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• W2335438718 abstract "Prostaglandin (PG) endoperoxide H synthase (PGHS)-2, also known as cyclooxygenase (COX)-2, can convert arachidonic acid (AA) to PGH2 in the committed step of PG synthesis. PGHS-2 functions as a conformational heterodimer composed of an allosteric (Eallo) and a catalytic (Ecat) monomer. Here we investigated the interplay between human (hu)PGHS-2 and an alternative COX substrate, the endocannabinoid, 2-arachidonoylglycerol (2-AG), as well as a stable analog, 2-O-arachidonylglycerol ether (2-AG ether). We also compared the inhibition of huPGHS-2-mediated oxygenation of AA, 2-AG, and 2-AG ether by the well-known COX inhibitor, ibuprofen. When tested with huPGHS-2, 2-AG and 2-AG ether exhibit very similar kinetic parameters, responses to stimulation by FAs that are not COX substrates, and modes of inhibition by ibuprofen. The 2-AG ether binds Ecat more tightly than Eallo and, thus, can be used as a stable Ecat-specific substrate to examine certain Eallo-dependent responses. Ibuprofen binding to Eallo of huPGHS-2 completely blocks 2-AG or 2-AG ether oxygenation; however, inhibition by ibuprofen of huPGHS-2-mediated oxygenation of AA engages a combination of both allosteric and competitive mechanisms. Prostaglandin (PG) endoperoxide H synthase (PGHS)-2, also known as cyclooxygenase (COX)-2, can convert arachidonic acid (AA) to PGH2 in the committed step of PG synthesis. PGHS-2 functions as a conformational heterodimer composed of an allosteric (Eallo) and a catalytic (Ecat) monomer. Here we investigated the interplay between human (hu)PGHS-2 and an alternative COX substrate, the endocannabinoid, 2-arachidonoylglycerol (2-AG), as well as a stable analog, 2-O-arachidonylglycerol ether (2-AG ether). We also compared the inhibition of huPGHS-2-mediated oxygenation of AA, 2-AG, and 2-AG ether by the well-known COX inhibitor, ibuprofen. When tested with huPGHS-2, 2-AG and 2-AG ether exhibit very similar kinetic parameters, responses to stimulation by FAs that are not COX substrates, and modes of inhibition by ibuprofen. The 2-AG ether binds Ecat more tightly than Eallo and, thus, can be used as a stable Ecat-specific substrate to examine certain Eallo-dependent responses. Ibuprofen binding to Eallo of huPGHS-2 completely blocks 2-AG or 2-AG ether oxygenation; however, inhibition by ibuprofen of huPGHS-2-mediated oxygenation of AA engages a combination of both allosteric and competitive mechanisms. Prostaglandin (PG) endoperoxide synthase (PGHS)-1 and PGHS-2 catalyze the formation of PGH2 from arachidonic acid (AA) in the committed step of PG biosynthesis (1Schneider C. Pratt D.A. Porter N.A. Brash A.R. Control of oxygenation in lipoxygenase and cyclooxygenase catalysis.Chem. Biol. 2007; 14: 473-488Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 2Tsai A.L. Kulmacz R.J. Prostaglandin H synthase: resolved and unresolved mechanistic issues.Arch. Biochem. Biophys. 2010; 493: 103-124Crossref PubMed Scopus (78) Google Scholar, 3Rouzer C.A. Marnett L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: Cross-talk between the eicosanoid and endocannabinoid signaling pathways.Chem. Rev. 2011; 111: 5899-5921Crossref PubMed Scopus (231) Google Scholar, 4Smith W.L. Urade Y. Jakobsson P.J. Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis.Chem. Rev. 2011; 111: 5821-5865Crossref PubMed Scopus (350) Google Scholar). PGHS-1 and PGHS-2 are considered to be the constitutive and inducible PGHS isoforms, respectively. PGHSs are often called cyclooxygenases (COXs). Both enzymes exhibit a bis-oxygenase or COX activity involved in the formation of the PG endoperoxide G2 and a peroxidase activity that reduces PG endoperoxide G2 to PGH2. COX activities of PGHSs are inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) that include COX-2-specific inhibitors, sometimes referred to as coxibs (5Grosser T. Yu Y. Fitzgerald G.A. Emotion recollected in tranquility: Lessons learned from the COX-2 saga.Annu. Rev. Med. 2010; 61: 17-33Crossref PubMed Scopus (188) Google Scholar). PGHSs are sequence homodimers composed of 72 kDa subunits. Despite the structural symmetry observed in crystal structures, both PGHS isoforms behave in solution as conformational heterodimers. One monomer (Eallo) acts as a regulatory allosteric monomer, and the other monomer (Ecat) binds heme and functions as the catalytic monomer (6Kulmacz R.J. Lands W.E. Prostaglandin H synthase. Stoichiometry of heme cofactor.J. Biol. Chem. 1984; 259: 6358-6363Abstract Full Text PDF PubMed Google Scholar, 7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Zou H. Yuan C. Dong L. Sidhu R.S. Hong Y.H. Kuklev D.V. Smith W.L. Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids.J. Lipid Res. 2012; 53: 1336-1347Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The COX activity of Ecat of human (hu)PGHS-2 is allosterically modulated by many common FAs, including saturated and monounsaturated FAs that are not COX substrates [e.g., palmitic acid (PA)] (9Yuan C. Sidhu R.S. Kuklev D.V. Kado Y. Wada M. Song I. Smith W.L. Cyclooxygenase allosterism, fatty acid-mediated cross-talk between monomers of cyclooxygenase homodimers.J. Biol. Chem. 2009; 284: 10046-10055Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Dong L. Zou H. Yuan C. Hong Y.H. Kuklev D.V. Smith W.L. Different fatty acids compete with arachidonic acid for binding to the allosteric or catalytic subunits of cyclooxygenases to regulate prostanoid synthesis.J. Biol. Chem. 2016; 291: 4069-4078Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and by some nonspecific NSAIDs, such as flurbiprofen and naproxen (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), that bind preferentially to Eallo of huPGHS-2. Marnett and coworker (3Rouzer C.A. Marnett L.J. Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: Cross-talk between the eicosanoid and endocannabinoid signaling pathways.Chem. Rev. 2011; 111: 5899-5921Crossref PubMed Scopus (231) Google Scholar) were the first to demonstrate that the endocannabinoid, 2-arachidonoylglycerol (2-AG), is an alternative substrate that is converted to 2-PGH2-glycerol by PGHS-2. This latter intermediate can, in turn, be converted to several different 2-prostanoyl-glycerol derivatives. A recent report has indicated that 2-AG binds with higher affinity to Ecat than Eallo of murine (mu)PGHS-2 (11Kudalkar S.N. Nikas S.P. Kingsley P.J. Xu S. Galligan J.J. Rouzer C.A. Banerjee S. Ji L. Eno M.R. Makriyannis A. et al.13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.J. Biol. Chem. 2015; 290: 7897-7909Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); in contrast, AA binds with much higher affinity to Eallo than Ecat (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Interestingly, a nonsubstrate (ns)FA, 13-methyl AA, increases the rate of oxygenation of 2-AG by muPGHS-2 by increasing the Vmax, but not the Km, toward 2-AG (11Kudalkar S.N. Nikas S.P. Kingsley P.J. Xu S. Galligan J.J. Rouzer C.A. Banerjee S. Ji L. Eno M.R. Makriyannis A. et al.13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.J. Biol. Chem. 2015; 290: 7897-7909Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The 2-AG is unstable and readily rearranges to 1-AG and hydrolyzes to AA and glycerol (12Vecchio A.J. Malkowski M.G. The structural basis of endocannabinoid oxygenation by cyclooxygenase-2.J. Biol. Chem. 2011; 286: 20736-20745Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This instability presents experimental difficulties in studying the interactions of 2-AG with PGHSs. The 2-O-arachidonylglycerol ether (2-AG ether) is a stable analog of 2-AG. In the first part of the present study, we report the characterization of 2-AG ether as a substrate of huPGHS-2. We find that the 2-AG ether behaves very much like 2-AG with huPGHS-2. Because of its stability, 2-AG ether can serve as a surrogate for 2-AG in enzyme studies. In related work described here, we examined the ability of the commonly used NSAID, (S)-(+)-ibuprofen (IBP), to interact with Eallo and Ecat to inhibit huPGHS-2. We confirm results of earlier studies that IBP is an allosteric inhibitor of 2-AG oxygenation (13Prusakiewicz J.J. Duggan K.C. Rouzer C.A. Marnett L.J. Differential sensitivity and mechanism of inhibition of COX-2 oxygenation of arachidonic acid and 2-arachidonoylglycerol by ibuprofen and mefenamic acid.Biochemistry. 