to a hydrophilic region where the p h o s p h o e n z y m e will be h y d r o l y z e d . Thus 2 H~ O and Me~ SO would act more effectively than H~ O to favor the shift of E, to the E~ f o rm o f the en zym e , which would result in reduced steady-state level of Na ÷d e p e n d e n t phosphoen.zyme f o r m a t i o n in the presence of ~ H2 O or Me~ SO. This will also be c o m m e n s u r a t e with increased affinity of K ÷ for the K ÷ stimulated p - n i t r o p h e n y l phosphatase activity in the presence of ~ H~ O [}1,11] and Me~ SO [ 6 , 7 ] , and a stimulation of K÷-mediated breakdown o f p h o s p h o e n z y m e [ 3 4 , 3 5 ] . The result in Table II suggests t hat ATP m ay act to cause a dissociation o f the K-E2 c o m p l e x [24] to release K ÷, by producing a shift of E~ c o n f o r m a t i o n to E, c o n f o r m a t i o n (Fig. 6). Further, the removal of H~ O from Na ÷ sites in the ATPase by solvents such as alcohols, acetone, ether, etc. would facilitate the binding of Na ÷ (E, c o n f o r m a t i o n , Fig. 6), and increase the phosp h o e n z y m e f o r m e d due to stabilization of the com pl ex [ 9 ] , whereas K ÷mediated h y d r o l y t i c steps would be inhibited [5, 9]. The increased affinity of K ÷ for the ATPase at r e duc e d temperatures [41,42] m ay also be explained if the e n z y m e c o m p l e x favors the E~ c o n f o r m a t i o n at lower temperatures due to an increase in b o u n d H~ O a n d / o r stronger H bondings, while the reverse is the case at higher temperatures, i.e. the E, c o n f o r m a t i o n is favored.
270 T o c o n c l u d e , the effects o f : H: O and Me: SO on the (Na ~ + K~)-ATPase, and possibly o t h e r m e m b r a n e - a s s o c i a t e d f u n c t i o n s [ 3 6 - - 4 0 ] , m a y be due t o conf o r m a t i o n a l changes caused by r e p l a c e m e n t o f H: O in the m e m b r a n e s t r u c t u r e and f o r m a t i o n o f stronger H (or : H) bonding. F u r t h e r , the affinity o f Na ÷ and K ÷ f o r their sites in the o p e r a t i o n o f the (Na ~ + K÷)-ATPase m a y be regulated by the b o u n d H: O al] the active center. The points t h a t favor this conclusion, and argue against a simple e f f e c t o f 2 H~ O on cation h y d r a t i o n alone t o explain the present and the previous w o r k b y us are as follows: (1) : H 2 0 inhibits (Na ÷ + K÷)-ATPase, b u t stimulates K*-stimulated p - n i t r o p h e n y l p h o s p h a t a s e ; the stimulated activity is b l o c k e d b y o l i g o m y c i n [4,11] ; (2) I n h i b i t i o n o f p h o s p h o e n z y m e f o r m a t i o n by : H: O or Me2 SO is n o t altered over a time course, b u t changes with Na ÷ c o n c e n t r a t i o n ; (3) : H: O acts differentially o n multiple nonequivalent Na ÷ sites; (4) The o r d e r o f addition o f r e a c t a n t s influences the e f f e c t o f solvents; (5) 2 H 2 0 (and Me: SO) a c t i o n on K÷-stimulated p - n i t r o p h e n y l p h o s p h a t a s e is r e f l e c t e d in the stimulation o f a K~-mediated b r e a k d o w n o f a slow c o m p o n e n t o f p h o s p h o e n z y m e b y these solvents [34,35] ; and, (6) : H 2 0 stimulates the rate, and steady-state level o f Mg 2÷ + Pi or Mn:÷-supported ouabain binding to the ATPase, whereas it inhibits the rate, b u t n o t the steadystate level, o f (Na ÷ + A T P ) - d e p e n d e n t ouabain binding [ 4 5 ] . Appendix T h e o r y f o r data expressed in Figs. 3 and 4
We m a k e the following identities and assumptions: [E-P] represents specific Na" binding; r t = moles Na ÷ b o u n d / u n i t o f e n z y m e = t [ E - P ] / [ E ] ; [E] = t o t a l e n z y m e c o n c e n t r a t i o n = [E-P] m ax + 0.15 × [E-P] max in the absence o f K ÷ [43] ; f u r t h e r , the value for [E] in 80% 2 H2 O was t a k e n as 93% o f t h a t in H2 O since a 7% inhibition o f the p h o s p h o e n z y m e at 64 mM Na ÷ was observed in the presence o f 80% : H2 O; [Na] = free Na ÷ c o n c e n t r a t i o n = [Na] W t[E-P] ~ [Na] T, the total c o n c e n t r a t i o n , since [Na] T is in units o f mM and t[E-P] is in units o f nM; t = total n u m b e r o f Na ÷ b o u n d t o the p h o s p h o e n z y m e ; [E-P] = [Nat • E-P], the N a - p h o s p h o e n z y m e c o m p l e x ; e = free e n z y m e c o n c e n t r a t i o n = [El - - [E-P] ; K a = [ E - P ] / ( [ N a ] [e] ), the intrinsic association c o n s t a n t . Therefore, "
t[E-P]
_
rt = [E-P] + [e]
tKa[Na] 1 + Ka[Na ]
(1)
In general, Ft =
~ N'~i[Na] i=1 1 + K i [ N a ]
(2)
where t = X ~ I Ni = the t o t a l n u m b e r o f sites, and where N i and K i are the n u m b e r o f identical sites and the intrinsic association c o n s t a n t , respectively, f o r each class o f sites i. By holding the total e n z y m e c o n c e n t r a t i o n [E] c o n s t a n t , the o n l y d e p e n d e n t variable is [E-P] and the only i n d e p e n d e n t variable is [ N a ] . F o r e q u a t i o n (2) it is a p p r o p r i a t e t o p l o t r t vs. [Na ÷] (Fig. 4). F u r t h e r , the
271 following form of Scatchard equation [17,18] graph of rt/[Na] vs. rt (Fig. 3). rt/[Na]
is employed to construct a
(3)
= tK -- rtK
In plotting the experimental data by this method, it was first assumed that t = 1; therefore, r t = [E-P]/[E]. The general solution of equation (3) states that the intercept on the abscissa gives the total number of binding sites, t, and the intercept on the ordinate gives the product of t and the average of all the association constants, (K) [15]. In Fig. 3, the intercept on the [E-P]/[E] axis is essentially 1, which, in terms of p h o s p h o e n z y m e formation as expressed in equation (3), means that as Na ÷-~ ¢*, all of the enzyme is converted into phosphoenzyme. However, by letting the phosphoenzyme represent bound Na ÷, the intercept on the abscissa simply gives the value of t that would be the assumed stoichiometry between the phosphoenzyme and Na ÷ binding. The uppermost intercept on the ordinate as shown in Fig. 4a, gives the c o n s t a n t / ~ , such that
~ /~ = t(K) where (K) =
m N~K~/~ N i
i=1
(4)
i=l
and where the Ki values are the intrinsic association constants as given in equation (2). For t = 1 , / ~ = 1.24 = N1 K1 as shown in Fig. 3A; for t, = 2, ~ = 2.48 = N ~ K ~ + N 2 K 2 ; a n d for t = 3,/~ = 3.72 = N ~ K 1 + N 2 K 2 + N 3 K 3 , etc. By assuming different values for t, Ni and K~, and using the experimental values of Na ÷ concentration employed, theoretical values of r~ for 1-, 2- and 3-site models were generated from equation (2~), with the appropriate values o f / f as a constraint. For equivalent site models K was divided equally among all the K~ values. Initial estimates for the Ki values in the non-equivalent site models may be obtained by substituting experimental values of rt, [Na], (K), and assumed values of Ni and t into equations (2) and (4). These equations are solved for a value of K~ which is substituted in the equations and the process is repeated until estimates of all the K,- values are obtained. These initial estimates of K; values are then varied by trial and error until the best fit is obtained. Since the values of r t calculated from equation (2) represent theoretical values for [EP ] / [ E ] , theoretical values for [E-P]/[Na] [E] were obtained by dividing the calculated values of r t by the experimental values of [Na], and are used to test the various models in a Scatchard plot as shown in Fig. 3. The K i values that gave the best fit for a three non-equivalent site model (Fig. 4A) were designated as the intrinsic association constants (Ka) for Na ÷. When plotting points from Fig. 3 to Fig. 4 and vice versa, the obvious division or multiplication by t was performed. Calculation of the standard error
The experimental values of [E-P]/[E] and the error associated with each are given in Table I. From equation (9) of Johansen and L u m r y [44], we define
272
the variance for
rt =
t[E-P]/[E] as follows:
from which S.E. = ( ~ r t / n I ] 2 was calculated, based on the assumption that the experimental determination of [E-P] and [E] are stochastically independent. Although [E] is calculated from [E-P] m ax, the measurement of [E-P] made at one concentration of Na ÷ is independent of that made at another.
Acknowledgements We are greatly indebted to Professor Rufus Lumry, Laboratory of Biophysical Chemistry, Department of Chemistry and Dr. R. Sawchuk, Department of Pharmaceutics, University of Minnesota, for valuable discussions which guided us to the relevant literature, and for evaluation of the application of the mathematical expressions in the appendix used for plots presented in Figs. 3 and 4. We thank Mr. Gregory Quarfoth and Mr. Alan Davis for their help in this work.
