ATPase activity of purified and reconstituted multidrug resistance proteinMRP1 from drug-selected H69AR cells

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which e xpress 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 immunoaff inity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 mu g of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gel s. After reconstitution of purified MRP1 into proteoliposomes, kinetic anal yses indicated that its K-m for ATP hydrolysis was 104 +/- 22 mu M with max imal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was fu rther characterized with various inhibitors and exhibited an inhibition pro file that distinguishes it from P-glycoprotein and other ATPases. The ATPas e activity of reconstituted MRP1 was stimulated by the conjugated organic a nion substrates leukotriene C-4 (LTC4) and 17 beta-estradiol 17-(beta-D-glu curonide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 mu M, 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 d escribed in which the ATPase activity of the reconstituted MRP1 retains kin etic characteristics with respect to ATP-dependence and substrate stimulati on that are very similar to those deduced from transport studies using MRP1 -enriched plasma membrane vesicles. (C) 1999 Elsevier Science B.V. All righ ts reserved.