PURIFICATION AND RECONSTITUTION OF FUNCTIONAL HUMAN P-GLYCOPROTEIN

The overexpression of the P-glycoprotein, the MDR1 gene product, has b een linked to the development of resistance to multiple cytotoxic natu ral product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (AB C) superfamily of transporters, is believed to function as an ATP-depe ndent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purificat ion and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug res istant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Pr oteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [H-3]vinblas tine accumulation. Both the ATPase and vinblastine transport activitie s of the reconstituted P-glycoprotein were inhibited by vanadate. In a ddition, the vinblastine transport was inhibited by verapamil and daun orubicin. These studies provide strong evidence that the human P-glyco protein functions as an ATP-dependent drug transporter. The developmen t of the reconstitution system and the availability of recombinant pro tein in large amounts due to recent advances in overexpression of P-gl ycoprotein in a heterologous expression system should facilitate a bet ter understanding of the function of this novel protein.