Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein.
P-glycoprotein, the multidrug transporter, is proposed to act as an ATP-driven drug efflux pump. We previously reported the partial purification of P-glycoprotein from multidrug-resistant cells (Doige, C. A., Yu, X., and Sharom, F. J. (1992) Biochim. Biophys. Acta 1109, 149-160). We now report the reconstitution of this preparation into phospholipid bilayers using rapid detergent removal by gel filtration chromatography. The resulting proteoliposomes displayed ATP-dependent [3H]colchicine uptake over a time period of 0-4 min. No drug uptake was observed for liposomes of lipid alone, or liposomes reconstituted with a similar extract from drug-sensitive cells. Drug uptake was osmotically sensitive, and abolished by detergent permeabilization, indicating that it represented true transport into the vesicle lumen. Steady-state levels of drug uptake increased with drug concentration, approaching saturation at approximately 150 microM colchicine, with half-maximal accumulation at 50 microM. Drug was accumulated actively against a 5.6-fold concentration gradient. Multidrug resistance spectrum drugs and chemosensitizers inhibited colchicine uptake by P-glycoprotein proteoliposomes, whereas cytosine arabinoside and methotrexate had no effect. Reconstituted liposomes showed high levels of ATPase activity, which was stimulated over 2-fold by verapamil and trifluoperazine. These results suggest that P-glycoprotein functions as an active drug transporter with constitutive ATPase activity.
Sharom
F J
FJ
Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Ontario, Canada.
Yu
X
X
Doige
C A
CA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003078:Colchicine / Q000378:metabolism*; D006224:Cricetinae; D003630:Daunorubicin / Q000494:pharmacology; D004347:Drug Interactions; D004351:Drug Resistance; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D011510:Proteolipids; C020485:proteoliposomes; C022571:adenosine 5'-O-(3-thiotriphosphate); D014747:Vinblastine; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003078:Colchicine; D003630:Daunorubicin