Reconstitution and purification by "transport specificity fractionation" of an ATP-dependent calcium transport component from synaptosome-derived vesicles

Abstract

A synaptosomal ATP-dependent Ca uptake system was reconstituted into artificial vesicles by a cholate dialysis procedure using an 80-fold excess of exogenous phospholipid. Under these conditions, most of these vesicles would be expected to have only one or, at most, a few membrane proteins. The vesicles containing an ATP-dependent Ca transport system were purified from the bulk of the preparation on density gradients by increasing their density by the ATP-dependent intravesicular precipitation of Ca oxalate; a approximately 100-fold purification resulted. The purified Ca-transporting vesicles contained two major protein components, of Mr 94,000 and 140,000 according to sodium dodecyl sulfate gel electrophoresis. These components are believed to be responsible for Ca transport in this synaptosome-derived membrane fraction.