Calmodulin-sensitive calcium-pumping ATPase of plasma membranes: isolation, reconstitution, and regulation.
The calmodulin-sensitive Ca2+ -pumping ATPase was purified to virtual homogeneity from erythrocytes. The purified enzyme exists in two functional states, having low and high Ca2+ affinity. Transition from low to high affinity is induced by 1) calmodulin; 2) acidic phospholipids, long-chain polyunsaturated fatty acids, polyphosphoinositides; and 3) a controlled proteolytic treatment with trypsin or chymotrypsin. The ATPase can be reconstituted into liposomes, where it pumps Ca2+ in exchange for H+ with a stoichiometry to ATP approaching 1. The purified enzyme can be fragmented by trypsin into a number of transient and of limit polypeptides, of which the most interesting from the functional standpoint are the following: 1) a limit polypeptide of Mr 76,000 that contains the active site (i.e., the sequence where the acyl-phosphate is formed); 2) a limit polypeptide of Mr 33,500 that binds the hydrophobic photoactivable label 3-trifluoromethyl-3-(m-(125I-iodophenyl]-diazirine, and is thus presumably the most hydrophobic portion of the molecule; and 3) a transient polypeptide of Mr 90,000 and a limit polypeptide of Mr 25,000-28,000, which specifically bind azido-modified, 125 I-labeled calmodulin. The transient 90,000-dalton calmodulin receptor is rapidly degraded to the 81,000-76,000 limit polypeptide. It can be isolated from the other proteolysis products on calmodulin affinity chromatography columns. The isolated 90,000-dalton fragment is a fully competent, calmodulin-sensitive ATPase that pumps Ca2+ into reconstituted liposomes.
D001693:Biological Transport, Active; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D008081:Liposomes; D008563:Membrane Lipids / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis; D010743:Phospholipids / Q000502:physiology; D013329:Structure-Activity Relationship
D002147:Calmodulin; D008081:Liposomes; D008563:Membrane Lipids; D010446:Peptide Fragments; D010743:Phospholipids; D000252:Calcium-Transporting ATPases