Partial purification and functional identification of a calmodulin-activated, adenosine 5'-triphosphate-dependent...

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.