K+-transporting ATPase by strophanthidin

Biochimica et Biophysica Acta, 1159 (1992) 109-112 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 30300

109

Rapid Report

Inhibition of H+-transporting ATPase, Ca2+-transporting ATPase and H+/K÷-transporting ATPase by strophanthidin Kai-yuan Xu Department of Cell Biology and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT (USA) (Received 15 June 1992)

Key words: ATPase, H+-; ATPase, Ca2+-; ATPase H+/K÷-; ATPase, Na+/K+-; Strophanthidin; Positive inotropic effect

Studies of the effect of strophanthidin on H +-transporting ATPase, Ca 2+-transporting ATPase and H +/K+-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H+-transporting ATPase, CaZ+-transporting ATPase, as well as H+/K+-transporting ATPase and Na+/K+-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca2+-transporting ATPase and Na+/K+-trans porting ATPase.

Digitalis glycosides and aglycones have been commonly used as drugs for the treatment of congestive heart failure for more than 200 years. Although it has been assumed that Na+/K+-transporting ATPase [1] is the sole receptor of digitalis glycosides and aglycones, which efficiently inhibit the active transport of potassium and sodium [2,3], yet the mechanism of the pharmacological action of these drugs still remains obscure. The results reported here show that H+-transporting ATPase [4] of fungal plasma membranes (Neurospora crassa) [5], CaZ+-transporting ATPase of the sarcoplasmic reticulum [6,7] and gastric H+/K+-transporting ATPase [8,9] can all be inhibited by strophanthidin, which is the aglycone of a digitalis glycoside. These observations suggest that the binding side for digitalis aglycones may be a common feature of all P-type [10] ATPases. The present data add further support to the assumption that Na+/K+-transporting ATPase, Ca 2+transporting ATPase, H + / K +-transporting ATPase and H +-transporting ATPase are closely related both structurally and functionally [11]. Furthermore, these results raise the possibility that digitalis intoxication may be the result of the multiple inhibition of P-type ATPases. This may lead to a better understanding of the action of these drugs and their positive inotropic effect.

Correspondence to: K.-y. Xu, Department of Cell Biology and Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.

The enzymatic activities of H +-transporting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase were assayed by modification of the method of Kyte [12]. The enzymatic activity is defined as the strophantidin-sensitive hydrolysis of MgATP in the presence of K + (20 mM) and absence of Na + for H+/K+-transporting ATPase, in the presence of Ca 2+ (10 /zM) and absence of both Na + and K + for Ca 2+transporting ATPase and in the absence of Ca 2+, K + and Na + for H+-transporting ATPase. In order to obtain the maximum access of substrates to active sites, the surfactant, saponin, (0.67%) which is known not to inhibit the enzymatic activity [13] of Na+/K+-trans porting ATPase was routinely present during the assay for Ca2+-transporting ATPase and H+/K+-transport ing ATPase. To limit Na+/K+-transporting ATPase activity, ouabain, (10 p.M) which has no inhibitory effect at this concentration on the enzymatic activity of H+/K+-transporting ATPase (data not shown), was also routinely present during the assay for H + / K +transporting ATPase. The final concentration of dimethylformamide (solvent for strophanthidin) for all samples were 0.5 (Fig. 1) and 0.2% (Figs. 2, 3) and no inhibition effect was observed at these concentrations of dimethylformamide on the enzymatic activities of H+-transporting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase (data not shown). The specific activities of H+-transporting ATPase, Ca2+-transporting ATPase, and H+/K+-transporting ATPase were 244 /zmol Pi mg -~ h - I , 360 p~mol Pi

110 100

m g - ] h - ~ and 107 ~mol P~ m g - 1 h - z, respectively, when only strophanthidin-sensitive hydrolysis of MgATP was considered. The percentage of the total ATPase activity in each of these purified preparations that was inhibited by strophanthidin at concentrations >/0.1 mM was 80, 90 and 65%, respectively. When only these strophanthidin-sensitive ATPase activities were measured, inhibition curves for the drug could be obtained (Fig. 1). Half-maximal inhibition of H+-trans porting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase were achieved at 50, 70 and 80 /xM strophanthidin and no further inhibition

