ABCMdb

PMID: 7665554

Loo TW, Clarke DM
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.
J Biol Chem. 1995 Sep 15;270(37):21449-52., 1995-09-15 [PubMed]

Sentences

No. Mutations Sentence Comment

64 ABCB1 p.Phe335Ala ABCB1 p.Gly830Val ABCB1 p.Gly141Val For example, mutants G141V or G830V conferred increased resistance to colchicine (about 3-fold) relative to that of wild-type enzyme while mutant F335A conferred decreased resistance to vinblastine. Login to comment 65 ABCB1 p.Phe978Ala By contrast, mutant F978A conferred decreased resistance to all drugs. Login to comment 66 ABCB1 p.Gly185Val In this study, we also included for comparison mutant G185V, which was recently shown by Rao (1995) to have increased verapamiland colchicine-stimulated ATPase activities (2and 3.3-fold, respectively). Login to comment 69 ABCB1 p.Gly185Val ABCB1 p.Gly830Val ABCB1 p.Gly141Val The maximal verapamil-stimulated ATPase activities of mutants G141V, G185V, and G830V were all slightly increased (1.4-1.7-fold) relative to that of wild-type enzyme (Fig. 2). Login to comment 81 ABCB1 p.Gly185Val ABCB1 p.Phe978Ala ABCB1 p.Phe335Ala ABCB1 p.Gly830Val ABCB1 p.Gly141Val Wild-type (E) and mutants G141V (å), G185V (Ⅺ), G830V (q), F335A (f), and F978A (Ç) P-glycoproteins-(His)10 were purified using Ni-NTA spin columns and reconstituted with sheep brain phosphatidylethanolamine. Login to comment 83 ABCB1 p.Phe978Ala Mutant F978A, which confers little resistance to vinblastine, colchicine, doxorubicin, or actinomycin D in transfected cells, also showed little drug-stimulated ATPase activity, except at very high concentrations of verapamil (1.04 ␮mol/min/mg of P-glycoprotein at 800 ␮M verapamil). Login to comment 88 ABCB1 p.Phe335Ala The basal ATPase activity of mutant F335A was also about 3-fold higher (0.32 ␮mol/min/mg of P-glycoprotein) than that of wild-type enzyme (0.11 ␮mol/ min/mg of P-glycoprotein). Login to comment 95 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met ABCB1 p.Gly432Ser ABCB1 p.Gly1075Ser To determine the contribution of either nucleotide-binding domain to drug-stimulatable ATPase activity, mutations were made to the core amino acids (G432S, K433M, G1075S, and K1076M, respectively) of the homology A consensus sequences. Login to comment 111 ABCB1 p.Gly185Val ABCB1 p.Phe978Ala ABCB1 p.Gly830Val ABCB1 p.Gly141Val For mutants G141V, G185V, G830V, and F978A, the pattern of drug-stimulated ATPase correlated with their relative drug-resistant profiles in transfected cells. Login to comment 114 ABCB1 p.Phe978Ala Mutant F978A conferred little resistance to all drug substrates in transfected cells, and the purified protein also showed ex- FIG. 3. Login to comment 122 ABCB1 p.Gly432Ser E, wild type; q, Cys-less; f, mutant G432S. Login to comment 123 ABCB1 p.Lys1076Met ABCB1 p.Lys433Met ABCB1 p.Gly1075Ser Mutants K433M, G1075S, and K1076M had no detectable ATPase activities and are omitted for clarity. Login to comment 126 ABCB1 p.Phe335Ala Purified mutant F335A P-glycoprotein, however, showed large increases in ATPase activity in the presence of all three drug substrates but conferred decreased relative resistance to vinblastine and only a small increase in resistance to colchicine in transfected cells (Loo and Clarke, 1993b). Login to comment 127 ABCB1 p.Phe335Ala One explanation for this discrepancy is that mutation F335A alters the dissociation of vinblastine from P-glycoprotein such that the enzyme is slow in effluxing vinblastine. Login to comment 129 ABCB1 p.Phe335Ala These possibilities could explain the fact that purified mutant F335A has a higher basal as well as drug-stimulated ATPase activity compared with wild-type enzyme. Login to comment 131 ABCB1 p.Gly185Val Acknowledgments-We thank Dr. Randal Kaufman (Boston) for pMT21 and Dr. Michael M. Gottesman (NIH) for the cDNA coding for P-glycoprotein mutant G185V. Login to comment