Active calcium transport and calcium-dependent membrane phosphorylation in human peripheral blood lymphocytes

The characteristics of the calcium pump were investigated in intact human peripheral blood lymphocytes /PBL/ and in inside-out vesicles prepared from their plasma membranes. Intact PBL were loaded with calcium by a short exposure to A23187 ionophore. After the elimination of the ionophore, calcium-loaded PBL produced an ATP-dependent, external lanthanum sensitive, uphill calcium extrusion. Calcium pump in intact PBL was insensitive to ouabain and /until cellular ATP was provided/ to oligomycin and dinitrophenol. Maximum calcium extrusion rate and the alkali cation sensitivity of the process were similar to those in human red cells. Calcium was partially sequestered by PBL, and this calcium could be released by A23187 ionophore only. Inside-out plasma membrane vesicles prepared from hypotonically lysed PBL showed an ATP + Mg2+-dependent uphill calcium uptake. This calcium transport was insensitive to ouabain, oligomycin, or dinitrophenol, while blocked by lanthanum and quercetin. Calmodulin significantly stimulated calcium pumping in EDTA-washed vesicles. ATP-dependent and -independent calcium uptake rates, respectively, showed different calcium concentration dependences. When PBL membrane vesicles were phosphorylated by gamma 32P-ATP, a calcium-induced, hydroxylamine-sensitive incorporation of 32P was found in 120-150 000 molecular weight proteins. Depending on the way of membrane preparation, the molecular weight of the phosphoprotein was shifted. Similarly to that found in red cell membranes, sensitivity to calmodulin stimulation and partial proteolysis of the calcium pump molecule showed an inverse relationship.