CHARACTERIZATION AND FUNCTIONAL RECONSTITUTION OF THE MULTIDRUG TRANSPORTER

P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CH(R)C5 cells using a selective two-step detergent extrac tion procedure. The partially purified protein displays a high level o f ATPase activity, which has a high K-M for ATP, is stimulated by drug s, and can be distinguished from that of other membrane ATPases by its unique inhibition profile. Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures. P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity. Proteoliposomes containing P -glycoprotein display osmotically sensitive ATP-dependent accumulation of H-3-colchicine in the vesicle lumen. Drug transport is active, gen erating a stable 5.6-fold concentration gradient, and can be blocked b y compounds in the multidrug resistance spectrum. Reconstituted P-glyc oprotein also exhibits a high level of ATPase activity which is furthe r stimulated by various drugs. P-Glycoprotein therefore functions as a n active drug transporter with constitutive ATPase activity.