Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

Article Details

Citation

Sharom FJ, Yu X, Chu JW, Doige CA

Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

Biochem J. 1995 Jun 1;308 ( Pt 2):381-90.

PubMed ID
7772017 [ View in PubMed
]
Abstract

P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

DrugBank Data that Cites this Article

Drug Targets
DrugTargetKindOrganismPharmacological ActionActions
Concanamycin AP-glycoprotein 1ProteinHumans
Yes
Inhibitor
Details
Drug Transporters
DrugTransporterKindOrganismPharmacological ActionActions
Concanamycin AP-glycoprotein 1ProteinHumans
Unknown
Inhibitor
Details