Reconstitution of purified P-glycoprotein into liposomes.
To study the molecular function of the multidrug-resistance gene product P-glycoprotein, we purified and reconstituted it into liposomes. Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein. We found that transport activity was effective reconstituted after solubilization with cholate, glycocholate and taurocholate. Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity. The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The reconstituted P-glycoprotein specifically bound [3H]azidopine and had an ATPase activity that was slightly stimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.
Naito
M
M
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Tsuruo
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D001692:Biological Transport; D003902:Detergents; D004095:Dihydropyridines / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D011510:Proteolipids / Q000737:chemistry; D014750:Vincristine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D003902:Detergents; D004095:Dihydropyridines; D011510:Proteolipids; C020485:proteoliposomes; D014750:Vincristine; C041130:azidopine; D000251:Adenosine Triphosphatases