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• W1991504728 abstract "Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [14C]ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [14C]ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [14C]ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a “closed conformation” involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event. Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [14C]ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [14C]ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [14C]ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a “closed conformation” involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event. P-glycoprotein (Pgp) 1The abbreviations used are: Pgp, P-glycoprotein; NBD, nucleotide-binding domain; DTT, dithiothreitol; Vi, vanadate. is a prominent member of the ABC-transporter family of membrane proteins that uses the energy of ATP hydrolysis to exclude hydrophobic compounds from cells. In human, it was first recognized as a major potential obstacle to successful chemotherapy of cancer because of its expression in cancer cells and was later realized to function physiologically in such strategic locations as the blood-brain barrier, intestine, placenta, and elsewhere as an important agent in protection from environmental and dietary toxins. More recent appreciation of its role in impeding the action of anti-AIDS therapy and its potentially general role in reducing the efficacy of many hydrophobic drugs, new and old, have made it a target of intense investigation. Recent reviews of the biochemistry and pharmacology of Pgp may be found in Refs. 1Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3567) Google Scholar, 2Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar, 3Stein W.D. Physiol. Rev. 1997; 77: 545-590Crossref PubMed Scopus (241) Google Scholar, 4Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1929) Google Scholar, 5Loo T.W. Clarke D.M. Biochim. Biophys. Acta. 1999; 1461: 315-325Crossref PubMed Scopus (88) Google Scholar, 6Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1355) Google Scholar. Our research has focused on the catalytic mechanism by which ATP is hydrolyzed and the energy transduced into drug transport. In 1995, we presented schemes for the hydrolysis of ATP at a single catalytic site (7Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), also for the interaction of the two ATP-binding sites in catalysis and transduction of the energy of hydrolysis to the drug binding site(s) situated in the membrane bilayer (8Senior A.E. Al-Shawi M.K. Urbatsch I.L. FEBS Lett. 1995; 377: 285-289Crossref PubMed Scopus (429) Google Scholar). Both ATP-binding sites were shown to have catalytic capability. Release of product Pi was thought to occur before the release of ADP, which was rate-limiting. Interaction of the two ATP-binding sites was concluded to be an integral and a necessary facet of catalysis with hydrolysis of ATP occurring sequentially and alternately at each site. Formation and collapse of the transition state of catalysis were postulated as critical events in driving the changes in drug-binding site affinity and orientation (inward versus outward facing) necessary for binding and extrusion of drug with a stoichiometry of one ATP hydrolyzed per change in drug-binding site affinity and orientation. Later studies of Pgp and also of ABC transporter homologs such as bacterial LmrA and the maltose transporter have supported and extended the earlier schemes (9Sauna Z.E. Smith M.M. Muller M. Kerr K.M. Ambudkar S.V. J. Bioenerg. Biomembr. 2001; 33: 481-491Crossref PubMed Scopus (165) Google Scholar, 10Van Veen H.W. Margolles A. Muller M. Higgins C.F. Konings W.N. EMBO J. 2000; 19: 2503-2514Crossref PubMed Scopus (241) Google Scholar, 11Davidson A.L. J. Bacteriol. 2002; 184: 1225-1233Crossref PubMed Scopus (95) Google Scholar). New ideas incorporated into more recent schemes include the concept of reciprocating pairs of drug-binding sites (10Van Veen H.W. Margolles A. Muller M. Higgins C.F. Konings W.N. EMBO J. 2000; 19: 2503-2514Crossref PubMed Scopus (241) Google Scholar) and of the requirement for a second ATP hydrolysis event to “reset” the drug-binding site for drug extrusion (9Sauna Z.E. Smith M.M. Muller M. Kerr K.M. Ambudkar S.V. J. Bioenerg. Biomembr. 