A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

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.