Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein.
Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. al-Shawi, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously. Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.
Urbatsch
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IL
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Senior
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AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000187:drug effects*; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003630:Daunorubicin / Q000494:pharmacology; D004095:Dihydropyridines / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D008055:Lipids / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry; D011510:Proteolipids / Q000378:metabolism; D012756:Sheep; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D004095:Dihydropyridines; D008055:Lipids; D011510:Proteolipids; C020485:proteoliposomes; D014747:Vinblastine; C041130:azidopine; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003078:Colchicine; D003630:Daunorubicin