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 mumol/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.
Loo
T W
TW
Department of Medicine, University of Toronto, Ontario, Canada.
Clarke
D M
DM
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003078:Colchicine / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009532:Nickel; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011994:Recombinant Proteins; D014747:Vinblastine; D009532:Nickel; D014700:Verapamil; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D003078:Colchicine