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.
Dong
M
M
Insitut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France. lgb@ibcp.fr
Penin
F
F
Baggetto
L G
LG
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