DRUG-BINDING AND NUCLEOTIDE HYDROLYZABILITY ARE ESSENTIAL REQUIREMENTS IN THE VANADATE-INDUCED INHIBITION OF THE HUMAN P-GLYCOPROTEIN ATPASE

P-glycoprotein (Pgp) mediates drug transport utilizing the energy rele ased from ATP hydrolysis. However, the mechanism by which Pgp couples these two reactions remains unclear. The present work is undertaken to describe kinetically the first step, which is the interdependence of nucleotide and drug binding to the Pgp by the use of vanadate. Preincu bation of human Pgp expressed in Sf9 insect cells with vanadate in the presence of Mg2+, ATP, and verapamil resulted in nearly complete and stable inhibition of the dug-stimulated ATPase function. In contrast, the Pgp ATPase function was nearly unaffected when Mg2+, ATP, or verap amil was omitted. Inhibition was highly specific for divalent cations that support ATP hydrolysis, for nucleotides that serve as substrates of hydrolysis, and for those drugs/compounds that interact with the dr ug-binding/transport sites of the Pgp. Kinetic analysis indicated that vanadate inhibition was MgATP concentration-dependent with an apparen t K-i value similar to the apparent K-m, suggesting that MgATP was bou nd to a similar ATP-binding site in both the ATPase inhibition and act ivation reactions. In support of this conclusion, vanadate, in the pre sence of Mg2+ and verapamil, caused selective trapping of 8-azido [alp ha-P-32] ATP and covalent labeling of ATP-binding site in the Pgp. Dif ferences were observed in the vanadate-induced inhibition of wild-type and Val(185) mutant Pgp's with different drug/compounds. These result s suggested that the affinity of the interacting drug/compound is a co nstant and influences the overall stability of the inhibited Pgp speci es. Possible implications of these observations for the coupling of AT P hydrolysis to drug transport are discussed.