ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

Biochim Biophys Acta. 1992 Aug 24;1109(2):149-60. doi: 10.1016/0005-2736(92)90078-z.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Adenosine Triphosphatases / metabolism*
  • Animals
  • CHO Cells
  • Ca(2+) Mg(2+)-ATPase / metabolism
  • Cation Transport Proteins
  • Cell Membrane / chemistry
  • Cholic Acids
  • Cricetinae
  • Detergents
  • Drug Resistance
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Kinetics
  • Membrane Glycoproteins / isolation & purification
  • Membrane Glycoproteins / metabolism*
  • Sulfhydryl Reagents

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Cation Transport Proteins
  • Cholic Acids
  • Detergents
  • Membrane Glycoproteins
  • Sulfhydryl Reagents
  • Adenosine Triphosphatases
  • Ca(2+) Mg(2+)-ATPase
  • potassium transporting ATPase
  • 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate