Purification of a low affinity Mg2+ (Ca2+)-ATPase from the plasma membranes of a human oat cell carcinoma.

Plasma membranes of many mammalian cells contain a Mg2+-dependent ATPase activity which is easily inactivated by detergents. This activity is the combined expression of at least two ATP-hydrolyzing enzymes (Knowles, A.F., Isler, R.E., and Reece, J.F. (1983) Biochim. Biophys. Acta 731, 88-96). We have purified one of these enzymes from the plasma membranes of a human oat cell carcinoma xenograft. The enzyme was extracted from the membranes by 0.5% digitonin and purified on a DE52 column. The purified enzyme contained a major protein band of Mr = 30,000 when dissociated by sodium dodecyl sulfate. It hydrolyzed all nucleoside triphosphates in the presence of Mg2+ or Ca2+, but showed little activity toward nucleoside diphosphates. The enzyme was inhibited by p-chloromercuriphenyl sulfonate, slowly inactivated by p-fluorosulfonylbenzoyl-5'-adenosine and dithiothreitol at room temperature, and lost activity readily in solutions containing low concentrations of several detergents. This knowledge of the macromolecular structure of the Mg2+(Ca2+)-ATPase and its catalytic properties is important in determining the orientation of the enzyme in the membrane and its physiological function.