Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase.
170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000032:analysis
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases