FUNCTIONAL RECONSTITUTION OF P-GLYCOPROTEIN REVEALS AN APPARENT NEAR STOICHIOMETRIC DRUG TRANSPORT TO ATP HYDROLYSIS

We have recently described an ATP-driven, valinomycin-dependent Rb-86( +) uptake into proteoliposomes reconstituted with mammalian P-glycopro tein (Eaten, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (199 4) J. Biol. Chem, 269, 26058-26065), P-glycoprotein mediated the ATP-d ependent uptake of Rb-86(+)-ionophore complex into the proteoliposomes , where the radioactive cation was accumulated, thus, circumventing th e obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies, Taking advantage of this assay and of the high lev els of P-glycoprotein expression in multi-drug-resistant Chinese hamst er ovary cells, we measured simultaneously both the ATPase and transpo rt activities of P-glycoprotein under identical conditions and observe d 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed, The amount of Rb-86(+) ions transported within 1 min via the ATP and vali nomycin-dependent P-glycoprotein was equivalent to an intravesicular c ation concentration of 8 mM. Thus, this stoichiometry and transport ca pacity of P-glycoprotein resemble various ion-translocating ATPases, t hat handle millimolar substrate concentrations. This constitutes the f irst demonstration of comparable rates of P-glycoprotein-catalyzed sub strate transport and ATP hydrolysis.