Inhibition by orthovanadate of ATP-dependent Ca2+ transport in microsomes isolated from rat liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5'-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at approximately 10 microM vanadate, corresponding to approximately 12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.