2001; 33: 481-491Crossref PubMed Scopus (165) Google Scholar). Another concept, recently introduced, envisages that the ATP-binding event provides the primary driving force for transport (12Rosenberg M.F. Velarde G. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (264) Google Scholar). Electron microscopy studies provide evidence for conformational changes in the membrane domains observed upon nucleotide binding (12Rosenberg M.F. Velarde G. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (264) Google Scholar); however, biochemical evidence has been provided showing that the drug-binding site also changes conformation in response to ATP hydrolysis per se (13Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Loo T.W. Clarke D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3511-3516Crossref PubMed Scopus (73) Google Scholar). Recent work (15Qu Q. Russell P.L. Sharom F.J. Biochemistry. 2003; 42: 1170-1177Crossref PubMed Scopus (64) Google Scholar) demonstrated that, at concentrations of MgATP and MgADP present in the cytoplasm of mammalian cells, both nucleotide-binding domains (NBD) in “resting” Pgp would be expected to bind MgATP (15Qu Q. Russell P.L. Sharom F.J. Biochemistry. 2003; 42: 1170-1177Crossref PubMed Scopus (64) Google Scholar). Our original scheme for ATP-driven drug transport (8Senior A.E. Al-Shawi M.K. Urbatsch I.L. FEBS Lett. 1995; 377: 285-289Crossref PubMed Scopus (429) Google Scholar) envisaged that upon attainment of a conformation in which two ATP were bound in the NBDs concurrent with occupation of the drug-binding site, an intimate interaction of the two NBDs would be engendered as a prerequisite for ATP hydrolysis. In later work we provided evidence for the interaction of the two NBDs in Pgp in catalysis and suggested that formation of a single transition state complex involved liganding from catalytic side-chains of both NBDs (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) in what we termed a closed conformation of the catalytic sites. Support for this idea has since come from cross-linking studies showing that catalytic sites of the two NBDs in Pgp can be very close (17Urbatsch I.L. Gimi K. Wilke-Mounts S. Lerner-Marmarosh N. Rousseau M.-E. Gros P. Senior A.E. J. Biol. Chem. 2000; 276: 26980-26987Abstract Full Text Full Text PDF Scopus (71) Google Scholar, 18Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 19435-19438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Loo T.W Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 20Loo T.W Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) from low resolution electron microscopy studies of Pgp (12Rosenberg M.F. Velarde G. Ford R.C. Martin C. Berridge G. Kerr I.D. Callaghan R. Schmidlin A. Wooding C. Linton K.J. Higgins C.F. EMBO J. 2001; 20: 5615-5625Crossref PubMed Scopus (264) Google Scholar, 21Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 22Lee J.Y. Urbatsch I.L. Senior A.E. Wilkens S. J. Biol. Chem. 2002; 277: 40125-40131Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and from fluorescence resonance energy transfer studies of the two Pgp catalytic sites (23Qu Q. Sharom F.J. Biochemistry. 2001; 40: 1413-1422Crossref PubMed Scopus (85) Google Scholar). In other ABC transporters the NBDs also interact with each other, as shown by photocleavage studies of the maltose transporter (24Fetsch E.E. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9685-9690Crossref PubMed Scopus (118) Google Scholar) and most recently from x-ray structural characterization of the ABC transporters MJ0796 (25Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) and BtuCD (26Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (934) Google Scholar). This x-ray work showed in detail how the two NBD subunits in ABC transporters can interdigitate to form a “dimer interface” at which the nucleotides are bound. 2The ABC transporter MsbA did not show such close association of the two NBDs (27Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (586) Google Scholar). However, use of OsCl3 to improve resolution may well have dictated orientation of the NBDs seen in this structure (Fig. 2 of Ref. 27Chang G. Roth C.B. Science. 