title: ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.
abstract: The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.
authors.affiliation: Cancer Research Laboratories, Room 328, Botterell Hall, Queen's University, Kingston, Ont., Canada.
authors.forename: Q
authors.initials: Q
authors.lastname: Mao
authors.forename: E M
authors.initials: EM
authors.lastname: Leslie
authors.forename: R G
authors.initials: RG
authors.lastname: Deeley
authors.forename: S P
authors.initials: SP
authors.lastname: Cole
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D018432:Drug Resistance, Multiple; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017997:Leukotriene C4 / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011510:Proteolipids / Q000737:chemistry; D014407:Tumor Cells, Cultured
chemical_list: D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011510:Proteolipids; C020485:proteoliposomes