CHARACTERIZATION OF THE ATPASE ACTIVITY OF P-GLYCOPROTEIN FROM MULTIDRUG-RESISTANT CHINESE-HAMSTER OVARY CELLS

P-Glycoprotein (Pgp) was isolated from CH(R)C5 membranes by selective detergent extraction and further purified by lentil lectin affinity ch romatography. The purified product displayed a very high basal ATPase activity (1.65 mu mol/min per mg protein in the absence of added drugs or lipids) with an apparent K-m for ATP of 0.4 mM. There was no evide nce of cooperativity, suggesting that the two ATP sites operate indepe ndently of each other. Pgp ATPase activity was stimulated by verapamil , trifluoperazine and colchicine, and inhibited by daunomycin and vinb lastine. All drugs and chemosensitizers acted as mixed activators or i nhibitors, producing changes in both the V-max of the ATPase and the K -m for ATP. ADP competitively inhibited Pgp ATPase, with a K-i of 0.2 mM. The macrolide antibiotics bafilomycin A(1), concanamycin A and con canamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 mu M, an d at an inhibitor:protein stoichiometry of 0.65-1.0 mu mol/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 mann er, whereas others produced inhibition. Metabolic labelling showed tha t the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. P-32-Labelling studies indicate d that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPas e whose catalytic ac tivity can be modulated by both drugs and phospho lipids.