Protein Kinase-Dependent Phosphorylation of Cardiac Sarcolemmal Ca2+ as Studied with a Specific Monoclonal Antibody1

Abstract

Phosphorylation of the Ca²⁺ ATPase of cadiac sarcolemmal vesicles by exogeneously added protein kineses was examined to elucidate the molecular basis for its regulation. The Ca²⁺-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca²⁺ Protein kinase C (C-kinase) was found to phosphorylate the Ca²⁺ The stoichiometry of this phosphor ylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca²⁺ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca²⁺ pump for Ca²⁺ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca²⁺ preparation isolated from cardiac sarcolemma with cal modulin-Sepharose and a high salt-washing procedure. Thus, C-klnase appears to stimu late the activity of the sarcolemmal Ca²⁺ pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca²⁺ protein kinase II phosphorylated the Ca²⁺ in the sarcoleinmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca²⁺ uptake by sarcolemmal vesicles.