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Reconstitution of purified P-glycoprotein into liposomes

Abstract

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 effectively 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 slightlystimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.

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Correspondence to Mikihiko Naito.

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Work dedicated to Dr. Haruo Sugano on the occasion of his 70th birthday

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Naito, M., Tsuruo, T. Reconstitution of purified P-glycoprotein into liposomes. J Cancer Res Clin Oncol 121, 582–586 (1995). https://doi.org/10.1007/BF01197774

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Key words

  • P-glycoprotein
  • Reconstitution
  • Liposome
  • Transport
  • Vincristine