References 1 S k o u , J.C. ( 1 9 7 2 ) B i o e n e r g e t i c s 4, 2 0 3 - - 2 3 2 2 S c h w a r t z , A., L i n d e n m a y e r , G.E. a n d Allen, J. ( 1 9 7 2 ) in C u r r e n t T o p i c s in M e m b r a n e T r a n s p o r t ( B o n n e t , F. a n d K l e i n z e l l e r , A., e d s . ) Vol. 3, p p . 1 - - 8 2 , A c a d e m i c Press, N.Y. 3 D a h l , J . L . a n d H o k i n , L.E. ( 1 9 7 4 ) A n n u . Rev. B i o c h e m . 4 3 , 3 2 7 - - 3 5 6 4 A h m e d , K., Riggs, C. a n d I s h i d a , H. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 6 1 9 7 - - 6 2 0 3 5 Israel, Y. a n d S a l a z a r , I. ( 1 9 6 7 ) A r c h . B i o c h e m . B i o p h y s . 1 2 2 , 3 1 0 ~ 3 1 7 6 A l b e r s , R.W. a n d K o v a l , G.J. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 3 0 8 8 - - 3 0 9 2 7 Robinson, J.D. (1972) Biochim. Biophys. Acta 274, 542--550 8 K a n i i k e , K., E r d m a n n , E. a n d S c h o n e r , W. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 5 2 , 2 7 5 ~ 2 8 6 9 H e g y v a ~ y , C. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 2 7 2 - - 2 9 1 1 0 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 5 ) F e d . P r o c . 3 4 , 3 2 8 11 A h m e d , K. a n d F o s t e r , D. ( 1 9 7 4 ) A n n . N.Y. A c a d . Sci. 2 4 2 , 2 8 0 - - 2 9 2 1 2 A h m e d , K. a n d T h o m a s , B.S. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 0 3 - - 1 0 9 13 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 4 ) B i o c h i m . B i o p h y s . A c t a 9 3 , 6 0 3 - - 6 1 3 1 4 A h m c d , K., J u d a h , J . D . a n d S c h o l e f i e l d , P.G. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 0 , 3 5 1 - - 3 6 0 1 5 K l o t z , I.M. a n d H u n s t o n , D.L. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 3 0 6 5 - - 3 0 6 9 1 6 S c h e l l m a n , J . A . , L u m r y , R. a n d S a m u e l s , L.T. ( 1 9 5 4 ) J . A m e r . C h e m . Soc. 76, 2 8 0 8 - - 2 8 1 3 17 Walter, C. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 9 9 - - 7 0 3 1 8 S c a t c h a r d , G. ( 1 9 4 9 ) A n n . N.Y. A c a d . Sci. 5 1 , 6 6 0 - - 6 7 2 19 L i n d e n m a y e r , G.E., S c h w a r t z , A. a n d T h o m p s o n , Jr., H . K . ( 1 9 7 4 ) J. P h y s i o l . 2 3 6 , 1 - - 2 8 2 0 V o n S t e d i n g k , L.V. a n d J a r n e f e l t , J. ( 1 9 6 3 ) A c t a P h y s i o l . S c a n d . 5 7 , 3 2 8 - - 3 3 8 21 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 1 2 , 5 8 - - 6 2 2 2 O s t r o y , F., J a m e s , T . L . , N o g g l e , J . H . , Sarrif, A. a n d H o k i n , L.E. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 2 , 421--425 2 3 J u d a h , J . D . a n d A h m e d , K. ( 1 9 6 4 ) Biol. R e v . C a m b r i d g e 3 9 , 1 6 0 - 1 9 3 2 4 P o s t , R . L . , H e g y v a r y , C. a n d K u m e , S. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 6 5 3 0 - - 6 5 4 0 2 5 A s k a r i , A. (ed.) ( 1 9 7 4 ) A n n a l s N . Y . A c a d . Sci. Vol. 2 4 2 26 K a n a z a w a , T., Y a m a d a , S., Y a m a m o t o , T. a n d T o n o m t t r a , Y. ( 1 9 7 1 ) J. B i o c h e m . T o k y o 7 0 , 9 5 - - 1 2 3 27 D a v i s o n , C. ( 1 9 7 1 ) in F u n d a m e n t a l s of D r u g M e t a b o l i s m a n d D r u g D i s p o s i t i o n ( L a D u , B.N., M a n d e l , H . G . a n d W a y , E.L., eds.) p p . 6 3 - - 7 4 , T h e Williams a n d Wilkins Press, N . Y . 2 8 R e p k e , K . R . H . , S c h / i n , R., H e n k e , W., S c h S n f e l d , W., S t r e c h e n b a c h , B. a n d D i t t r i c h , R. ( 1 9 7 4 ) A n n . N.Y. A c a d . Sci 2 4 2 , 2 0 3 - - 2 1 9 2 9 B a k e r , P . F . , B l a u s t e i n , M.P., K e y n e s , R . D . , Manil, J., S h a w , T.I. a n d S t e i n h a r d t , R . A . ( 1 9 6 9 ) J. Physiol. 200, 459--496
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30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
G a t a y , R.P. a n d G a x r a h a n , P.J. ( 1 9 7 3 ) J. P h y s i o l . L o n d . 2 3 1 , 2 9 7 - - 3 2 5 G l y n n , I.M. a n d K a r l i s h , S . J , D . ( 1 9 7 5 ) A n n u . R e v . P h y s i o l . 3 7 , 1 3 - - 5 5 R a m m l e r , D . H . a n d Z a f f a r o n i , A. ( 1 9 6 7 ) A n n . N . Y . A c a d . Sci. 1 4 1 , 1 3 - - 2 3 M a c G r e g o r , W.S. ( 1 9 6 7 ) A n n . N.Y. A c a d . Sci. 1 4 1 , 3 - - 1 2 F o s t e r , D. a n J A h m e d , K. ( 1 9 7 4 ) P h a r m a c o l o g i s t 1 6 (2), 2 9 3 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 4 ) F e d . P r o c . 3 3 (5), 1 3 3 2 De B r u i j n e , A.W., V a n 1~ees, H. a n d V a n S t e v e n i n c k , J. ( 1 9 7 1 ) B i o c h e m . P h a r m a c o l . 20, 2 6 8 7 - - 2 6 9 3 V o n E u l e r , U.S. a n d L i s h a j k o , F. ( 1 9 7 1 ) A c t a P h y s i o l . S c a n d . 8 2 , 1 3 1 - - 1 3 4 S v e n s m a x k , O. ( 1 9 6 1 ) A c t a P h y s i o l . S c a n d . 5 3 , 7 5 - - 8 4 K a m i n e r , B. a n d K i m u r a , J. ( 1 9 7 2 ) S c i e n c e 1 7 6 , 4 0 6 - - 4 0 7 K i h a r a , T. a n d M c C r a y , J . A . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 2 9 2 , 2 9 7 - - 3 0 9 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 5 ) C a n . J. B i o c h e m . 4 3 , 8 7 7 - - 8 8 0 S c h S n , R., S c h S n f e l d , W., M e n k e , K . - H . a n d R e p k e , K . R . H . ( 1 9 7 2 ) A c t a Biol. M e d . Get. 2 9 , 6 4 3 - - 6 5 9 K a n a z a w a , T., S a i t o , M. a n d T o n o m u r a , Y. ( 1 9 7 0 ) J. B i o c h e m . T o k y o 6 7 , 6 9 3 - - 7 1 1 J o h a n s e n , G. a n d L u m r y , R. ( 1 9 6 1 ) C . R . T r a y . L a b . C a r l s b e r g 3 2 , 1 8 5 - - 2 1 4 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 5 ) P h a r m a c o l o g i s t 1 7 , 1 9 9
Biochimica et Biophysica Acta, 429 ( 1 9 7 6 ) 2 5 8 - - 2 7 3 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 67713
Na÷-DEPENDENT P H O S P H O R Y L A T I O N OF T H E RAT BRAIN (Na + + K+)-ATPase POSSIBLE NON-EQUIVALENT ACTIVATION SITES FO R Na ÷
DONALD FOSTER and KHALIL AHMED
Toxicology Research Laboratory, Minneapolis Veterans Hospital, Minneapolis, Minn. 55417 *, and the Department o f Laboratory Medicine and Pathology, University o f Minnesota 55455 (U.S.A.) (Received July 28th, 1975)
S u m mar y 1. The steady state levels of Na÷-dependent p h o s p h o e n z y m e (E-P) in the (Na + + K÷)-ATPase (EC 3.6.1.3) of rat brain, obtained from a time course study of p h o s p h o e n z y m e f or m a t i on at 4 ° C, were d e p e n d e n t on the concent rat i on of Na ÷ in the reaction and were maximal in the presence of 64 mM Na ÷. The plot of p h o s p h o e n z y m e vs. Na* c onc e nt r at i on gave a curve which on conversion to a double reciprocal plot {1/E-P vs. 1/Na +) gave a line with two breaks, yielding apparently three linear segments. This may be taken to indicate the presence of multiple Na ÷ sites for the formation of the p h o s p h o e n z y m e . To test this hypothesis further, the following approach was taken. By making the assumption t h a t the p h o s p h o e n z y m e may represent bound Na ÷, it was possible to subject the data to rigorous multiple-site analysis by utilizing steady-state binding equations described by Klotz and Hunston (1971) (Biochemistry 10, 3065--3069), and by Scatchard (1949) (Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 ) . The analysis of the data by these methods suggests that there may be three non-equivalent Na ÷ activation sites for the f or m a t i on of Na÷-dependent p h o s p h o e n z y m e in the (Na ÷ + K÷)-ATPase. The estimated intrinsic association constants (K a) for activation by Na ÷ at each o f the three sites were 3.4, 0.295, and 0.025 mM -1 , respectively. 2. The steady-state level of Na÷-dependent p h o s p h o e n z y m e was reduced by 2 H2 O (deuterated water) and Me2 SO {dimethylsulfoxide). This inhibition was reversed by increasing the c onc e nt r at i on of Na ÷ in the reaction but remained constant over a time course at any given Na + concentration. An analysis of the effect of : H2 O on Na÷-dependent p h o s p h o e n z y m e form at i on in the presence * Please address all communications to this address. Abbreviations: 2H 20, deuterated water; Me2SO. dimethyl sulfoxide; (Na + + K÷)-ATPase, (Na + + K+)-stimulated adenosine triphosphatase.
259 o f gradually increasing Na ÷ c o n c e n t r a t i o n also revealed the presence o f t h r e e n o n - e q u i v a l e n t sites for Na t. The intrinsic association c o n s t a n t s (K a) for the activation o f Na * at each o f the t h r e e sites in the p r e s e n c e o f 2 H2 O were changed to 1.4, 0.232, and 0 . 0 3 3 mM -1 , respectively, which suggests a differential e f f e c t o f 2 H~ O o n the t h r e e n o n - e q u i v a l e n t Na t sites. 3. On statistical g r o u n d s (-+2 S.E. o f m e a n ) a t w o n o n - e q u i v a l e n t site m o d e l also fits the data. In this case, the intrinsic association c o n s t a n t s ( g a ) were 2.44 and 0.041 mM -~ in H~ O m e d i u m , and 1.062 and 0 . 0 4 8 m M - ' , in 2 H~ O med i u m , respectively, showing a differential e f f e c t o f 2 H~ O on the t w o none q u i v a l e n t sites. 4. The i n h i b i t o r y e f f e c t o f ~ H~ O and Me~ SO on the f o r m a t i o n o f Na ÷d e p e n d e n t p h o s p h o e n z y m e was m a x i m a l w h e n the e n z y m e was allowed a cont a c t with these agents p r i o r to the a d d i t i o n o f Na t in the reaction. On the o t h e r h a n d , p r i o r c o n t a c t o f the e n z y m e with Na ÷ r e d u c e d or abolished the i n h i b i t o r y e f f e c t o f 2 H~ O or Me~ SO. Prior c o n t a c t o f the e n z y m e with ATP also abolished the i n h i b i t i o n o f Me~ SO. These results s u p p o r t the view t h a t H~ O plays a r e g u l a t o r y role in the active c e n t e r o f the (Na ÷ + K÷)-ATPase so t h a t its presence t e n d s to favor the E2 (Kt-accepting) f o r m o f the e n z y m e whereas Na t binds to at least t w o o f its activation sites, in an a p p a r e n t l y c o m p e t i t i v e m a n n e r with r e s p e c t to H~ O, yielding the c o n f o r m a t i o n suitable f o r the p h o s p h o r y l a tion ( E l ) . At the l o w e s t affinity Na ÷ activation site for e i t h e r the 2 or 3 n o n - e q u i v a l e n t site case the p r e s e n c e o f H 2 0 m a y facilitate its binding. The results also suggest t h a t b o t h Na * or A T P can i n d e p e n d e n t l y shift the e n z y m e c o n f o r m a t i o n to E l .
Introduction (Na ÷ + K÷)-stimulated t r a n s p o r t ATPase (EC 3.6.1.3) p r e s e n t in m o s t m a m malian p l a s m a cell m e m b r a n e s , is c o n s i d e r e d t o r e p r e s e n t the e n z y m i c basis for the m o v e m e n t o f cations across the cell m e m b r a n e [ 1 ] . Evidence f r o m several l a b o r a t o r i e s has suggested t h a t the o p e r a t i o n o f this e n z y m e p r o c e e d s t h r o u g h a series o f i n t e r m e d i a r y steps involving c o n f o r m a t i o n a l changes related t o Na ÷d e p e n d e n t p h o s p h o r y l a t i o n o f the e n z y m e and the K÷-dependent b r e a k d o w n o f the p h o s p h o e n z y m e . In this c o n t e x t , the use o f modifiers or inhibitors o f the partial r e a c t i o n s o f this e n z y m e s y s t e m has p r o v i d e d a considerable a m o u n t o f i n f o r m a t i o n o n the ionic i n t e r a c t i o n s in the ATPase, and possible m e c h a n i s m ( s ) o f its o p e r a t i o n (for r e c e n t reviews see e.g. refs. 1--3). In previous work, we d e m o n s t r a t e d t h a t s u b s t i t u t i o n o f 2 H 2 0 ( d e u t e r a t e d w a t e r ) f o r H 2 0 in the ATPase r e a c t i o n r e s u l t e d in an i n h i b i t i o n o f the Na ÷ activation o f the ATPase in an a p p a r e n t l y c o m p e t i t i v e m a n n e r , while the associated K ÷ - d e p e n d e n t p - n i t r o p h e n y l p h o s p h a t a s e was s t i m u l a t e d b y 2 H: O [ 4 ] . These observations based o n a s t u d y o f the kinetics o f (Na ÷ + Kt)-ATPase led us to p o s t u l a t e t h a t the binding o f Na ÷ t o t h e (Na t + K÷)-ATPase p r o d u c e d a c o n f o r m a t i o n which was r e q u i r e d f o r the initiation o f the p h o s p h o e n z y m e f o r m a t i o n in the p r e s e n c e o f ATP, and f u r t h e r , t h a t H 2 0 p l a y e d a role in this i n t e r a c t i o n b e t w e e n Na t and t h e e n z y m e at this site. Several studies o n t h e effects o f a variety o f solvents o n
260 the (Na t + K~)-ATPase have been r e por t ed recently | 5 - - 9 ] . Among the various solvents examined, dimethylsulfoxide (Me2 SO) appears to have some features in c o m m o n with 2 H~ O, in that it inhibits the Na%dependent activation of the e n z y m e while c o n c o m i t a n t l y stimulating the K%dependent p - n i t r o p h e n y l phosphatase [6,7]. However, the results reported on the effect of Me~ SO on the Na%dependent p h o s p h o e n z y m e formation in the (Na ~ + K+)-ATPase are at variance [6,8]. We have e x t e n d e d these studies and have examined in detail the action of ~H~O and Me2 SO on the formation of Na%dependent phosphoe n z y m e in the (Na t + K÷)-ATPase complex. These experiments offer some clues to the possible nature of the interaction of Na ÷ and H~ O at the phosphorylation sites in the (Na t + Kt)-ATPase. Further, we have measured the steady-state levels of p h o s p h o e n z y m e formed when the concentrations of Na t are gradually increased (in K%free reaction media), and have devised an approach to analyze and relate these data in terms of the possible activation sites for Na ÷ for the formation of Na%dependent p h o s p h o e n z y m e in the ATPase complex. On the basis of these studies, we suggest the presence of three non-equivalent sites for Na t interaction with the (Na ÷ + Kt)-ATPase. A preliminary account of this work has been given [ 1 0 ] . Experimental procedure
Materials 2 H 2 0 (99.5%, lot TTS) was purchased from Mallinckrodt Chemical works, and was distilled twice in an all-glass apparatus before use. Me~ SO of the analytical grade was purchased from Baker Chemicals. Sucrose and Tris were of the ultrapure grade from Schwartz-Mann, New York; NaC1, KC1, and MgC12 were spectroscopically pure. Solutions of ATP, including those containing [~,~ ~ P] ATP, were passed through large columns of Tris form of AG-50 × 8 cation exchange resin, to eliminate any contamination from Na t, K + or NH~. EDTA was dissolved in Tris base, and treated the same way as ATP, described above. All other details concerning the materials used have been given previously [4,11--13]. Methods Preparatio~ of (Na ~ ÷ K÷)-A TPase. The details concerning the preparation of rat brain membrane (Na t + Kt)-ATPase and its properties are the same as described in previous work [4,14]. The e n z y m e preparation was suspended in a medium consisting of 0.25 M sucrose, 10 mM imidazole-HC1 and 1 mM EDTA, pH 7.4, following two additional washes in this medium. It was stored frozen in small aliquots and was stable over several weeks. The specific activity of the (Na t + K* )-stimulated ATPase on the average was 125 pm ol / m g of protein per h while the basic Mg2÷-stimulated c o m p o n e n t was generally between 5--10% of the total (Mg 2÷ + Na ÷ + K÷)-dependent ATPase. Preparation of phosphoenzyrne. The standard reaction medium, in a final volume of 2 ml, maintained at 4 ° C, consisted of 30 mM Tris • HC1, pH 7.45 (at 4°C), 0.3 mM MgC12,8 mM NaC1, 0.05 mM [~/.32 p] ATP {6 • 104 d p m / n m o l of ATP) and between 350 to 500 pg of rat brain membrane (Na t + K÷)-ATPase preparation. In general, the reaction time was 4 s which gave the steady-state
261 level p h o s p h o r y l a t i o n , and unless o t h e r w i s e stated, the r e a c t i o n was initiated by the a d d i t i o n o f [7 -3 a P] ATP and was t e r m i n a t e d by adding 25 ml o f ice cold 5% (w/v) t r i c h l o r o a c e t i c acid c o n t a i n i n g 15 mM NaH~ PO4 and 0.6 mM ATP. The r a d i o a c t i v i t y d u e to 3 ~p i n c o r p o r a t e d in the p r o t e i n was m e a s u r e d as described before [ 11]. T o d e t e r m i n e the a c c u r a c y o f the p h o s p h o e n z y m e activity m e a s u r e d at low a d d e d Na * c o n c e n t r a t i o n s (e.g. at 0.5 mM Na*), c o n t r o l e x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e the c o n t r i b u t i o n o f the e n d o g e n o u s Na ÷ in the ATPase. F o r this, p h o s p h o e n z y m e f o r m e d in the presence o f 0.3 mM Mg ~÷ alone, and 0.3 mM Mg 2÷ + 16 mM K ÷ was measured. T h e d i f f e r e n c e b e t w e e n these t w o values should r e p r e s e n t t h e a m o u n t o f p h o s p h o e n z y m e activity due to the e n d o g e n o u s c o n t a m i n a t i o n o f the e n z y m e b y Na÷; in several e x p e r i m e n t s the m e a n values o f p h o s p h o e n z y m e activity were precisely the same in b o t h cases. F u r t h e r , b y a t o m i c a b s o r p t i o n s p e c t r o s c o p y , the e n z y m e p r e p a r a t i o n was f o u n d to c o n t a i n n o d e t e c t a b l e NH~ and K *, while the a m o u n t o f Na ÷ p r e s e n t was 60 n m o l / m g p r o t e i n , which would c o n t r i b u t e negligible a m o u n t s of Na ÷ in t h e reaction. T h e value o f the p h o s p h o e n z y m e o b t a i n e d in the presence o f (Mg ~÷ + K ÷) was s u b t r a c t e d f r o m t h a t in the presence o f (Mg 2÷ + Na÷), the diff e r e n c e being the Na÷-dependent p h o s p h o e n z y m e f o r m e d in the (Na÷+ K÷) ATPase system. T h e value o f p h o s p h o e n z y m e in the presence o f (Mg ~÷ + K ÷) was no m o r e t h a n 6% o f t h a t o b t a i n e d in the presence o f (Mg 2÷ + 64 mM Na÷). All d a t a are calculated as p m o l 3 ~p per mg o f p r o t e i n or as p e r c e n t p h o s p h o r y l a t i o n ( c o m p a r e d with m a x i m a l p h o s p h o e n z y m e f o r m e d in the presence o f 64 mM Na ÷) w h e n d i f f e r e n t e n z y m e p r e p a r a t i o n s were used. Other procedures. M e t h o d s for the e s t i m a t i o n o f p r o t e i n , and p r e p a r a t i o n o f [7 -3~ P] A T P were the same as given b e f o r e [ 1 1 , 1 2 ] . P r e p a r a t i o n o f the solutions in 2 H~ O was the same as r e p o r t e d previously [ 4 , 1 1 ] . Results
Effect o f 2 H2 0 and Me2 SO on Na%dependent p h o s p h o e n z y m e formation. It has been r e p o r t e d t h a t the c o n c e n t r a t i o n s at which 2 H 2 0 [4] and Me~ SO [6,7] p r o d u c e maximal, reversible, i n h i b i t o r y effects on the (Na + + K÷)-ATPase are 80---90 and 30%, respectively. T h e m a x i m a l inhibition o f Na÷-dependent p h o s p h o e n z y m e f o r m a t i o n by 2 H~ O was also observed at c o n c e n t r a t i o n s o f 8 0 - - 9 0 % [ 1 1 ] . In the p r e s e n c e o f 80% 2 H~ O or 30% Me~ SO, the i n h i b i t i o n o f the f o r m a t i o n o f Na÷-dependent p h o s p h o e n z y m e was m o d i f i e d by the level o f Na ÷ in the r e a c t i o n . The i n h i b i t i o n at 0.5, 1.0, 8.0, and 64 mM Na ÷ in the p r e s e n c e o f 2 H 2 0 was 50, 28, 23, and 7%, respectively. H o w e v e r , even at 128 mM Na ÷, a small b u t r e p r o d u c i b l e inhibition ( a b o u t 7%) by ~ H 2 0 was still apparent. Similarly, i n h i b i t i o n b y Me2 SO was r e d u c e d f r o m 69% at 8 mM Na ÷ t o 20% at 64 m M Na ÷. It m a y be m e n t i o n e d t h a t in previous work, it was established t h a t the i n h i b i t i o n o f (Na ÷ + K÷)-ATPase [4] or N a % d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n [11] b y ~ H 2 0 was n o t due t o alterations in the p H o p t i m a f o r these activies in the p r e s e n c e o f ~ H~ O. Time course o f the effect o f 2 H~ 0 on the formation o f p h o s p h o e n z y m e . T h e d a t a given in Fig. 1 d e p i c t the variations o f steady-state p h o s p h o e n z y m e with varying c o n c e n t r a t i o n s o f Na ÷. H o w e v e r , at any given c o n c e n t r a t i o n o f
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263 Na ÷, the level of phosphoenzyme formed remains steady over a time course of 30 s as shown in the figure; in other control experiments the steady-state level of phosphoenzyme formed in the presence of 8 mM Na ÷ did not change significantly ovel a period of 80 s (data not shown). The inhibition produced by 2 H2 O in the presence of 8 mM Na ÷ was also constant over a reaction period of 30 s. Formation of p h o s p h o e n z y m e in the presence of varying Na ÷. The effect of gradually increasing concentrations of Na ÷ on the formation of steady-state Na÷-dependent phosphoenzyme in the H~ O reaction media is shown in Fig. 2 (open circles). Maximal steady-state levels of Na÷-dependent phosphoenzyme were obtained at some 64 mM Na ÷. As is evident from Fig. 2 (inset A), a double reciprocal plot of the data showed a break at two points, yielding three linear segments designated in Fig. 2 inset A, as 1, 2 and 3. These segments are depicted as the expanded plots in the insets B1, C2, and D3 to illustrate that the slopes and the points of intersection on the ordinate are different for each of the three segments. The non-linearity of the plot in Fig. 2, inset A, may possibly be interpreted as empirical evidence for the presence of multiple nonequivalent Na ÷ sites for the formation of phosphoenzyme in the (Na ÷ + K÷)ATPase. However, this plot alone is considered inadequate to determine the number of sites and their constants [ 1 5 - - 1 7 ] . Therefore, the procedures described by Klotz and Hunston [15] and by Scatchard [18] to determine the number of binding sites and their kinetic constants, were considered. Since the steadystate phosphoenzyme varies with Na ÷ concentration (Fig. 1), we let the phosphoenzyme represent Na ÷ binding for its formation (see also the Discussion).
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Fig. 3. S c a t c h a r d p l o t a n a l y s i s o f N a + a c t i v a t i o n sites. Panel A , t h e v a l u e s f o r [ E - P ] a n d N a + are t h e s a m e as in T a b l e I. E, as d e f i n e d in t h e A p p e n d i x , is t h e t o t a l c o n c e n t r a t i o n o f e n z y m e in t h e r e a c t i o n ( 3 0 4 p m o l / m g o f p r o t e i n ) . T h e t h e o r e t i c a l v a l u e s ( × ) w e r e c a l c u l a t e d as d e s c r i b e d u n d e r t h e A p p e n d i x ; o, experimental values, where [E-P]/[El represents the moles of Na + bound per unit of the enzyme which h a s o n e c o v a l e n t l y l i n k e d p h o s p h a t e . P a n e l B, a t e s t f o r t w o n o n - e q u i v a l e n t s i t e s is s h o w n , o, e x p e r i m e n t a l v a l u e s ; + o r - - , t w o p o s s i b l e t h e o r e t i c a l fits w h i c h p r e s u m a b l y b r a c k e t t h e b e s t fit for this m o d e l . Panel C, t h r e e n o n - e q u i v a l e n t site m o d e l a p p l i e d t o N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n in t h e p r e s e n c e o f 2 H 2 0 . T h e v a l u e s o f t h e N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m e d in t h e p r e s e n c e o f 2 H 2 0 are as s h o w n in Fig. 2; t h e a m o u n t o f p h o s p h o e n z y m e at 6 4 m M N a + in t h e p r e s e n c e o f 8 0 % 2 H 2 0 w a s 2 4 6 . 1 p m o l / m g o f p r o t e i n , o, e x p e r i m e n t a l p o i n t s ; X , c a l c u l a t e d p o i n t s o n t h e basis o f t h r e e n o n - e q u i v a l e n t sites f o r N a +. All l i n e s are d r a w n t o fit t h e e x p e r i m e n t a l p o i n t ~ .
264
By doing so it is possible to plot the data in terms of a Scatchard plot as shown in Fig. 3 (panel A) to describe Na ÷ binding. The actual values of phosphoenzyme activity and standard errors are given in Table I. The three intercepts on the abscissa may suggest three classes of binding sites while the uppermost intercept on the ordinate gives a stoichiometric binding constant designated in the Appendix as/~. Based on the general equation (2) given in the Appendix, models for two and three equivalent and non-equivalent binding sites were tested by plotting r t = t[E-P]/[E] vs. [Na t] , as shown in Fig. 4, where [E-P] represents the phosphoenzyme concentration. The theoretical values for data points in Fig. 4, panels A and B, for the three and two non-equivalent site models were also plotted in Fig. 3, for comparison of experimental values with the theoretical values for three non-equivalent site (Fig. 3, panel A) or two non-equivalent site models (Fig. 3, panel B). As shown in Figs. 3 and 4, the two and three equivalent site models showed considerable deviation between the theoretical and experimental points; the result with a one-site model was so deviant as not to merit inclusion in the figure. The model with two non-equivalent sites also appeared to be incapable of fitting the experimental points within -+1 S.E. on both ends of the curve in Fig. 4. The deviation was minimal {within + 1 S.E.) when the data were fitted to a three non-equivalent site model, and a good agreement was noted between the calculated and the experimental points (Fig. 3, panel A and Fig. 4, panel A). The intrinsic association constants (K a) for the activation by Na ÷ were estimated from the three non-equivalent site model shown in Fig. 4, panel A, as described under the Appendix; the values of these constants in the H 2 0 system were 3.4, 0.295, and 0.025 mM -~ Other theoretical details for the above plots are given in the Appendix. Effect of 2H2 0 on the formation of phosphoenzyme in the presence o f varying Na ÷. The data given in Fig. 2 also show the effect of gradually increas-
TABLE I VALUES
O F D A T A P O I N T S I N F I G S . 2, 3 A N D 4
E-P r e f e r s t o t h e s t e a d y - s t a t e N a + - d e p e n d e n t p h o s p h o e n z y m e as p m o l / m g o f p r o t e i n , i n t h e p r e s e n c e o f t h e v a r i o u s c o n c e n t r a t i o n s o f N a + s h o w n . E r e f e r s t o t h e t o t a l c o n c e n t r a t i o n o f e n z y m e i n t h e r e a c t i o n as d e f i n e d i n t h e A p p e n d i x , a n d was 3 0 4 p m o l / m g o f p r o t e i n . T h e c a l c u l a t i o n s o f t h e s t a n d a r d e r r o r ( S . E . ) i n t h e v a r i o u s t r a n s f o r m a t i o n s o f E-P w e r e b a s e d o n t h e p r o c e d u r e o f J o h a n s e n a n d L u m r y [ 1 4 ] as d e scribed in the Appendix. No. of deterrninations
Na+ (raM)
[E-P] -+ S.E.
