CHARACTERIZATION OF THE ATPASE ACTIVITY OF PURIFIED CHINESE-HAMSTER P-GLYCOPROTEIN

A Simple and rapid procedure is described for purification of P-glycop rotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell lin e (CR1R12) in which the plasma membranes are highly enriched in Pgp (A l-Shawl, M. K., Senior A, E. (1993) J. Biol. Chem. 268, 4197-4206). Th e procedure consisted of octylglucoside solubilization of Pgp from pla sma membranes and chromatography on Reactive Red 120 agarose. The puri fied Pgp displayed substantial verapamil-stimulated MgATPase activity (k(cat) = 9.2 s(-1), K-M(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characteriz ed in terms of pH dependence, magnesium versus calcium specificity, ki netic parameters, nucleotide specificity, and inhibitors. There was po tent inactivation of MgATPase activity by NEM and NBD-Cl, which was di minished greatly by MgATP protection. Vanadate was also an effective i nhibitor. Predominantly, the catalytic features seen resembled those r eported previously for the plasma membrane-bound form of Pgp. The cata lytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.