Efficient purification and reconstitution of P-glycoprotein for functional and structural studies.
Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein-overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.
Insitut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France. lgb@ibcp.fr
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D011487:Protein Conformation; D051381:Rats; D013329:Structure-Activity Relationship; D014316:Tritium; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014316:Tritium; D014747:Vinblastine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases