P-GLYCOPROTEIN IS STABLY INHIBITED BY VANADATE-INDUCED TRAPPING OF NUCLEOTIDE AT A SINGLE CATALYTIC SITE

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimul ated ATPase activity, The catalytic sites are known to be of low affin ity and specificity for nucleotides, From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a m ultidrug-resistant Chinese hamster ovary cell line, which are highly e nriched in Pgp, we show that vanadate-induced trapping of nucleotide a t a single catalytic site produces stably inhibited Pgp, with t(1/2) f or reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C. Reactivation of ATPase correlated with release of tra pped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 mu M, respectively, thus the apparent af finity for nucleotide is greatly increased by vanadate-trapping. The t rapped nucleotide species was ADP, Divalent cation was required, with magnesium, manganese, and cobalt all effective; cobalt yielded a very stable inhibited species, t(1/2) at 37 degrees C = 18 h, No photocleav age of Pgp was observed after vanadate trapping with MgATP, nor was UV -induced photolabeling of Pgp by trapped adenine nucleotide observed, Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused p ermanent inactivation and specific labeling of Pgp, Vanadate-induced i nhibition was also shown with pure, reconstituted Pgp, with similar ch aracteristics to those in plasma membranes, Vanadate trapping overcome s technical difficulties posed by lack of high affinity nucleotide-bin ding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site /Pgp is sufficient to give full inhibition of ATPase activity shows th at the two pre dieted nucleotide sites can not function independently as catalytic sites.