11 2 5 4 6 4 6 6 2 2 12 2
0.5 0.8 1.0 1.2 2.0 4.0 8.0 16.0 32.0 48.0 64.0 96.0
79.8 92.0 106.0 109.3 129.0 175.0 198.2 209.3 236.5 251.8 264.5 242.8
[E-PI/[El + S.E.
-+ 3 . 8 3 ± 5.67 + 8.20 -+ 8 . 4 0 -+ 7 . 3 3 -+ 7 . 9 7 -+ 7 . 1 4
-+ 6 . 0 8
0 . 2 6 2 3 -+ 0 . 0 1 4 0.3026 0.3486 + 0.022 0 . 3 5 9 5 -+ 0 . 0 3 1 0 . 4 2 4 3 -+ 0 . 0 3 1 0 . 5 7 5 6 -+ 0 . 0 3 4 0.6519 ± 0.034 0 . 6 8 8 4 -+ 0 . 0 3 3 0.7779 0.8282 0.870 + 0.028
265 3D
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A
B
./
25
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3
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)
J 24
~0
,5~ 72
0,5~
i I
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Na*¢me)
Fig. 4. Multiple site analysis of Na ÷ a c t i v a t i o n o f p h o s P h o e n z y m e f o r m a t i o n . Panel A, t h e c o n c e n t r a t i o n of N a +, v a l u e s o f [ E ] , [ E - P ] , a n d c a l c u l a t e d [ E - P ] / [ E ] are t h o s e given in T a b l e I a n d Fig. 3. T h e e q u a t i o n rt = >?i=lm N i K i [ N a +] ]1 + K i [ N a +], ( m = 3), w a s s o l v e d for t h r e e e q u i v a l e n t (~) or n o n - e q u i v a l e n t ( × ) a s s o c i a t i o n c o n s t a n t s f o r Na + (see the A p p e n d i x for details); ©, e x p e r i m e n t a l p o i n t s for t × [E-P] / [ E ] . Panel B, t h e s a m e e q u a t i o n as in p a n e l A e x c e p t t h a t m = 2 was solved for the t w o e q u i v a l e n t (A) or n o n - e q u i v a l e n t (+ or - - ) a s s o c i a t i o n c o n s t a n t s for Na+; o, e x p e r i m e n t a l points. All lines are d r a w n to c o n n e c t t h e t h e o r e t i c a l p o i n t s . T h e a p p a r e n t intrinsic a s s o c i a t i o n c o n s t a n t s ( K a ) f o r t h e a c t i v a t i o n b y Na ÷ at e a c h o f the t h r e e n o n - e q u i v a l e n t sites w e r e 3.4, 0 . 2 9 5 , a n d 0 . 0 2 5 m M -1, r e s p e c t i v e l y , in t h e p r e s e n c e of H 2 0 , a n d w e r e 1.4, 0 . 2 3 2 , a n d 0 , 0 3 3 m M -l, r e s p e c t i v e l y , in t h e p r e s e n c e o f 80% 2 H 2 0 .
ing co n cen tr atio ns of Na ÷ on the f o r m a t i o n of steady-state Na÷-dependent p h o s p h o e n z y m e in the reaction media containing 80% 2 H~ O. A double reciprocal p lo t o f the data (i.e. 1/E-P vs. 1/Na ÷) gave three linear segments as in the case o f the p l o t obtained in the presence of H~ O (not shown in the inset A, but shown in the e x p a n d e d scale segments B1, C2, and D3 of Fig. 2). Further, it appeared th at the segments designated as no. 1 and as no. 2 in bot h H~ O and 2 H~ O in ter cep te d the ordinate at the same point in each case, whereas the segments designated as no. 3 did n o t m e e t at the same p o i n t on the ordinate in the presence of H~ O c o m p a r e d with ~ H~ O. The result suggests that over the range o f c o n c e n t r a t i o n of Na ÷ given in the insets B1 and C2, the maximal p h o s p h o e n z y m e activity attainable is essentially the same in the presence or absence o f ~ H2 O, and t ha t the r educt i on in the p h o s p h o e n z y m e levels in the presence o f 2 H~ O at these concentrations of Na ÷ may be due to a reduced affinity o f Na ÷. We f u r t h e r subjected the Na÷-dependent p h o s p h o e n z y m e data obtained in the presence of ~ H~ O to multiple-site analysis as in the case of the data obtained in the presence o f H~ O described above (Fig. 3, panel C), which also indicated three Na ÷ activation sites. Again, using the intercept on the ordinate o f Fig. 3, panel C, and assuming different ratios of Na ÷ binding per p h o s p h o e n z y m e molecule, tests o f the n u m b e r of Na ÷ sites and their equiva-
266 lency s h o w e d t h a t an equivalent site m o d e l would n o t fit the data (plots n o t included in Fig. 4). The intrinsic association c o n s t a n t s for Na ÷ ( g a ) i n the presence o f 2 H2 O for a three n o n - e q u i v a l e n t site m o d e l were e s t i m a t e d as 1.4, 0.232 and 0.033 mM -1, respectively. These values, c o m p a r e d with the constants o b t a i n e d f r o m the e x p e r i m e n t s with H2 O r e a c t i o n media, indicate a differential e f f e c t o f 2 H~ O on the three sites. The decrease in the first t w o constants (K a ) by 2 H~ O was 59 and 21%, respectively, while for the third one there was an increase o f 32%. F u r t h e r , a c o m p a r i s o n o f the i n t e r c e p t s on the [ E - P ] / [ N a ÷] [E] axis for H~O (Fig. 3, panel A) and ~ H~O (Fig. 3, panel C) shows an overall r e d u c t i o n o f 47% in the affinity o f Na*. It m a y be recalled t h a t 80% 2 H2 O gives an average o f 40% inhibition o f the (Na + + K ÷)-ATPase [4]. Consideration o f two non-equivalent site models. We have p r o p o s e d above t h a t a t h r e e n o n - e q u i v a l e n t Na ÷ activation site m o d e l shows the best fit to o u r d a t a w h e n all points are c o n s i d e r e d within +1 S.E. o f each mean. However, on the basis o f +2 S.E. o f each mean, the t w o n o n - e q u i v a l e n t site m o d e l (Fig. 4, panel B, b r o k e n line) is n o t distinguishable f r o m the three n o n - e q u i v a l e n t site model. If this were the case the K a values (derived b y the same p r o c e d u r e s as described above) for Na ÷ activation o f the e n z y m e in the presence o f H~ O m e d i u m were e s t i m a t e d at 2.440 and 0.041 mM -~ , respectively. In the presence o f ~ H~ O, these values were changed to 1.062 and 0 . 0 4 8 mM -1, respectively. The relative i n h i b i t o r y e f f e c t o f 2 H2 O on the high affinity n o n - e q u i v a l e n t site w o u l d thus be 56% with an increase o f 17% o n the low affinity site.
Effect o f the order o f addition o f ligands on the inhibition by ~H~ 0 and Me, SO. R e c e n t l y , Albers and Koval [6] s h o w e d t h a t Me~ SO did n o t inhibit the Na÷-dependent p h o s p h o e n z y m e f o r m a t i o n , whereas Kaniike et al. [8] were
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15
20
Time (s) Fig. 5. T i m e c o u r s e o f t h e e f f e c t o f M e 2 S O o n N a + - d e p e n d e n t p h o s p h o e n z y m e f o r m a t i o n a n d t h e e f f e c t o f t h e o r d e r o f t h e a d d i t i o n o f v a r i o u s c o m p o n e n t s . T h e e n z y m e w a s a l l o w e d a c o n t a c t w i t h N a + o r K +, a n d Me 2 S O i n t h e f o l l o w i n g o r d e r p ~ i o r t o t h e s t a x t o f t h e r e a c t i o n : ©, e n z y m e + N a + i n H 2 0 s y s t e m ; ~, e n z y m e + N a +, f o l l o w e d b y M e 2 S O ; A, e n z y m e + K +, f o l l o w e d b y M e 2 S O ; t , e n z y m e + K + i n H 2 0 s y s t e m ; u, N a + + M e 2 S O , f o l l o w e d b y t h e e n z y m e ; m, K + + M e 2 S O , f o l l o w e d b y t h e e n z y m e . A l l o t h e r e x p e r i m e n t a l d e t a i l s w e r e t h e s a m e as f o r e x p e r i m e n t s n o . 1 - - 6 i n T a b l e II.
267 O ATP__ E2
No+.-.
(~) E 1 • (No o r ATP)
--~IMgATP'Na~'~--
E~ • MgATP • NO×
-- H20~
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°
® E 2- Ky
@ ~o -H2~
~ + ~ , H~O E2-P-Ky
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Fig. 6. Sites o f a c t i o n o f s o l v e n t s a n d H 2 0 in t h e o p e r a t i o n o f t h e ( N a + + K+)-ATPase. ( 1 ) N a + or A T P s h i f t t h e e q u i l i b r i u m w i t h r e s p e c t to t h e e n z y m e c o n f o r m a t i o n f r o m E 2 to E l , w h e r e a s H 2 0 , 2 H 2 0 , a n d Me 2 SO will r e v e r s e it; o r g a n i c s o l v e n t s s u c h as a c e t o n e , e t h e r , a l c o h o l s [ 9 ] a n t a g o n i z e t h e a c t i o n o f H 2 O, a n d m a y s h i f t t h e e q u i l i b r i u m to t h e right~ ( 2 ) E 1 N a or E 1 A T P r e a c t s w i t h s u b s t r a t e M g A T P a n d Na~ to f o r m t h e i n t e r m e d i a t e [E 1 • M g A T P - N a x ] w h i c h l e a d s to t h e f o r m a t i o n o f E2-P • N a x ; ( 3 ) t h e i n t e r a c t i o n o f K~ is f a c i l i t a t e d b y H 2 O, 2 H2 O a n d Me 2 SO, a n d a n t a g o n i z e d b y o r g a n i c s o l v e n t s ; ( 4 ) t h e i n t e r a c t i o n o f H 2 0 on t h e b r e a k d o w n o f K + - m e d i a t e d p h o s p h o e n z y m e is e v i d e n c e d b y s t i m u l a t o r y e f f e c t o f 2 H 2 0 or M e 2 S O , a n d i n h i b i t o r y e f f e c t o f o r g a n i c s o l v e n t s ; (5) t h e p r e s e n c e o f N a + or A T P leads t o t h e d i s s o c i a t i o n o f E 2 • Ky b e c a u s e o f t h e a c t i o n o f t h e s e a g e n t s at s t e p 1, a b o v e .