2001; 293: 1793-1800Crossref PubMed Scopus (586) Google Scholar). Thus, “dimerization” of the NBDs appears now to be a well supported feature. In Fig. 1 we show a proposed mechanism of Pgp incorporating the concept of NBD dimerization. A diagram of this kind encourages the thinking that the catalytically active site is actually a composite entity consisting of the two NBDs and two bound ATP, only one of which will by hydrolyzed during one catalytic event. Movement of the NBDs between open and closed forms (Fig. 1) will occur in synchrony with drug transport. However, it is clear that many features of the Pgp catalytic mechanism remain to be elucidated, such as the nature of interdomain communication between the drug-binding site(s) and the NBDs, the structural and functional character of the ATP binding pockets and the transition state, and the time sequence of the dimerization phenomenon in relation to the progression of catalysis through intermediate species. Earlier, we introduced the approach of vanadate (Vi)-induced trapping of nucleotide as a tool to study the catalytic sites in Pgp (7Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Incubation of MgATP with Vi and Pgp was shown to cause strong inhibition of Pgp ATPase brought about by tenacious trapping of MgADP in stoichiometry of just 1 mol ADP/mol Pgp. As was discussed (7Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), biochemical evidence from other ATPase enzymes, notably myosin and dynein, had led to the idea that the pentacovalent Vi in the trapped MgADP·Vi complex mimicked the transition state structure around the γ-phosphate of MgATP in an associative trigonal bipyramidal transition state structure (37Mildvan A.S. Proteins. 1997; 29: 401-416Crossref PubMed Scopus (255) Google Scholar). Smith and Rayment (38Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar) reported an x-ray structure of myosin bound to MgADP-vanadate, which confirmed this geometry. Biochemical and mutagenesis studies of Pgp have supported such an assignment in Pgp (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 39Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar, 40Urbatsch I.L. Julien M. Carrier I. Rousseau M.E. Cayrol R. Gros P. Biochemistry. 2000; 39: 14138-14149Crossref PubMed Scopus (79) Google Scholar, 41Szabo K. Welker E. Bakos E. Muller M. Roninson I. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 10132-10138Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 42Szakacs G. Ozvegy C. Bakos E. Sarkadi B. Varadi A. Biochem. Biophys. Res. Commun. 2000; 276: 1314-1319Crossref PubMed Scopus (28) Google Scholar). The rate of onset of Vi-induced inhibition and trapping of nucleotide in the presence of MgATP was shown to be accelerated by drugs (40Urbatsch I.L. Julien M. Carrier I. Rousseau M.E. Cayrol R. Gros P. Biochemistry. 2000; 39: 14138-14149Crossref PubMed Scopus (79) Google Scholar, 41Szabo K. Welker E. Bakos E. Muller M. Roninson I. Varadi A. Sarkadi B. J. Biol. Chem. 1998; 273: 10132-10138Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Since acceleration of the rate of ATP hydrolysis is a well known property of drugs (43Sarkadi B. Price E.M. Boucher R.C. Germann U. Scarborough G.A. J. Biol. Chem. 1992; 267: 4854-4858Abstract Full Text PDF PubMed Google Scholar, 44Al-Shawi M.K. Senior A.E. J. Biol. Chem. 1993; 268: 4197-4206Abstract Full Text PDF PubMed Google Scholar) this provided one explanation of the accelerated inhibition, however, it is not yet clear whether the presence of drugs specifically stabilizes the transition state or not. This question also relates to the broader question of the nature of interdomain communication between drug-binding sites and catalytic sites in Pgp. We showed earlier that incubation of Pgp with Vi and either MgADP or the analog Mg-8-azido-ADP led to strong inhibition of ATPase and tenacious trapping of MgADP·Vi (or Mg-8-azido-ADP) in catalytic sites (7Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), this occurring in the absence of any catalytic turnover. Evidence indicated that whether Pgp was initially incubated with MgATP or MgADP together with Vi, the resultant inhibited Pgp·MgADP·Vi showed similar properties, suggesting