38] ATPase activity of Chinese hamster P-glycoprotein

Abstract

We have developed two defined experimental systems for biochemical investigation of P-glycoprotein, namely, plasma membranes highly enriched in Pgp, obtained from the CR1R12 Chinese hamster ovary cell line, and pure, reconstituted Pgp, obtained by solubilization of Pgp from CR1R12 plasma membranes, Reactive Red 120 chromatography, and reconstitution in liposomes. Studies of the ATPase catalytic mechanism by kinetic methods and covalent inactivation have been greatly facilitated by the availability of these experimental systems. The technique of vanadate trapping of nucleotide has been particularly useful. As a result of these studies, we now have explicit, testable, proposals for (1) the normal catalytic pathway of ATP hydrolysis, (2) a postulated alternating catalytic site cycle, and (3) coupling of ATP hydrolysis to drug transport. The experimental methods described here should prove valuable for future studies of Pgp and of ABC transporters in general.