RESUMEN

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

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RESUMEN

The activation of the Ca(2+)-ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half-maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concentrations when the pH is increased. In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.

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RESUMEN

(1) We have reexamined the effects of calmodulin and of the calmodulin antagonist, compound 48/80 on the interaction of ATP at its low-affinity site in the Ca2(+)-ATPase from human red cells. (2) At variance with our earlier proposal (Biochim. Biophys. Acta (1985) 816, 379-386) calmodulin increased the maximum effect of ATP without changing the apparent affinity for ATP at the low-affinity site. Accordingly, ATP increased the maximum activation by calmodulin without altering the apparent affinity of the Ca2(+)-ATPase for calmodulin. (3) Confirming our previous observation (Biochim. Biophys. Acta (1985) 816, 379-386) compound 48/80 lowered the apparent affinity of the Ca2(+)-ATPase for ATP at the low-affinity site. This has to be attributed to a direct effect of this compound on the enzyme rather than to its effect as calmodulin antagonist.

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