Partial purification and functional identification of a calmodulin-activated, adenosine 5'-triphosphate-dependent calcium pump from synaptic plasma membranes.
Synaptic plasma membranes isolated from rat brain contain a calmodulin-activated Ca2+ pump. It has been purified 80- to 160-fold by solubilization with Triton X-100 and affinity chromatography on a calmodulin-Sepharose 4B column. After reconstitution into phospholipid vesicles, the affinity-purified pump efficiently catalyzed ATP dependent Ca2+ transport, which was activated 7- to 9-fold by calmodulin. The major protein component of the affinity-purified preparation had a Mr = 140,000; it was virtually the only band visualized on a Coomassie blue-stained SDS polyacrylamide gel. It has been identified as the Ca2+ pump by two functional criteria. First, it was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner; the phosphorylated protein had the chemical reactivity of an acyl phosphate, characteristic of the phosphorylated intermediates of ion-transporting ATPases. Second, the protein was enriched by transport-specific fractionation, a density gradient procedure which uses the transport properties of the reconstituted Ca2+ pump as a physical tool for its purification. By analogy with calmodulin-activated (Ca2+ + Mg2+) ATPases of other cell types, and because of its presence in a synaptic plasma membrane fraction, we hypothesize that the calmodulin-activated Ca2+ pump functions in vivo to extrude Ca2+ from nerve terminals.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D013570:Synaptic Membranes / Q000201:enzymology*
D002147:Calmodulin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases