The purified Ca2+ pump of human erythrocyte membranes catalyzes an electroneutral Ca2+-H+ exchange in reconstituted liposomal systems.

Abstract

395-401) transports Ca2+ after reconstitution inasolectin liposomes, and the ATPase activity i s stimulated 9to 10-fold by A 23187.This indicates tight coupling between ATP hydrolysis and Ca2+ transport.In this system "extra" protons (i.e. in excess of these produced during the hydrolysis of ATP) appear in the medium, with a ratio of about 2 per ATP hydrolyzed.Evidently, the ATP-linked Ca2+ uptake induces the ejection of protons from the vesicles.In the presence of the protonophore carbonyl cyanide-m-chlorophenyl hydrazone and of valinomycin, the proton ejection is decreased by about 50%, indicating that a transmembrane proton gradient is indeed formed during Ca2+ transport.In the presence of the Ca'+ ionophore A 23187, no extra protons are produced during ATP hydrolysis.In addition, evidence that Caz* transport is electroneutral is provided by the fact that no accumulation of tetraphenylboron, a lipophilic anion, takes place during Ca2+ transport.Moreover, valinomycin (in the presence of K') has no stimulatory effect on Ca" transport rates.In the presence of the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, which should inhibit anion transport through residues of band I11 possibly contaminating the Ca2'-ATPase preparation, valinomycin was equally ineffective.It is therefore proposed that the Ca2+ pump of human erythrocyte membranes operates as an electroneutral Ca2+-H+ antiporter.ATP-dependent Ca" pumps have now been identified in a variety of cell membranes (1) and two of them have even been purified to near homogeneity (2, 3 ) .The mechanism of these pumps, however, has not yet been clarified.For instance, the problem of whether Ca2+ transport is electrogenic, or obligatorily compensated by co-or countertransport of other ions has not yet been solved.As discussed by Sarkadi (41, there is some evidence, based on experiments with resealed ghosts, that Ca2+ uptake is not dependent upon Na', K', and not coupled to the countertransport of any divalent cation.Recently, Waisman et al. have presented some evidence that Ca2+ transport in inside-out resealed ghosts is stimulated by