Isolation of frog heart sarcolemma possessing (Ca2+ + Mg2+)-ATPase and Ca2+ pump activities

Abstract

The presence of a (Ca2+ + Mg2+)-ATPase and Ca2+ pump in frog heart sarcolemma was investigated. Frog cardiac sarcolemma was isolated by using a modification of a recent procedure for isolating sarcolemma from dog heart (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 17-39). Despite the sparseness of sarcoplasmic reticulum in frog heart, this tissue possessed (Ca2+ + Mg2+)-ATPase activity which appeared endogenous to the sarcolemma. The purified sarcolemmal vesicles also possessed ouabain-sensitive (Na+ + K+)-ATPase, Na+ -Ca2+ exchange activity and Ca2+ pump activity. Inclusion of ionophore A23187 into the assay medium resulted in an approximately 5-fold increase in (Ca2+ + Mg2+)-ATPase which may be due to removal of inhibitory concentrations of Ca2+ accumulated within the membrane vesicles by an ATP-dependent process. Vesicles pre-loaded with 45Ca2+, via Na+-Ca2+ exchange, lost 92% of their Ca2+ when ATP was present and only 60% in the absence of ATP. The results indicate that frog heart sarcolemma possesses a (Ca2+ + Mg2+)-ATPase and an ATP-dependent Ca2+ pump which may be involved in the outward pumping of Ca2+ from the heart cell.