Relation between the turnover number for vinblastine transport and for vinblastine-stimulated ATP hydrolysis by human P-glycoprotein

Considerable uncertainty surrounds the stoichiometry of coupling of ATP hydrolysis to drug pumping by P-glycoprotein, the multidrug transporter. To estimate relative turnovers for pumping of the drug vinblastine and ATP hydrolysis, we began by measuring the number of P-glycoprotein molecules on the surface of murine NIH3T3 cells expressing the human MDR1 gene. Fluorescence of cells treated with monoclonal antibody UIC2 was determined as a function of (i) amount of antibody at a fixed number of cells and (ii) increasing cell number at constant antibody. The two together gives 1.95 x 10⁶ P-glycoprotein molecules/cell. Initial uptake rates of vinblastine ± verapamil measure the ability of P-glycoprotein to extract vinblastine from the plasma membrane before it enters the cell. As a function of [vinblastine] at 37 °C, they give the maximum rate of this component of outward pumping as 2.1 x 10⁶ molecules s^-1 cell^-1 or a turnover number of 1.1 s^-1. Initial rates of one-way efflux as a function of [vinblastine] at 25 °C ± glucose give the maximum rate of this component of pumping as 0.59 x 10⁶ molecules s^-1 cell^-1. The ratio of ATPase activity of P-glycoprotein at 37 and 25 °C is 4.6. Appropriating this ratio for pumping, maximum one-way efflux at 37 °C is 4.6 x 0.59 = 2.7 x 10⁶ molecules s^-1 cell 1^-1, a turnover number of 1.4 s^-1. The vinblastine-stimulated ATPase activity of P-glycoprotein has a turnover number of 3.5 s^-1 at 37 °C, giving 2.8 molecules of ATP hydrolyzed for every vinblastine molecule transported in a particular direction. These calculations involve several approximations, but turnover numbers for pumping of vinblastine and for vinblastine-stimulated ATP hydrolysis are comparable. Thus, ATP hydrolysis is probably directly linked to drug transport by P-glycoprotein.