Purification and reconstitution of the Ca2+-ATPase from plasma membrane of pig erythrocytes.

Abstract

The Ca2+-ATPase from plasma membranes of pig erythrocytes was purified by mixed micelle gel chromatography (Wolf, H.U., Diekvoss, G., and Lichtner, R. (1977) Acta Biol. Med. Germ. 36, 847-858). The enzyme was activated at high concentrations of Tween 20 (10 mg/ml) or by appropriate mixtures of Triton X-100 and phospholipids. It was highly unstable in the absence of Ca2+ and activator protein. The Ca2+-ATPase was incorporated into liposomes by freeze-thaw sonication. After removal of non-ionic detergent by passage through a phenyl Sepharose 4B column, the reconstituted vesicles catalyzed a rapid ATP-dependent uptake of Ca2+. Modulator protein from brain substituted for the natural activator protein and stimulated Ca2+ uptake in reconstituted vesicles.