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Fig. 1. Inhibition of H+-transporting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase activities as function of the concentration of strophanthidin. Purified Neurospora crassa H+-transporting ATPase [29] (e), Ca2+-transporting ATPase of rabbit sarcoplasmic reticulum [30] (a) and a microsomal preparation of pig gastric H+/K+-transporting ATPase [9] (D) were incubated at 36°C for 30 rain in 60 mM histidinium chloride (pH 6.8) for H ÷transporting ATPase and in 30 mM histidinium chloride (pH 7.1) for Ca2+-transporting ATPase and H+/K+-transporting ATPase with the indicated concentrations of strophanthidin before measuring enzymatic activity• Final concentration of MgATP was 3 mM for all assays of enzymatic activity. The strophanthidin-sensitive ATPase activities are expressed relative to the activity in the absence of inhibitor. Data represent average of three experiments.

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Fig. 2. Inhibition of H+-transporting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase by strophanthidin as a function of the concentration of MgATP. At various fixed concentrationsofMgATP, t h e d a t a a r e plottedinthe formof V ' against [MgATP]-] at various fixed concentrations of strophanthidin (A, B and C)" V represents the absOrbance at 700nmOfstrOphanthidinsensitive MgATP hydrolysis. The fixed concentrations of substrate or inhibitor (I) associated with each line are indicated in each panel. The apparent values of K i of strophanthidin for H+-transporting ATPase (A), Ca2+-transporting ATPase (B) and H+/K+-transport ing ATPase (C) were determined by replot of intercepts as show in the inset. All the experiments were performed at 36°C for 10 min at pH 6.8 for H+-transporting ATPase, pH 7.1 for Ca2+-transporting ATPase and H+/K+-transporting ATPase. The data are presented from one of three similar independent experiments in each case. The lines are computer-generated linear regression 'best-fit' lines.

transporting ATPase and H+/K+-transporting ATPase, the effect of varying the concentrations of various substrates and cations on the inhibition was determined. At fixed concentrations of K + and Ca e+ and variable concentrations of MgATP, strophanthidin was found to be a noncompetitive inhibitor with respect to MgATP of H+-transporting ATPase (Fig. 2A), Ca e+-

111 transporting ATPase (Fig. 2B), and H + / K + - t r a n s p o r t ing ATPase (Fig. 2C). These data indicate that MgATP and strophanthidin bind to the enzyme reversibly, randomly, and independently at different sites. Values of K m and K i (Table I) were calculated from double reciprocal plots (Fig. 2A-C). The values of Krn for MgATP were 2.8, 0.16 and 0.22 mM for H+-transport L

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Kinetics of inhibition of H +-transporting ATPase, Ca 2 +-transporting ATPase and H + / K + - t r a n s p o r t i n g A T P a s e by strophanthidin Enzymatic assays were performed as described in the legends to Figs. 2 and 3. Kinetic constants were determined by Dixon plots and Lineweaver-Burk plots. The mode of inhibition is noncompetitive with respect to M g A T P and competitive with respect to potassium. The data represent the m e a n of 3 - 6 independent determinations. Enzyme

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Fig. 3. Inhibition of H+-transporting ATPase, Ca2+-transporting ATPase and H + / K + - t r a n s p o r t i n g A T P a s e by strophanthidin as a function of the concentration of K +. At constant concentrations of MgATP, 3 m M for H+-transporting ATPase, 2 m M for CaZ+-trans porting ATPase and H + / K + - t r a n s p o r t i n g ATPase, the concentrations of potassium and stophanthidin were varied in the standard assay as indicated. The fixed concentration of either strophanthidin (I) or K + associated with each line is indicated in each panel. Dixon plots are presented for H+-transporting ATPase (A) and CaZ+-trans porting ATPase (B) and a Lineweaver-Burk plot is presented for H + / K + - t r a n s p o r t i n g ATPase (C). The data are presented from 1 of 3 similar independent experiments. All the experiments were performed at 36°C for 10 min at pH 6.8 for H+-transporting ATPase and pH 7.1 for Ca2+-transporting ATPase and H + / K + - t r a n s p o r t i n g ATPase. The lines are computer-generated linear regression 'best-fit' lines.

