ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells.

P-glycoprotein was purified from multidrug-resistant Chinese hamster ovary CHRB30 cells by a combination of anion exchange and immunoaffinity chromatography. The P-glycoprotein was about 90% pure and had a Vmax for ATP hydrolysis in detergent solution of 321 nmol/min/mg with a Km of 0.94 mM. The ATPase activity was inhibited by low concentrations of vanadate and N-ethylmaleimide, but unaffected by azide or ouabain. When the purified P-glycoprotein was reconstituted into phospholipid bilayer membranes, the ATPase activity became highly stimulated by several chemosensitizers and drugs involved with multidrug resistance. Verapamil, a potent chemosensitizer, increased the Vmax for ATP hydrolysis by 22-fold and the Km for ATP by 5.4-fold. This effect of verapamil on P-glycoprotein has not previously been observed. These results demonstrate that purified P-glycoprotein has an intrinsic ATPase activity with unique properties. This activity appears sufficient to account for the ATP-dependent reduction in intracellular drug accumulation of P-glycoprotein-expressing multidrug-resistant cells.