THE FUNCTIONAL PURIFICATION OF P-GLYCOPROTEIN IS DEPENDENT ON MAINTENANCE OF A LIPID-PROTEIN INTERFACE

P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which ca n confer the multi-drug resistance phenotype on tumor cells. We have e xamined the factors required to preserve activity of P-gp during its p urification. The starting material for purification was plasma membran es from Chinese hamster ovary (CH(r)B30) cells, overexpressing P-glyco -protein. These membranes displayed drug stimulated ATPase activity (V -m = 897 +/- 55 nmol min(-1) mg(-1); K-m = 1.8 +/- 0.4 mM) and high af finity binding of [H-3]vinblastine (K-d = 36 +/- 5 nM; B-m = 161 +/- 1 1 pmol/mg). Several non-ionic detergents which readily solubilized P-g lycoprotein significantly inhibited ATPase activity and drug binding a t concentrations well below their respective CMC values. This inactiva tion was prevented by excess crude lipid mixtures, with the greatest p rotection afforded against dodecyl-maltoside. Furthermore, the signifi cantly reduced binding affinity and capacity of solubilized P-gp was p artly reversed by the addition of lipids. A combination of anion-excha nge and hydroxyapatite chromatography were used to purify P-gp with hi gh yield to greater than 90%, The purified, reconstituted P-gp display ed high ATPase activity (V-m = 2137 +/- 309; K-m = 2.9 +/- 0.9 mM) whi ch was stimulated by verapamil (EC50 = 3.8 +/- 0.6 mu M) and inhibited by orthovanadate (3.1 +/- 0.8 mu M). Pure P-gp also displayed high af finity vinblastine binding (K-d = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp act ivity reported to date, and indicates a dependence of function on main taining a lipid-protein interface. (C) 1997 Elsevier Science B.V.