ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.
In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Canada.
C A
CA
Doige
X
X
Yu
F J
FJ
Sharom
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids; D006224:Cricetinae; D003902:Detergents; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013439:Sulfhydryl Reagents
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027682:Cation Transport Proteins; D002793:Cholic Acids; D003902:Detergents; D008562:Membrane Glycoproteins; D013439:Sulfhydryl Reagents; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C022761:potassium transporting ATPase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate