title: Effect of modulators on the ATPase activity and vanadate nucleotide trapping of human P-glycoprotein.
abstract: P-Glycoprotein (Pgp) is responsible for the energy-dependent efflux of many natural product oncolytics. Overexpression of Pgp may result in multidrug resistance (MDR). Modulators can block Pgp efflux and sensitize multidrug resistant cells to these oncolytics. To study the interaction of modulators with Pgp, Pgp-ATPase activity was examined, using plasma membranes isolated from the multidrug-resistant cell line CEM/VLB100. A survey of modulators indicated that verapamil, trifluoperazine, and nicardipine stimulated ATPase activity by 1.3- to 1.8-fold, whereas two others, trimethoxybenzoylyohimbine (TMBY) and vindoline, had no effect. Further evaluation showed that TMBY completely blocked the stimulation by verapamil of ATPase activity by competitive inhibition, with a Ki of 2.1 microM. When the effects of these two modulators on the formation of the enzyme-nucleotide complex important in the catalytic cycle were examined, verapamil increased the amount of vanadate-trapped 8-azido-[alpha-32P]ATP bound to Pgp by two-fold, whereas TMBY had no effect. Moreover, TMBY blocked the verapamil stimulation of vanadate-8-azido-[alpha-32P]ATP. Together, these data indicate that verapamil and TMBY bind to Pgp at a common site or overlapping sites, but only verapamil results in enhanced Pgp-ATP hydrolysis and formation of the vanadate-nucleotide-enzyme complex.
authors.affiliation: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0424, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Shepard
authors.forename: M A
authors.initials: MA
authors.lastname: Winter
authors.forename: S C
authors.initials: SC
authors.lastname: Hsaio
authors.forename: H L
authors.initials: HL
authors.lastname: Pearce
authors.forename: W T
authors.initials: WT
authors.lastname: Beck
authors.forename: A H
authors.initials: AH
authors.lastname: Dantzig
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000187:drug effects* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004347:Drug Interactions; D018432:Drug Resistance, Multiple; D006801:Humans; D006868:Hydrolysis; D007945:Leukemia, Lymphoid / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011487:Protein Conformation; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology; D015016:Yohimbine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D001386:Azides; D002121:Calcium Channel Blockers; D010761:Phosphorus Radioisotopes; C114675:trimethoxybenzoylyohimbine; D015016:Yohimbine; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D014747:Vinblastine; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases