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

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte memb rane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-d iisothiocyanatostilbene-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 mu M DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolys is by DIDS is equally effective for both ghosts and purified enzyme, and th erefore is independent of its effect as an anion transport inhibitor. Bindi ng of the activator does not change the Ca2+ dependence of the pNPPase acti vity. Stimulation is partially additive to the activation of the pNPPase ac tivity elicited by calmodulin and appears to involve a strong affinity bind ing or covalent binding to sulfhydryl groups of the enzyme, since activatio n is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS dec reases 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 mu M DIDS the observed K-m 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, respectivel y). However, the pNPPase rate is always increased las 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 Ca2and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the p NPPase activity in the absence of Ca2+5, both in the presence and in the ab sence of DIDS. Therefore, kinetic evidence indicates that DIDS does more th an shift the enzyme to the E-2 conformation. We propose that the transition from E-2 to E-1 is decreased and a new enzyme conformer, denoted E-2*, is accumulated in the presence of DIDS.