Purification and reconstitution of functional human P-glycoprotein.
The overexpression of the P-glycoprotein, the MDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [3H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.
Ambudkar
S V
SV
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D018432:Drug Resistance, Multiple / Q000235:genetics; D006801:Humans; D007624:KB Cells; D008081:Liposomes; D011510:Proteolipids / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D014747:Vinblastine; D000251:Adenosine Triphosphatases