Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma.
The Ca2+-pumping ATPase has been isolated from calf heart sarcolemma by calmodulin affinity chromatography (Caroni, P., and Carafoli, E. (1981) J. Biol. Chem. 256, 3263-3270) as a polypeptide of Mr about 140,000. The purified enzyme has high affinity for Ca2+ in the presence of calmodulin (Km about 0.4 microM) but shifts to a low affinity state (Km about 20 microM) 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.
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D004910:Erythrocyte Membrane / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D012508:Sarcolemma / Q000201:enzymology*; D014357:Trypsin
D002147:Calmodulin; D008081:Liposomes; D010446:Peptide Fragments; D000001:Calcimycin; D014357:Trypsin; D000252:Calcium-Transporting ATPases