Characterization of the ATPase Activity of Purified Chinese Hamster P-glycoprotein

Abstract

A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

“…4), PgP had been purified using conditions optimized for its own recovery, and the preparation showed a basal ATPase activity and response to substrate consistent with what one expects for the fully functioning molecule (Table I). As a consequence, we conclude that nucleotide binding by CFTR is comparable to that shown by Pgp [36,38]. We also note that CFTR catalyzes a net hydrolysis of ATP (Fig.…”

“…Indeed, when the data were expressed on a molar basis (with correction for protein content and molecular weight), we concluded that CFTR and Pgp bind nucleotide (Az-ATP) with approximately equal efficiency (109% vs. 100%, respectively). Since the biochemical characteristics of Pgp purified from Sf9 cells (e.g., V max , K M , and substrate stimulation) match values cited in the literature for the fully functioning molecule [33,[35][36][37][38] (Table I), we conclude that nucleotide processing by purified CFTR exhibits characteristics similar to that of Pgp.In mammalian systems, R domain phosphorylation must occur before CFTR chloride channel function is observed. However, in Sf9 cells, the CFTR-mediated anion conductance appears without addition of exogenous stimuli that might activate intracellular protein kinases and promote R domain phosphorylation [13].…”

“…We also evaluated recovery of functional CFTR in a quantitative manner by comparing nucleotide trapping of CFTR in parallel with that of its well-characterized relative, Pgp, which had been expressed in Sf9 cells and purified and reconstituted using conditions optimized for its own recovery [33,[35][36][37]38] (Table I, Fig. 4c-e).…”

“…4ab). This behavior, noted by others in studies of crude membranes [14,15], has not been reported for other ABC transporters [14,15,33] and may be a distinctive feature of CFTR.We also evaluated recovery of functional CFTR in a quantitative manner by comparing nucleotide trapping of CFTR in parallel with that of its well-characterized relative, Pgp, which had been expressed in Sf9 cells and purified and reconstituted using conditions optimized for its own recovery [33,[35][36][37]38] (Table I, Fig. 4c-e).…”

Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

“…Both halves of P-gp can hydrolyze ATP (14,15), but drug-stimulated ATPase activity (14) and conferring of drug resistance (16) requires interaction between the two halves of P-gp. Both halves of P-gp are required for activity because drug binding requires interaction between the NH 2 and COOHterminal TM domains (16) whereas both ATP binding sites are needed for ATPase activity (17)(18)(19)(20)(21) and the nucleotide-binding sites appear to function in an alternating mechanism (22).…”