ing ATPase, Ca2+-transporting ATPase, and H + / K +transporting ATPase, respectively. These are similar to those reported previously [24,25]. The apparent values for K i w e r e 0.09, 0.25 and 0.16 mM for H+-transport ing ATPase, CaZ+-transporting ATPase and H + / K +transporting ATPase, respectively. It is well-established that the inhibition of Na+/K+-transporting ATPase by cardiac glycosides is antagonized by potassium [26,27]. Further kinetic experiments were performed to determine the effect of ions on the inhibition of the other ATPases by strophanthidin. At a fixed concentration of MgATP, with varying concentrations of K + and inhibitor, K + is a competitive antagonist of the inhibition caused by strophanthidin (Fig. 3) of H+-transporting ATPase (Fig. 3A), CaZ+-transporting ATPase (Fig. 3B) and H + / K +transporting ATPase (Fig. 3C). These kinetic studies suggest that there is a binding site for K + on both H+-transporting ATPase and CaZ+-transporting ATPase. The kinetics of strophanthidin inhibition also suggest (Fig. 3A) that there is a binding site for Na + on H+-transporting ATPase. It is unclear whether the site at which K + binds is actually involved in the active transport catalyzed by these enzymes or if it is just a remnant of evolution. The results in Fig. 3, however, further support the existence of a common cardiac glycoside binding-site located on the extracellular surface of the enzyme which is affected competitively by the binding of K + to H+-transporting ATPase, Ca 2+transporting ATPase, as well as H+/K+-transporting ATPase and Na+/K+-transporting ATPase. Certain kinetic constants for H+-transporting ATPase, Ca 2+ transporting ATPase and H+/K+-transporting ATPase are summarized in Table I. From the values of K i it can be seen that the other three ATPases are less sensitive to stophanthidin than dog, pig and sheep Na+/K+-transporting ATPase, but similar to N a + / K+-transporting ATPase from rat kidney [28].

112 T h e o b s e r v a t i o n s r e p o r t e d here d e m o n s t r a t e t h r e e i m p o r t a n t points. First, the b i n d i n g site for s t r o p h a n thidin may be a c o m m o n f e a t u r e of all P-type A T P a s e s . Second, kinetic data suggest that there is a b i n d i n g site for K ÷ on H + - t r a n s p o r t i n g A T P a s e a n d Ca2÷-trans p o r t i n g A T P a s e . Third, K ÷ a n t a g o n i z e s the b i n d i n g of s t r o p h a n t h i d i n competitively, p r e s u m a b l y by b i n d i n g to an extracellular site c o m m o n to all P-type A T P a s e . F o r a b e t t e r u n d e r s t a n d i n g of the m e c h a n i s m of action of cardiac glycosides, the possibility that positive inotropic action might result from the i n h i b i t i o n of both Ca e+transporting ATPase and N a ÷ / K + - t r a n s p o r t i n g ATPase to lead to a t r a n s i e n t m e m b r a n e d e p o l a r i z a t i o n a n d a rise in i n t r a c e l l u l a r Ca 2÷ should be explored. I am grateful to B. F o r b u s h , J. H o f f m a n , J. Kyte, C.W. S l a y m a n a n d C.L. S l a y m a n for their e n c o u r a g e m e n t , advice a n d helpful discussions. I also t h a n k R. L e v e n s o n for the support. Purified H ÷ - A T P a s e a n d C a 2 + - A T P a s e were from C.W. Slayman. H + / K +A T P a s e was from B. F o r b u s h . This work was supported by grants from the N a t i o n a l I n s t i t u t e s of H e a l t h to R. L e v e n s o n a n d B. F o r b u s h .

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