title: Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis.
abstract: We have recently described an ATP-driven, valinomycin-dependent 86Rb+uptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (1994) J. Biol. Chem. 269, 26058-26065). P-glycoprotein mediated the ATP-dependent uptake of 86Rb+-ionophore complex into the proteoliposomes, where the radioactive cation was accumulated, thus, circumventing the obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies. Taking advantage of this assay and of the high levels of P-glycoprotein expression in multidrug-resistant Chinese hamster ovary cells, we measured simultaneously both the ATPase and transport activities of P-glycoprotein under identical conditions and observed 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed. The amount of 86Rb+ ions transported within 1 min via the ATP- and valinomycin-dependent P-glycoprotein was equivalent to an intravesicular cation concentration of 8 mM. Thus, this stoichiometry and transport capacity of P-glycoprotein resemble various ion-translocating ATPases, that handle millimolar substrate concentrations. This constitutes the first demonstration of comparable rates of P-glycoprotein-catalyzed substrate transport and ATP hydrolysis.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: G D
authors.initials: GD
authors.lastname: Eytan
authors.forename: R
authors.initials: R
authors.lastname: Regev
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004640:Emetine / Q000633:toxicity; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D009840:Oligomycins / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007476:Ionophores; D009840:Oligomycins; D010455:Peptides; D011510:Proteolipids; C099320:polytryptophan; C020485:proteoliposomes; D014634:Valinomycin; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D012413:Rubidium; D004640:Emetine