Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein.
A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.
Urbatsch
I L
IL
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
al-Shawi
M K
MK
Senior
A E
AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity; D014227:Triazines; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D009711:Nucleotides; D014227:Triazines; C012842:Cibacron Blue F 3GA; D014700:Verapamil; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium