EFFECT OF MODULATORS ON THE ATPASE ACTIVITY AND VANADATE NUCLEOTIDE TRAPPING OF HUMAN P-GLYCOPROTEIN

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 sensit ize multidrug resistant cells to these oncolytics. To study the intera ction of modulators with Pgp, Pgp-ATPase activity was examined, using plasma membranes isolated from the multidrug-resistant cell line CEM/V LB100. A survey of modulators indicated that verapamil, trifluoperazin e, and nicardipine stimulated ATPase activity by 1.3- to 1.8-fold, whe reas 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 K-i of 2.1 mu M. When the effects of these two modulators on t he formation of the enzyme-nucleotide complex important in the catalyt ic cycle were examined, verapamil increased the amount of vanadate-tra pped 8-azido-[alpha-P-32]ATP bound to Pgp by two-fold, whereas TMBY ha d no effect. Moreover, TMBY blocked the verapamil stimulation of vanad ate-8-azido-[alpha-P-32]ATP. Together, these data indicate that verapa mil and TMBY bind to Pgp at a common site or overlapping sites, but on ly verapamil results in enhanced Pgp-ATP hydrolysis and formation of t he vanadate-nucleotide-enzyme complex. (C) 1998 Elsevier Science Inc.