RAPID PURIFICATION OF HUMAN P-GLYCOPROTEIN MUTANTS EXPRESSED TRANSIENTLY IN HEK-293 CELLS BY NICKEL-CHELATE CHROMATOGRAPHY AND CHARACTERIZATION OF THEIR DRUG-STIMULATED ATPASE ACTIVITIES

P-glycoprotein containing 10 tandem histidine residues at the COOH end of the molecule was transiently expressed in HEK 293 cells and purified by nickel chelate chromatography. The purified protein had an apparent mass of 170 kDa, and its verapamil-stimulated ATPase activity in the presence of phospholipid was 1.2 mu mol/min/mg of P-glycoprotein. We then characterized P-glycoprotein mutants that exhibited altered drug-resistant phenotypes and analyzed the contribution of the two nucleotide binding folds to drug-stimulated ATPase activity, Mutation of residues in either nucleotide binding fold abolished drug-stimulated ATPase activity. The pattern of drug-stimulated ATPase activities of mutants, which conferred increased relative resistance to colchicine (G141V, G185V, G830V) or decreased relative resistance to all drugs (F978A), correlated with their drug-resistant phenotypes, By contrast, the ATPase activity of mutant F335A was significantly higher than that of wild-type enzyme when assayed in the presence of verapamil (3.4 fold), colchicine (9.1-fold), or vinblastine (3.7-fold), even though it conferred little resistance to vinblastine in transfected cells. These results suggest that both nucleotide-binding domains must be intact to couple drug binding to ATPase activity and that the drug-stimulated ATPase activity profile of a mutant does not always correlate with its drug-resistant phenotype.