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

Abstract

A [rat brain] 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 1 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 calcium oxalate; approximately 100-fold purification resulted. The purified Ca-transporting vesicles contained 2 major protein components, of Mr [relative MW] 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.