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

Cell morphology, protein/DNA ratio, ganglioside analysis, taurine uptake, and the activity of neurone specific enolase showed that neuronal cells mature faster when grown on polyethyleneimine coated plates compared to cells grown on polylysine coated plates. Our results also show higher protein/DNA ratio and total and neuron specific enolase activities in cells grown in serum supplemented medium, when compared to their counterparts grown in synthetic medium. Moreover, our results show that only some specific markers can be used to determine the early and late events of cell maturation, whereas other markers continuously vary with time.

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