Purification of the synaptosomal plasma membrane (Ca2+ + Mg2+)-ATPase from pig brain

Abstract

The Ca2+-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca2+-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr 140000 that can be phosphorylated by [γ-32P]ATP in the presence of La3+ and recognized by specific antibodies against the plasma membrane Ca2+-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5–1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.