able to d e m o n s t r a t e an i n h i b i t i o n o f the p h o s p h o e n z y m e f o r m a t i o n b y Me2 SO. T h e results s u m m a r i z e d in Table II clearly illustrate t h a t the p r i o r c o n t a c t o f the e n z y m e with Na ÷ or A T P c o n s i d e r a b l y alters the i n h i b i t o r y e f f e c t o f Me2 SO or ~ H 2 0 on the steady-state level o f p h o s p h o e n z y m e . When Me: SO and Na +, or 2 H2 O and Na ÷, are a d d e d t o g e t h e r , a large i n h i b i t i o n is observed which is even greater w h e n Me~ SO or : H: O are in c o n t a c t with the e n z y m e
T A B L E II EFFECT
OF THE ORDER
O F A D D I T I O N O F L I G A N D S O N T H E I N H I B I T I O N BY 2 H 2 0 O R M e 2 S O
A d d i t i o n s in t h e s e q u e n c e l i s t e d w i t h e a c h e x p e r i m e n t w e r e m a d e to t h e s t a n d a r d r e a c t i o n m e d i u m (as g i v e n u n d e r M e t h o d s ) . F o r e x p e r i m e n t nos. 1 ~ 6 , w h e r e i n d i c a t e d , c a t i o n s a n d e n z y m e w e r e in c o n t a c t f o r 10 r a i n f o l l o w e d b y a 6 0 - r a i n c o n t a c t w i t h M e 2 S O o r 2 H 2 0 . W h e n c a t i o n s a n d M e 2 S O o r 2 H 2 0 w e r e a d d e d t o g e t h e r to t h e e n z y m e , t h e i n c u b a t i o n w a s c a r r i e d o u t f o r 60 rain. W h e n M e 2 S O a n d 2 H 2 0 w e r e a d d e d p r i o r to c a t i o n s , t h e c o n t a c t w i t h t h e e n z y m e w a s f o r 10 m i n , t h e n an a d d i t i o n a l 50 r a i n f o l l o w i n g c a t i o n s . F o l l o w i n g t h e s e t r e a t m e n t s , t h e r e a c t i o n w a s s t a r t e d by t h e a d d i t i o n o f [7-52P] A T P a n d w a s t e r m i n a t e d 4 s later. In e x p e r i m e n t n o . 7, t h e e n z y m e w a s in c o n t a c t w i t h A T P ( c o n t a i n i n g [ 7 - ~ 2 P ] A T P ) f o r 1 0 s f o l l o w e d b y 10 s w i t h M e 2 S O , a n d t h e n t h e r e a c t i o n w a s i n i t i a t e d b y t h e a d d i t i o n o f Na+ a n d w a s t e r m i n a t e d 10 s l a t e r . I n e x p e r i m e n t 8, M e 2 S O w a s i n c o n t a c t w i t h t h e e n z y m e f o r 10 s f o l l o w e d bY [ 7 - 3 2 P ] A T P a n d Na+ o r K"~ f o r an a d d i t i o n a l 10 s. T h e a m o u n t o f e n z y m e (E) w a s 50 # g , M e 2 S O , 2 H 2 0 , a n d N a+ w e r e p r e s e n t at a c o n c e n t r a t i o n o f 3 0 % , 80%, a n d 8 raM, r e s p e c t i v e l y . V a l u e s f b r N a + - i n d e p e n d e n t p h o s p h o e n z y m e (i.e. in t h e p r e s e n c e o f Mg 2+ + 16 m M K+) w e r e s u b t r a c t e d f x o m e x p e r i m e n t a l values, to o b t a i n N a + - d e p e n d e n t p h o s p h o e n z y m e , a n d o t h e r a p p r o p r i a t e c o n t r o l s w e r e i n c l u d e d t h r o u g h o u t , All o t h e r d e t a i l s w e r e t h e s a m e as g i v e n u n d e r M e t h o d s . Exp. no.
Sequence of additions
Percent inhibition of phosphoenzyme
1 2 3 4 5 6 7 8
2 H 2 0 + E + Na+ 2H20+Na ++ E Na++ E+2H20 M e 2 S O + E + Na + Me2SO + Na + + E Na + + E + M e 2 S O ATP + E + Me2SO Me2SO + E + ATP
44 36 24 77 69 2 1 34
268 p r i o r to the a d d i t i o n o f Na ÷. This suggests t h a t Me2 SO a n d 2 H 2 0 b l o c k the i n t e r a c t i o n of Na ÷ with its a c t i v a t i o n sites. F u r t h e r , if A T P is p r e s e n t on the e n z y m e , Na ÷ can also b i n d to its sites w i t h o u t i n t e r f e r e n c e f r o m Me2 SO. This e x p e r i m e n t gives clear evidence t h a t the r a n d o m m e c h a n i s m w o r k s in the (Na * + K*)-ATPase since either Na ~ or A T P m a y get on to their sites i n d e p e n d e n t l y and t h a t with b o t h p r e s e n t , p h o s p h o e n z y m e f o r m a t i o n will t a k e place. T h e f a c t t h a t p r i o r c o n t a c t of the e n z y m e with A T P leads to the f o r m a t i o n o f p h o s p h o e n z y m e in the p r e s e n c e o f Na * and Me2 SO suggests t h a t A T P b o u n d to the e n z y m e assists Na * to a t t a c h to its active sites. The d a t a in Fig. 5 s h o w t h a t the e f f e c t o f t h e o r d e r o f a d d i t i o n o f ligands is n o t m o d i f i e d over a t i m e course in the p r e s e n c e o f Me~ SO. Similar results were o b s e r v e d with ~ H~ O (Fig. 2, and o t h e r d a t a n o t shown).
Discussion Previous studies utilizing the analyses o f the kinetics o f a c t i v a t i o n of the (Na ÷ + K*)-ATPase b y varying c o n c e n t r a t i o n s o f Na ÷ in the p r e s e n c e of varied or fixed c o n c e n t r a t i o n s o f K ÷ [ 1 4 , 1 9 ] have i n d i c a t e d t w o or t h r e e e q u i v a l e n t Na ÷ a c t i v a t i o n sites. T h e s e m o d e l s do n o t clearly distinguish b e t w e e n the e q u i v a l e n t and n o n - e q u i v a l e n t a c t i v a t i o n sites [ 1 9 ] . This t y p e o f a p p r o a c h is also c o m p l i c a t e d b y c o m p l e x i n t e r a c t i o n o f cations at e x t r e m e s o f c o n c e n t r a t i o n ratios. A T P - d e p e n d e n t binding o f Na t to the A T P a s e has been investigated, b u t has n o t b e e n utilized for binding-site analysis [ 2 0 - - 2 2 ] . T h e e x p e r i m e n t a l design in the p r e s e n t w o r k relies on the f o r m a t i o n o f N a ÷ - d e p e n d e n t p h o s p h o e n z y m e (the b r e a k d o w n o f which is sensitive to K÷), w h e r e b y the c o m p l i c a t e d i n t e r a c t i o n s o f N a ÷ a n d K ÷ are avoided, since t h e r e a c t i o n s y s t e m c o n t a i n s o n l y Na *, and only the N a ÷ - d e p e n d e n t s t e a d y - s t a t e p h o s p h o e n z y m e f o r m e d is examined. If, as suggested b y a large b o d y o f evidence, the p h o s p h o e n z y m e is the i n t e r m e d i a t e in the o p e r a t i o n o f the (Na" + K ' ) - A T P a s e and m a y be r e g a r d e d as the Na÷-carrier in the p u m p cycle [ 2 , 3 , 2 3 - - 2 6 ] (see also 1), t h e n it m a y be possible to use the p h o s p h o e n z y m e as a m e a s u r e of Na t binding. In this case, the s t e a d y state e q u a t i o n s and graphical m e t h o d s for s t u d y i n g the binding of small m o l e c u l e s to m a c r o m o l e c u l e s m a y be applied to analyse Na ÷ b o u n d t o p h o s p h o e n z y m e (see e.g. 1 5 , 1 8 , 2 7 , and the A p p e n d i x ) . By assuming t h a t cation binding sites are i n d e p e n d a n t with fixed affinities and w i t h o u t defining the p h y s i c a l n a t u r e o f the m a c r o m o l e c u l a r state o f t h e carrier, the s t e a d y - s t a t e b i n d i n g e q u a t i o n s w o u l d a p p l y w h e t h e r the e n z y m e were p r e s e n t as d i f f e r e n t species each having a distinct a f f i n i t y c o n s t a n t or as a single species with all o f the d i s t i n c t binding sites on a single m a c r o m o l e c u l e . T h e p r e s e n t m o d e l a t t e m p t s to relate the s t o i c h i o m e t r y o f Na * binding p e r p h o s p h o e n z y m e m o l e c u l e b a s e d on the o b s e r v e d c o r r e s p o n d e n c e b e t w e e n Na ÷ c o n c e n t r a t i o n a n d s t e a d y - s t a t e level o f p h o s p h o e n z y m e . T h e binding o f Na ÷ to its sites w o u l d be a c o n s e q u e n c e o f an e q u i l i b r i u m involving r e a c t i o n 1 as s h o w n in Fig. 6. It w o u l d be i m p l i e d f r o m the law o f mass a c t i o n as discussed b y S c a t c h a r d [18] t h a t t h e p r o b a b i l i t y o f o c c u p a n c y o f m u l t i p l e Na ÷ binding sites w o u l d be d e p e n d e n t u p o n the Na ÷ c o n c e n t r a t i o n . T h e result f r o m experim e n t s 4 and 6 in T a b l e II suggests t h a t p h o s p h o r y l a t i o n w o u l d o c c u r o n l y
269 when the conditions of reaction 2 (Fig. 6) are met, including a sufficient a m o u n t o f b o u n d Na ÷ per potential p h o s p h o r y l a t i o n site. Accordingly, the steady-state level of p h o s p h o r y l a t i o n may be used as a measure of Na ÷ t h a t was b o u n d prior to p h os phor yl a t i on. This is in agreement with the c o n c e p t t hat the c o n cen tr atio n s of Na ÷ and K ÷ m a y shift the steady-state concentrations of carrier moieties to control the rates of cation transport [ 2 8 ] . Our suggestion of three non-equivalent Na ÷ activation sites for (Na ÷ + K÷}-ATPase is compatible with the data on the kinetics of transport in nerve where it was shown that three sites with different Na ÷ affinities were necessary to a c c o u n t for the Na + activation o f Na÷-Na ÷ exchange [ 2 9 ] . However, in red cells, three' sites of equal apparent affinity for Na ÷ are involved in Na+-Na + exchange [ 3 0 ] . It would be interesting to determine if the nature of Na ÷ activation sites (i.e. equivalent or non-equivalent) varies with tissues. The possibility of interaction between the multiple non-equivalent Na ÷ sites postulated above c a n n o t be ruled o u t at the present. Nor can we rule o u t t hat our results are due to the possible i n h o m o g e n e i t y of the e n z y m e system used. Further, based on strict statistical considerations, the two non-equivalent site model may also fit the data. The results in Table II have several implications. If b o u n d H2 O plays a role at the Na ÷ sites [ 4 , 1 1 ] , it would seem reasonable that the agents such as Me2 SO and 2 H 2 0 which form stronger h y d r o g e n (or deuterium) bonding by replacing H~ O [32,33] would tend to block the interaction of Na ÷ with its activation sites. We propose that H~ O shifts the c o n f o r m a t i o n of the e n z y m e f r o m E, to E~, while Na ÷ reverses this trend (Fig. 6). This would imply that Na ÷ must expel the b o u n d H~ O f r om its activation sites in order to bind to these sites, although this does n o t seem to be the case at one of the Na ÷ sites (i.e. the weak Na ÷ binding site with the lowest Ka). Removal of H~ O from the higher affinity Na ÷ sites could increase the h y d r o p h o b i c i t y in the region, thereby preventing the spontaneous br eakdow n o f the p h o s p h o e n z y m e [ 9 ] . However, H2 O and K ÷ will tend to move the [K-E~-P] com pl ex back to a hydrophilic region where the p h o s p h o e n z y m e will be h y d r o l y z e d . Thus 2 H~ O and Me~ SO would act more effectively than H~ O to favor the shift of E, to the E~ f o rm o f the en zym e , which would result in reduced steady-state level of Na ÷d e p e n d e n t phosphoen.zyme f o r m a t i o n in the presence of ~ H2 O or Me~ SO. This will also be c o m m e n s u r a t e with increased affinity of K ÷ for the K ÷ stimulated p - n i t r o p h e n y l phosphatase activity in the presence of ~ H~ O [}1,11] and Me~ SO [ 6 , 7 ] , and a stimulation of K÷-mediated breakdown o f p h o s p h o e n z y m e [ 3 4 , 3 5 ] . The result in Table II suggests t hat ATP m ay act to cause a dissociation o f the K-E2 c o m p l e x [24] to release K ÷, by producing a shift of E~ c o n f o r m a t i o n to E, c o n f o r m a t i o n (Fig. 6). Further, the removal of H~ O from Na ÷ sites in the ATPase by solvents such as alcohols, acetone, ether, etc. would facilitate the binding of Na ÷ (E, c o n f o r m a t i o n , Fig. 6), and increase the phosp h o e n z y m e f o r m e d due to stabilization of the com pl ex [ 9 ] , whereas K ÷mediated h y d r o l y t i c steps would be inhibited [5, 9]. The increased affinity of K ÷ for the ATPase at r e duc e d temperatures [41,42] m ay also be explained if the e n z y m e c o m p l e x favors the E~ c o n f o r m a t i o n at lower temperatures due to an increase in b o u n d H~ O a n d / o r stronger H bondings, while the reverse is the case at higher temperatures, i.e. the E, c o n f o r m a t i o n is favored.
270 T o c o n c l u d e , the effects o f : H: O and Me: SO on the (Na ~ + K~)-ATPase, and possibly o t h e r m e m b r a n e - a s s o c i a t e d f u n c t i o n s [ 3 6 - - 4 0 ] , m a y be due t o conf o r m a t i o n a l changes caused by r e p l a c e m e n t o f H: O in the m e m b r a n e s t r u c t u r e and f o r m a t i o n o f stronger H (or : H) bonding. F u r t h e r , the affinity o f Na ÷ and K ÷ f o r their sites in the o p e r a t i o n o f the (Na ~ + K÷)-ATPase m a y be regulated by the b o u n d H: O al] the active center. The points t h a t favor this conclusion, and argue against a simple e f f e c t o f 2 H~ O on cation h y d r a t i o n alone t o explain the present and the previous w o r k b y us are as follows: (1) : H 2 0 inhibits (Na ÷ + K÷)-ATPase, b u t stimulates K*-stimulated p - n i t r o p h e n y l p h o s p h a t a s e ; the stimulated activity is b l o c k e d b y o l i g o m y c i n [4,11] ; (2) I n h i b i t i o n o f p h o s p h o e n z y m e f o r m a t i o n by : H: O or Me2 SO is n o t altered over a time course, b u t changes with Na ÷ c o n c e n t r a t i o n ; (3) : H: O acts differentially o n multiple nonequivalent Na ÷ sites; (4) The o r d e r o f addition o f r e a c t a n t s influences the e f f e c t o f solvents; (5) 2 H 2 0 (and Me: SO) a c t i o n on K÷-stimulated p - n i t r o p h e n y l p h o s p h a t a s e is r e f l e c t e d in the stimulation o f a K~-mediated b r e a k d o w n o f a slow c o m p o n e n t o f p h o s p h o e n z y m e b y these solvents [34,35] ; and, (6) : H 2 0 stimulates the rate, and steady-state level o f Mg 2÷ + Pi or Mn:÷-supported ouabain binding to the ATPase, whereas it inhibits the rate, b u t n o t the steadystate level, o f (Na ÷ + A T P ) - d e p e n d e n t ouabain binding [ 4 5 ] . Appendix T h e o r y f o r data expressed in Figs. 3 and 4
We m a k e the following identities and assumptions: [E-P] represents specific Na" binding; r t = moles Na ÷ b o u n d / u n i t o f e n z y m e = t [ E - P ] / [ E ] ; [E] = t o t a l e n z y m e c o n c e n t r a t i o n = [E-P] m ax + 0.15 × [E-P] max in the absence o f K ÷ [43] ; f u r t h e r , the value for [E] in 80% 2 H2 O was t a k e n as 93% o f t h a t in H2 O since a 7% inhibition o f the p h o s p h o e n z y m e at 64 mM Na ÷ was observed in the presence o f 80% : H2 O; [Na] = free Na ÷ c o n c e n t r a t i o n = [Na] W t[E-P] ~ [Na] T, the total c o n c e n t r a t i o n , since [Na] T is in units o f mM and t[E-P] is in units o f nM; t = total n u m b e r o f Na ÷ b o u n d t o the p h o s p h o e n z y m e ; [E-P] = [Nat • E-P], the N a - p h o s p h o e n z y m e c o m p l e x ; e = free e n z y m e c o n c e n t r a t i o n = [El - - [E-P] ; K a = [ E - P ] / ( [ N a ] [e] ), the intrinsic association c o n s t a n t . Therefore, "
t[E-P]
_
rt = [E-P] + [e]
tKa[Na] 1 + Ka[Na ]
(1)
In general, Ft =
~ N'~i[Na] i=1 1 + K i [ N a ]
(2)
where t = X ~ I Ni = the t o t a l n u m b e r o f sites, and where N i and K i are the n u m b e r o f identical sites and the intrinsic association c o n s t a n t , respectively, f o r each class o f sites i. By holding the total e n z y m e c o n c e n t r a t i o n [E] c o n s t a n t , the o n l y d e p e n d e n t variable is [E-P] and the only i n d e p e n d e n t variable is [ N a ] . F o r e q u a t i o n (2) it is a p p r o p r i a t e t o p l o t r t vs. [Na ÷] (Fig. 4). F u r t h e r , the
271 following form of Scatchard equation [17,18] graph of rt/[Na] vs. rt (Fig. 3). rt/[Na]
is employed to construct a
(3)
= tK -- rtK
In plotting the experimental data by this method, it was first assumed that t = 1; therefore, r t = [E-P]/[E]. The general solution of equation (3) states that the intercept on the abscissa gives the total number of binding sites, t, and the intercept on the ordinate gives the product of t and the average of all the association constants, (K) [15]. In Fig. 3, the intercept on the [E-P]/[E] axis is essentially 1, which, in terms of p h o s p h o e n z y m e formation as expressed in equation (3), means that as Na ÷-~ ¢*, all of the enzyme is converted into phosphoenzyme. However, by letting the phosphoenzyme represent bound Na ÷, the intercept on the abscissa simply gives the value of t that would be the assumed stoichiometry between the phosphoenzyme and Na ÷ binding. The uppermost intercept on the ordinate as shown in Fig. 4a, gives the c o n s t a n t / ~ , such that
~ /~ = t(K) where (K) =
m N~K~/~ N i
i=1
(4)
i=l
and where the Ki values are the intrinsic association constants as given in equation (2). For t = 1 , / ~ = 1.24 = N1 K1 as shown in Fig. 3A; for t, = 2, ~ = 2.48 = N ~ K ~ + N 2 K 2 ; a n d for t = 3,/~ = 3.72 = N ~ K 1 + N 2 K 2 + N 3 K 3 , etc. By assuming different values for t, Ni and K~, and using the experimental values of Na ÷ concentration employed, theoretical values of r~ for 1-, 2- and 3-site models were generated from equation (2~), with the appropriate values o f / f as a constraint. For equivalent site models K was divided equally among all the K~ values. Initial estimates for the Ki values in the non-equivalent site models may be obtained by substituting experimental values of rt, [Na], (K), and assumed values of Ni and t into equations (2) and (4). These equations are solved for a value of K~ which is substituted in the equations and the process is repeated until estimates of all the K,- values are obtained. These initial estimates of K; values are then varied by trial and error until the best fit is obtained. Since the values of r t calculated from equation (2) represent theoretical values for [EP ] / [ E ] , theoretical values for [E-P]/[Na] [E] were obtained by dividing the calculated values of r t by the experimental values of [Na], and are used to test the various models in a Scatchard plot as shown in Fig. 3. The K i values that gave the best fit for a three non-equivalent site model (Fig. 4A) were designated as the intrinsic association constants (Ka) for Na ÷. When plotting points from Fig. 3 to Fig. 4 and vice versa, the obvious division or multiplication by t was performed. Calculation of the standard error
The experimental values of [E-P]/[E] and the error associated with each are given in Table I. From equation (9) of Johansen and L u m r y [44], we define
272
the variance for
rt =
t[E-P]/[E] as follows:
from which S.E. = ( ~ r t / n I ] 2 was calculated, based on the assumption that the experimental determination of [E-P] and [E] are stochastically independent. Although [E] is calculated from [E-P] m ax, the measurement of [E-P] made at one concentration of Na ÷ is independent of that made at another.
