BOTH P-GLYCOPROTEIN NUCLEOTIDE-BINDING SITES ARE CATALYTICALLY ACTIVE

The technique of vanadate trapping of nucleotide was used to study cat alytic sites of P-glycoprotein (Pgp) in plasma membranes from multidru g-resistant Chinese hamster ovary cells. Vanadate trapping of Mg- or C o-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37 degrees C of 1.4 x 10(-3) s(-1) (t(1/2) = 8 min) or 3.3 x 10(-4) s(-1) (t(1/2) = 35 min), respectively. UV irradiation of the inhibited Pgp yielded permanent i nactivation of ATPase activity and specific photolabeling of Pgp, Mild trypsin digestion showed that the two nucleotide sites were labeled i n equal proportion. The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucle otide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleoti de sites occurs non-selectively. A minimal scheme is presented to expl ain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway, The inhibited Pgp Mg-nucleotide vanadate com plex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two s ites may alternate in catalysis.