that the same complex was formed. Inhibition and trapping of the natural MgADP represents therefore an alternative approach to study the effect of drugs on the transition state, which has not so far been studied in detail. In this paper we have utilized pure, soluble, wild-type mouse Mdr3 Pgp to characterize the formation of the MgADP·Vi complex from MgADP and Vi and have additionally studied mutations that impair catalysis directly or that impair interdomain communication between drug sites and catalytic sites. Use of pure protein with the natural MgADP ligand facilitated accurate calculation of stoichiometries and avoids possible limitations inherent from use of photoactivated analogs. Wild-type and mutant mouse Mdr3 Pgp were expressed in Pichia pastoris and purified to homogeneity as described previously (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 45Lerner-Marmarosh N. Gimi K. Urbatsch I.U. Gros P. Senior A.E. J. Biol. Chem. 1999; 274: 34711-34718Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Expression and purification of human Mdr1 Pgp were as described previously (17Urbatsch I.L. Gimi K. Wilke-Mounts S. Lerner-Marmarosh N. Rousseau M.-E. Gros P. Senior A.E. J. Biol. Chem. 2000; 276: 26980-26987Abstract Full Text Full Text PDF Scopus (71) Google Scholar). Activation of Pgp with DTT and lipid was essentially as described previously (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 45Lerner-Marmarosh N. Gimi K. Urbatsch I.U. Gros P. Senior A.E. J. Biol. Chem. 1999; 274: 34711-34718Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Pgp was incubated with DTT (8 mm) and Escherichia coli lipid (Avanti, acetone/ether-precipitated) at a final ratio of 2/1 lipid/protein (w/w) for 20 min at room temperature followed by sonication for 30 s at 4 °C in a bath sonicator. Linked Enzyme Assay—This assay was used routinely unless otherwise stated in the text. This assay utilizing pyruvate kinase and phosphoenolpyruvate to regenerate ATP from ADP and lactate dehydrogenase to couple ATP hydrolysis to NADH oxidation by pyruvate was performed at 37 °C as described previously (36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). MgATP (10 mm) and verapamil (150 μm) were included. Charcoal Adsorption Assay—This assay was used for Ki(ADP) measurement and was performed essentially as described previously (46Tombline G. Fishel R. J. Biol. Chem. 2002; 277: 14417-14425Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Activated Pgp (0.5 μg in 25-μl volume) was added with mixing to 25 μl of 40 mm Tris-Cl, pH 7.5, 0.1 mm EGTA, 1 mCi of [γ-32P]ATP, varied concentration of MgATP, and 2 mm excess MgCl2. Incubation was at 37 °C for appropriate times. Reaction was stopped by addition of 0.75-ml ice-cold acid-washed charcoal (catalog C-5510, Sigma), which was suspended at 10% (w/v) in 10 mm EDTA immediately before use. After ≥3-h incubation in ice, samples were centrifuged (14,000 × g for 30 min at 4 °C) and the supernatant containing [32P]Pi was counted by the Cerenkov method. Background samples lacking protein but with all of the other components were subtracted at each MgATP concentration. For determination of Ki(ADP), MgADP was included in the assays at 250, 500, and 1000 μm and MgATP concentration was varied from 25 μm to 3 mm. All of the reactions were linear with time, and <10% of the added MgATP was hydrolyzed. 1-ml centrifuge columns of Sephadex G-50 in disposable syringes were equilibrated with 50 mm Tris-Cl, pH 7.5, and 0.001% N-dodecyl-β-d-maltoside at 4 °C. The pre-spin was at 1000 rpm in a clinical centrifuge for 2 min at 4 °C, after which Pgp (10–20 μg in 100 μl) was loaded onto the column and eluted by a second spin. Recovery of Pgp was 70–95% as judged by ATPase and/or protein assays. 