Acknowledgements We are greatly indebted to Professor Rufus Lumry, Laboratory of Biophysical Chemistry, Department of Chemistry and Dr. R. Sawchuk, Department of Pharmaceutics, University of Minnesota, for valuable discussions which guided us to the relevant literature, and for evaluation of the application of the mathematical expressions in the appendix used for plots presented in Figs. 3 and 4. We thank Mr. Gregory Quarfoth and Mr. Alan Davis for their help in this work.
References 1 S k o u , J.C. ( 1 9 7 2 ) B i o e n e r g e t i c s 4, 2 0 3 - - 2 3 2 2 S c h w a r t z , A., L i n d e n m a y e r , G.E. a n d Allen, J. ( 1 9 7 2 ) in C u r r e n t T o p i c s in M e m b r a n e T r a n s p o r t ( B o n n e t , F. a n d K l e i n z e l l e r , A., e d s . ) Vol. 3, p p . 1 - - 8 2 , A c a d e m i c Press, N.Y. 3 D a h l , J . L . a n d H o k i n , L.E. ( 1 9 7 4 ) A n n u . Rev. B i o c h e m . 4 3 , 3 2 7 - - 3 5 6 4 A h m e d , K., Riggs, C. a n d I s h i d a , H. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 6 1 9 7 - - 6 2 0 3 5 Israel, Y. a n d S a l a z a r , I. ( 1 9 6 7 ) A r c h . B i o c h e m . B i o p h y s . 1 2 2 , 3 1 0 ~ 3 1 7 6 A l b e r s , R.W. a n d K o v a l , G.J. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 3 0 8 8 - - 3 0 9 2 7 Robinson, J.D. (1972) Biochim. Biophys. Acta 274, 542--550 8 K a n i i k e , K., E r d m a n n , E. a n d S c h o n e r , W. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 5 2 , 2 7 5 ~ 2 8 6 9 H e g y v a ~ y , C. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 1 , 2 7 2 - - 2 9 1 1 0 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 5 ) F e d . P r o c . 3 4 , 3 2 8 11 A h m e d , K. a n d F o s t e r , D. ( 1 9 7 4 ) A n n . N.Y. A c a d . Sci. 2 4 2 , 2 8 0 - - 2 9 2 1 2 A h m e d , K. a n d T h o m a s , B.S. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 0 3 - - 1 0 9 13 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 4 ) B i o c h i m . B i o p h y s . A c t a 9 3 , 6 0 3 - - 6 1 3 1 4 A h m c d , K., J u d a h , J . D . a n d S c h o l e f i e l d , P.G. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 0 , 3 5 1 - - 3 6 0 1 5 K l o t z , I.M. a n d H u n s t o n , D.L. ( 1 9 7 1 ) B i o c h e m i s t r y 10, 3 0 6 5 - - 3 0 6 9 1 6 S c h e l l m a n , J . A . , L u m r y , R. a n d S a m u e l s , L.T. ( 1 9 5 4 ) J . A m e r . C h e m . Soc. 76, 2 8 0 8 - - 2 8 1 3 17 Walter, C. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 9 9 - - 7 0 3 1 8 S c a t c h a r d , G. ( 1 9 4 9 ) A n n . N.Y. A c a d . Sci. 5 1 , 6 6 0 - - 6 7 2 19 L i n d e n m a y e r , G.E., S c h w a r t z , A. a n d T h o m p s o n , Jr., H . K . ( 1 9 7 4 ) J. P h y s i o l . 2 3 6 , 1 - - 2 8 2 0 V o n S t e d i n g k , L.V. a n d J a r n e f e l t , J. ( 1 9 6 3 ) A c t a P h y s i o l . S c a n d . 5 7 , 3 2 8 - - 3 3 8 21 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 1 2 , 5 8 - - 6 2 2 2 O s t r o y , F., J a m e s , T . L . , N o g g l e , J . H . , Sarrif, A. a n d H o k i n , L.E. ( 1 9 7 4 ) A r c h . B i o c h e m . B i o p h y s . 1 6 2 , 421--425 2 3 J u d a h , J . D . a n d A h m e d , K. ( 1 9 6 4 ) Biol. R e v . C a m b r i d g e 3 9 , 1 6 0 - 1 9 3 2 4 P o s t , R . L . , H e g y v a r y , C. a n d K u m e , S. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 6 5 3 0 - - 6 5 4 0 2 5 A s k a r i , A. (ed.) ( 1 9 7 4 ) A n n a l s N . Y . A c a d . Sci. Vol. 2 4 2 26 K a n a z a w a , T., Y a m a d a , S., Y a m a m o t o , T. a n d T o n o m t t r a , Y. ( 1 9 7 1 ) J. B i o c h e m . T o k y o 7 0 , 9 5 - - 1 2 3 27 D a v i s o n , C. ( 1 9 7 1 ) in F u n d a m e n t a l s of D r u g M e t a b o l i s m a n d D r u g D i s p o s i t i o n ( L a D u , B.N., M a n d e l , H . G . a n d W a y , E.L., eds.) p p . 6 3 - - 7 4 , T h e Williams a n d Wilkins Press, N . Y . 2 8 R e p k e , K . R . H . , S c h / i n , R., H e n k e , W., S c h S n f e l d , W., S t r e c h e n b a c h , B. a n d D i t t r i c h , R. ( 1 9 7 4 ) A n n . N.Y. A c a d . Sci 2 4 2 , 2 0 3 - - 2 1 9 2 9 B a k e r , P . F . , B l a u s t e i n , M.P., K e y n e s , R . D . , Manil, J., S h a w , T.I. a n d S t e i n h a r d t , R . A . ( 1 9 6 9 ) J. Physiol. 200, 459--496
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G a t a y , R.P. a n d G a x r a h a n , P.J. ( 1 9 7 3 ) J. P h y s i o l . L o n d . 2 3 1 , 2 9 7 - - 3 2 5 G l y n n , I.M. a n d K a r l i s h , S . J , D . ( 1 9 7 5 ) A n n u . R e v . P h y s i o l . 3 7 , 1 3 - - 5 5 R a m m l e r , D . H . a n d Z a f f a r o n i , A. ( 1 9 6 7 ) A n n . N . Y . A c a d . Sci. 1 4 1 , 1 3 - - 2 3 M a c G r e g o r , W.S. ( 1 9 6 7 ) A n n . N.Y. A c a d . Sci. 1 4 1 , 3 - - 1 2 F o s t e r , D. a n J A h m e d , K. ( 1 9 7 4 ) P h a r m a c o l o g i s t 1 6 (2), 2 9 3 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 4 ) F e d . P r o c . 3 3 (5), 1 3 3 2 De B r u i j n e , A.W., V a n 1~ees, H. a n d V a n S t e v e n i n c k , J. ( 1 9 7 1 ) B i o c h e m . P h a r m a c o l . 20, 2 6 8 7 - - 2 6 9 3 V o n E u l e r , U.S. a n d L i s h a j k o , F. ( 1 9 7 1 ) A c t a P h y s i o l . S c a n d . 8 2 , 1 3 1 - - 1 3 4 S v e n s m a x k , O. ( 1 9 6 1 ) A c t a P h y s i o l . S c a n d . 5 3 , 7 5 - - 8 4 K a m i n e r , B. a n d K i m u r a , J. ( 1 9 7 2 ) S c i e n c e 1 7 6 , 4 0 6 - - 4 0 7 K i h a r a , T. a n d M c C r a y , J . A . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 2 9 2 , 2 9 7 - - 3 0 9 A h m e d , K. a n d J u d a h , J . D . ( 1 9 6 5 ) C a n . J. B i o c h e m . 4 3 , 8 7 7 - - 8 8 0 S c h S n , R., S c h S n f e l d , W., M e n k e , K . - H . a n d R e p k e , K . R . H . ( 1 9 7 2 ) A c t a Biol. M e d . Get. 2 9 , 6 4 3 - - 6 5 9 K a n a z a w a , T., S a i t o , M. a n d T o n o m u r a , Y. ( 1 9 7 0 ) J. B i o c h e m . T o k y o 6 7 , 6 9 3 - - 7 1 1 J o h a n s e n , G. a n d L u m r y , R. ( 1 9 6 1 ) C . R . T r a y . L a b . C a r l s b e r g 3 2 , 1 8 5 - - 2 1 4 F o s t e r , D. a n d A h m e d , K. ( 1 9 7 5 ) P h a r m a c o l o g i s t 1 7 , 1 9 9