CA2+ H+ COUNTERTRANSPORT AND ELECTROGENICITY IN PROTEOLIPOSOMES CONTAINING ERYTHROCYTE PLASMA-MEMBRANE CA-ATPASE AND EXOGENOUS LIPIDS/

A reconstituted proteoliposomal system was obtained with Ca-ATPase pur ified from human erythrocyte membrane (plasma membrane, PM ATPase), an d liposomes prepared by reverse-phase evaporation. The reconstituted P M ATPase behaved as an electrogenic Ca2(+)/H+ exchanger and, under opt imal conditions, utilization of 1 mol of ATP was accompanied by uptake of one Ca2+ by the vesicles, and ejection of one H+ from the lumen of the vesicles. Ca2+ uptake was greatly (5-fold) stimulated by the addi tion of calmodulin, and by collapsing the H+ gradient with the ionopho re carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of calmodulin and p-trifluoromethoxyphenylhydrazone, the reconstitute d system sustained transport rates of 1.00 +/- 0.12 mu mol of Ca2+/mg of protein min(-1) (30 degrees C), reaching asymptotic levels of 8.05 +/- 0.41 mu mol of Ca2+/mg of protein (i.e. 20 mM lumenal Ca2+). The c orresponding net charge transfer produced a maximal electrical gradien t of 40.5 +/- 1.8 mV at steady state. Demonstration of the electrogeni c behavior of the PM ATPase, obtained for the first time with these ex periments, was critically dependent on the detergent used in the recon stitution procedure. The lumenal pH rise had a much greater rate-limit ing effect on the pump, than the electrical potential developed by the pump.