Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis.

Abstract

Multidrug-resistant human tumor cells overexpress the MDRI gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/Vol) ocyl 3-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCi fraction, enriched in P-glycoprotein but devoid of NaK-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P- Several previous reports suggest the association ofATPase activity with P-glycoprotein: (i) the ATP analog 8-azido-[32-P]ATP binds to P-glycoprotein (8); (ii) the immunoaffinity-purified protein exhibits a low level of ATPase activity (9); and (iii) in vitro mutagenesis of the consensus sequences of either or both ATP-binding domains of the MDR] cDNA fails to confer drug resistance in transfected cells that express the altered protein (10, 11). In addition, the ATP-dependent transport of vinblastine by membrane vesicles of multidrugresistant cells has been demonstrated (12). Thus, it has been proposed that P-glycoprotein catalyzes ATP-dependent efflux of drugs from resistant cells. These previous studies, however, do not conclusively prove that the two functions (i.e., ATP hydrolysis and drug transport) are directly mediated by P-glycoprotein. To establish that P-glycoprotein is an ATP-dependent multidrug transporter, purification and functional reconstitution into phospholipid vesicles is essential. We describe here partial purification and reconstitution of P-glycoprotein. The data suggest that P-glycoprotein exhibits a high level of substrate-stimulatable ATPase activity similar to other ion-transporting pumps. MATERIALS AND METHODSPreparation of Plasma Membrane Vesides. The multidrugresistant human carcinoma KB-V1 cells, a subclone of KB3-1, were grown in the presence of vinblastine (1 ,g/ml) to confluence (13). The membrane vesicles were prepared by nitrogen cavitation and sucrose density gradient centrifugation as described (12). The final pellet of vesicles was resuspended and stored in vesicle buffer (10 mM Tris HCl, pH 7.4/50 mM NaCl/250 mM sucrose/0.5 mM phenylmethylsulfonyl fluoride) at -700C.Solubilization of P-Glycoprotein. The solubilization of membrane proteins followed the protocols established earlier (14,15

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

“…Reaction intermediates have affinities 10 10 to 10 15 times higher than the corresponding Michaelis-Menten complexes (16), reflected in the K m . The K m (ATP) for human Pgp is 0.3-1.0 mM (9,25,33,40). Following trapping of 2.5 M 8-azido-[␣-32 P]ATP in the mutant Pgp, we found that 75 mM ATP (a 30,000-fold excess) could only displace ϳ20% of the label (data not given).…”

“…The wild-type Pgp also occludes the nucleotide, but it is [␣-32 P]ADP that is trapped and only in the presence of Vi. It is well established that wild-type Pgp has a relatively low affinity for ATP (25,32,33), but the conformational changes that accompany ATP hydroly- The reaction was stopped by adding 0.1 ml of ice-cold ATPase buffer containing 10 mM ATP and 1 M NaCl. The nucleotides and proteoliposomes were separated using a G-50 column, as described under "Experimental Procedures."…”

“…This value could be sensitive to the orientation and functional status of Pgp molecules in the proteoliposomal bilayer. We have previously demonstrated that Ͼ95% (range 95-97%) of Pgp molecules are reconstituted with an inside-out orientation (25,26). For purification and reconstitution of the wild type and E556Q/E1201Q double mutant, we used protocol that allows Ͼ90% recovery of verapamil-stimulated ATPase activity (23,25,26).…”

“…We have previously demonstrated that Ͼ95% (range 95-97%) of Pgp molecules are reconstituted with an inside-out orientation (25,26). For purification and reconstitution of the wild type and E556Q/E1201Q double mutant, we used protocol that allows Ͼ90% recovery of verapamil-stimulated ATPase activity (23,25,26). The specific activity of verapamil-stimulated ATPase activity in proteoliposomes prepared with various batches of pure wild-type protein ranged between 1100 and 1300 nmol of P i /min/mg of protein as previously described (23).…”

“…Purified Pgp was reconstituted into proteoliposomes by dialysis using a lipid/protein ratio of 6:1 as described (24,25). We have previously experimentally determined that Pgp that is inserted in proteoliposomes is Ͼ95% in inside-out orientation (NBDs facing extravesicular space (25,26)). …”