10–20 μg of activated Pgp were preincubated with 200 μm sodium orthovanadate, 1 mm MgCl2, and 50 mm Tris-Cl, pH 7.5, in 100-μl total volume at 37 °C for varied time as described in the text. NaADP or NaATP was present at required concentration as described in the text. Incubations were started by the addition of Pgp and halted by transfer to ice, then passage through centrifuge columns to remove unbound ligand. Stock orthovanadate solutions (100 mm) were prepared from Na3VO4 (Fisher Scientific) at pH 10 and boiled for 2 min before each use. Other variations of the conditions are given in the text. Where MgADP and Vi were used, 100 μm [8-14C]ADP replaced ADP in the preincubation. Where MgATP and Vi were used, [α-32P]ATP was used. Control experiments showed that in absence of Pgp, elution of radioactive nucleotide was negligible. The amount of trapped radioactive ADP was proportional to the amount of Pgp applied in the range 5–20 μg (35–140 pmol). Stoichiometry of trapped nucleotide was calculated using a molecular mass of 142 kDa for Pgp (47Urbatsch I.L. Wilke-Mounts S. Gimi K. Senior A.E. Arch. Biochem. Biophys. 2001; 388: 171-177Crossref PubMed Scopus (29) Google Scholar). Pgp was inactivated as above in the presence of Vi plus ADP or Vi plus ATP and then passed through centrifuge columns to remove unbound ligands. Eluates were then incubated at 37 °C for various times and then assayed for ATPase by linked enzyme assay. Accurate Pgp concentration was calculated by reference to a standard pure wild-type preparation, which had been subjected to amino acid analysis. A range of amounts of wild-type or mutant Pgp were run on SDS-gels alongside this reference standard, stained with Coomassie Blue, and scanned. For rapid analysis, the Bradford assay was used with bovine serum albumin as standard normalized to the reference Pgp standard. Verapamil and other drugs were added as solution in Me2SO, keeping the final solvent concentration <2% (v/v), and control “no drug” samples contained the same amount of Me2SO. SDS gel electrophoresis was performed as described previously (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). NaADP was purchased from Sigma (Catalog A-2754) or Roche Applied Science (236 675). [8-14C]ADP and [α-32P]ATP were from PerkinElmer Life Sciences. E. coli lipids (acetone/ether-precipitated) were from Avanti Polar lipids. Inhibition of Wild-type Mouse Mdr3 Pgp after Preincubation with Vanadate and MgADP or Vanadate and MgATP—We previously reported that ATP (or 8-azido-ATP) in conjunction with Mg2+ and Vi could produce long-lived inhibition of Pgp caused by tenacious trapping of the Vi-nucleoside diphosphate complex at a stoichiometry of 1 mol/mol Pgp (7Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Similar long-lived inhibition was seen when ADP (or 8-azido-ADP) was substituted for ATP, although the stoichiometry of trapped nucleotide was not established in that case. For this earlier work we used primarily Chinese hamster ovary cell plasma membrane preparations enriched in Pgp. As discussed in Introduction, there is good support for the concept that the Pgp·MgADP·Vi complex mimics the natural transition state of ATP hydrolysis. Subsequently, we and others have shown that purified detergent-soluble mouse (Mdr3) Pgp forms this same complex when incubated with MgATP or Mg-8-azido-ATP (16Urbatsch I.L. Gimi K. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2000; 275: 25031-25038Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 39Urbatsch I.L. Beaudet L. Carrier I. Gros P. Biochemistry. 1998; 37: 4592-4602Crossref PubMed Scopus (126) Google Scholar). Here we used purified mouse Mdr3 Pgp to investigate the formation of the Pgp·MgADP·Vi complex in the absence of ATP hydrolysis using MgADP as the loading nucleotide. Fig. 2A shows the inhibition of Pgp ATPase activity as a function of time in the presence of verapamil. Rate of onset of inhibition was slow with a half-time of 37 min, and approximately 90% inhibition was reached after 120 min. Control experiments showed that inhibition was only seen if the ligands ADP, Mg2+, and Vi were present. An omission of any one prevented inhibition. From the first order rate constant (3.4 × 10–4 s–1), an apparent second order rate constant of 3.1 m–1 s–1 in respect to MgADP was calculated. This is low in comparison to simple protein-ligand association reactions and suggests that a slow protein isomerization step has to occur after initial MgADP binding to allow the Pgp·MgADP·Vi complex to form. 3As noted later, Ki(MgADP) under similar conditions = 263 μm. Making the assumption that Ki ≈ Kd and that turnover rate of MgAT-Pase (10Van Veen H.W. Margolles A. Muller M. Higgins C.F. Konings W.N. EMBO J. 2000; 19: 2503-2514Crossref PubMed Scopus (241) Google Scholar s–1) is limited by MgADP dissociation (see Introduction), MgADP association rate ≈ 4 × 104m–1 s–1. Qu et al. (15Qu Q. Russell P.L. Sharom F.J. Biochemistry. 