ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells.
P-glycoprotein was purified from multidrug-resistant Chinese hamster ovary CHRB30 cells by a combination of anion exchange and immunoaffinity chromatography. The P-glycoprotein was about 90% pure and had a Vmax for ATP hydrolysis in detergent solution of 321 nmol/min/mg with a Km of 0.94 mM. The ATPase activity was inhibited by low concentrations of vanadate and N-ethylmaleimide, but unaffected by azide or ouabain. When the purified P-glycoprotein was reconstituted into phospholipid bilayer membranes, the ATPase activity became highly stimulated by several chemosensitizers and drugs involved with multidrug resistance. Verapamil, a potent chemosensitizer, increased the Vmax for ATP hydrolysis by 22-fold and the Km for ATP by 5.4-fold. This effect of verapamil on P-glycoprotein has not previously been observed. These results demonstrate that purified P-glycoprotein has an intrinsic ATPase activity with unique properties. This activity appears sufficient to account for the ATP-dependent reduction in intracellular drug accumulation of P-glycoprotein-expressing multidrug-resistant cells.
Shapiro
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AB
Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Canada.
Ling
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V
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001386:Azides / Q000494:pharmacology; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002999:Clone Cells; D006224:Cricetinae; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D014462:Ultrafiltration; D014638:Vanadates / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D001386:Azides; D002352:Carrier Proteins; D008081:Liposomes; D008562:Membrane Glycoproteins; D011510:Proteolipids; C020485:proteoliposomes; D014638:Vanadates; D010042:Ouabain; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide