The purified heart sarcolemma Ca2+-pumping ATPase has high affinity for Ca2+ in the presence of calmodulin and can be reconstituted in liposomes to pump Ca2+ with a 1:1 stoichiometry to ATP.
as a polypeptide of Mr about 140,000.The purified enzyme has high affinity for Ca2+ in the presence of calmodulin ( K , about 0.4 pM) but shifts to a low affinity state ( K , about 20 PM) in its absence.Calmodulin increases also the Vmax of the enzyme.The effects of calmodulin are mimicked by phosphatidylserine and by a limited proteolytic treatment of the enzyme with trypsin.The purified ATPase can be reconstituted in asolectin liposomes, where it pumps Ca2+ with an approximate stoichiometry to ATP of 1.The purified (and reconstituted) enzyme is not phosphorylated by added ATP and CAMP-dependent protein kinase under conditions where the enzyme in situ is stimulated concomitant with the phosphorylation of the sarcolemmal membrane (Caroni, P., and Carafoli, E. (1981) J. Biol.Chem.256,9371-9373).Hence, the target of the regulatory phosphorylation system is not the ATPase molecule.The purified ATPase cross-reacts with an antibody raised against the erythrocyte Ca2+-pumping ATPase.Under the same conditions, the purified sarcoplasmic reticulum Ca2+-ATPase does not react.The proteolytic splitting pattern of the purified heart sarcolemma and erythrocyte enzymes are similar but not identical.' The abbreviations used are: DTT, dithiothreitol; EGTA, ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid Hepes, 4-(2-hydroxyethy1)-1-piperazineethanesulfonic acid SDS, sodium dodecyl sulfate; SL, sarcolemma.
Caroni et al. (Wed,) reported a other. The purified heart sarcolemma Ca2+-pumping ATPase has high affinity for Ca2+ in the presence of calmodulin and can be reconstituted in liposomes to pump Ca2+ with a 1:1 stoichiometry to ATP.