2003; 42: 1170-1177Crossref PubMed Scopus (64) Google Scholar) reported Kd(MgADP) as 0.41 mm in hamster Pgp, so that the assumption seems valid. It may also be noted that kcat/Km(MgATP), often considered to approximate MgATP association rate of an ATPase, similarly calculates to ≈ 4 × 104m–1 s–1 for Pgp. In previous work using hamster Pgp in plasma membrane preparations, an equivalent half-time of 4.8 min was found for inhibition (36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). A possible explanation for the difference could be the different lipid environment of pure Pgp activated with E. coli lipid versus Pgp in mammalian plasma membranes or insufficient lipids or the presence of detergent. We addressed these questions by varying the ratio of lipid to protein during activation of Pgp from 1/1 up to 50/1 by supplementing the E. coli lipids with phosphatidylcholine, phosphatidylserine, and cholesterol, which better mimics mammalian plasma membrane lipid composition (29Figler R.A. Omote H. Nakamoto R.K. Al-Shawi M.K. Arch. Biochem. Biophys. 2000; 376: 34-46Crossref PubMed Scopus (87) Google Scholar, 48Ambudkar S.V. Lelong I.H. Zhang J. Cardarelli C. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. (U. S. A.). 1992; 89: 8472-8476Crossref PubMed Scopus (382) Google Scholar), and by reconstituting Pgp into proteoliposomes by extensive dialysis to remove detergent. None of these treatments increased the rate of onset of inhibition. Thus, the rate is probably an intrinsic property of the species of Pgp under study. In contrast, the inhibition of Mdr3 Pgp was much more rapid with Vi and MgATP (Fig. 2B). The half-time was 27 s, which is almost two orders of magnitude faster than with MgADP. For comparison, the half-time for inhibition of hamster Pgp in plasma membranes by Vi plus MgATP was 10 s (36Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), which is similar to that seen here with Mdr3 Pgp. Hydrolysis of MgATP by Mdr3 Pgp reached 10 s–1 at saturating (10 mM) MgATP, and at the concentration of MgATP (100 μm) and other conditions used in the inhibition experiments, the measured rate of hydrolysis was 0.38 s–1. A comparison of the rate of hydrolysis with the first order rate constant for inhibition (0.026 s–1) demonstrates that not every turnover event leads to the formation of a trapped and inhibited intermediate, at least under the conditions used here. The calculated apparent second order rate constant for inhibition of 198 m–1 s–1 in respect to MgATP is consistent with this conclusion. Verapamil and a Range of Other Drug Substrates Accelerate the Rate of Inhibition by Vi plus MgADP—Fig. 3A shows the effect of verapamil on the inhibition of Pgp ATPase activity by Vi plus ADP. Verapamil has been shown earlier to be among the most effective stimulators of Mdr3 Pgp ATPase (49Beaudet L. Urbatsch I.L. Gros P. Methods Enzymol. 1998; 292: 397-413Crossref PubMed Scopus (35) Google Scholar). Little inhibition was seen in the absence of added drug or at verapamil concentrations <1 μm. Half-maximal inhibition was seen at 12 μm, and maximal inhibition (70% here because a 60-min incubation time was used, see Fig. 2A) was reached at around 150 μm verapamil, which is also the concentration at which maximal ATPase is elicited. Kinetic experiments utilizing varied concentrations of verapamil indicated that formation of the inhibited state was faster and more complete with increasing concentrations of verapamil (not shown). The data show that, in the absence of hydrolysis, binding of drug to the drug-binding site greatly enhances the formation of the transition state-like" @default.
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• W1991504728 date "2003-06-01" @default.
• W1991504728 modified "2023-09-26" @default.
• W1991504728 title "P-glycoprotein Catalytic Mechanism" @default.
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• W1991504728 doi "https://doi.org/10.1074/jbc.m301957200" @default.
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