2009; 48: 7353-7355Crossref PubMed Scopus (111) Google Scholar) and extend this finding to 2-AG ether. We also observed that IBP binding to Eallo of huPGHS-2 allosterically inhibits AA oxygenation, but does so only incompletely. Complete inhibition involves the binding of IBP to both Ecat and Eallo of huPGHS-2. Complete protease inhibitor was from Roche Applied Science. Ni-NTA Superflow resin and Ni-nitrilotriacetic acid were from Qiagen. PA (16:0), oleic acid (18:1ω9), stearic acid (18:0), 11-eicosaenoic acid (20:1ω9), FLAG peptide, and FLAG affinity resin were from Sigma-Aldrich. AA, 2-AG, and 2-AG ether were from Cayman Chemical (Ann Arbor, MI). Hemin was from Frontier Scientific, Logan, UT. IBP was from Tocris Bioscience. The [1-14C]AA (1.85 GBq/mmol) was from American Radiolabeled Chemicals. Decyl maltoside, n-octyl β-D-glucopyranoside, and C10E6, used in protein purification, were purchased from Anatrace (Maumee, OH). BCA protein reagent was from Pierce. Hexane, isopropyl alcohol, and acetic acid were HPLC grade from Thermo Fisher Scientific, Inc. Anti-PGHS-2 antibodies directed against the 18-amino acid insert unique to PGHS-2 were as described (14Mbonye U.R. Yuan C. Harris C.E. Sidhu R.S. Song I. Arakawa T. Smith W.L. Two distinct pathways for cyclooxygenase-2 protein degradation.J. Biol. Chem. 2008; 283: 8611-8623Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Anti-FLAG antibodies were from LifeTein, South Plainfield, NJ. Horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) were from Bio-Rad. Procedures for the expression and purification of recombinant native huPGHS-2 and mutant huPGHS-2 heterodimer variants from insect cells were as described previously (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 15Dong L. Sharma N.P. Jurban B.J. Smith W.L. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.J. Biol. Chem. 2013; 288: 28641-28655Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The purity of the recombinant huPGHS-2 was determined by SDS-PAGE and Western blot analysis (9Yuan C. Sidhu R.S. Kuklev D.V. Kado Y. Wada M. Song I. Smith W.L. Cyclooxygenase allosterism, fatty acid-mediated cross-talk between monomers of cyclooxygenase homodimers.J. Biol. Chem. 2009; 284: 10046-10055Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In most cases, COX activity was determined using measurements of O2 consumption with an O2 electrode (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). One unit of COX activity is defined as 1 μmol of O2 consumed per minute at 37°C in the standard assay mixture. The average specific activity of purified huPGHS-2 with 100 μM AA was 40 units per milligram protein. This specific activity is similar to that reported in earlier studies from our laboratory using different lots of purified huPGHS-2 (i.e., within ±5%) (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Dong L. Zou H. Yuan C. Hong Y.H. Kuklev D.V. Smith W.L. Different fatty acids compete with arachidonic acid for binding to the allosteric or catalytic subunits of cyclooxygenases to regulate prostanoid synthesis.J. Biol. Chem. 2016; 291: 4069-4078Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 15Dong L. Sharma N.P. Jurban B.J. Smith W.L. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.J. Biol. Chem. 2013; 288: 28641-28655Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, COX assays were performed at high enzyme/[1-14C]AA ratios in order to quantify [1-14C]AA binding to Eallo of huPGHS-2. Unlabeled ligands (e.g., PA, 2-AG, and 2-AG ether) were tested for their abilities to displace unreacted [1-14C]AA remaining bound to Eallo. Reaction mixtures (100 μl final volume) containing 1 μM [1-14C]AA, 0.10–2.0 μM huPGHS-2, 5 μM hematin, and 1 mM phenol in 0.1 M Tris-HCl (pH 8.0) were incubated at 37°C for 1–8 min, and the products were separated and quantified by radio-reverse-phase (RP)-HPLC, as detailed previously (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Dong L. Zou H. Yuan C. Hong Y.H. Kuklev D.V. Smith W.L. Different fatty acids compete with arachidonic acid for binding to the allosteric or catalytic subunits of cyclooxygenases to regulate prostanoid synthesis.J. Biol. Chem. 2016; 291: 4069-4078Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The principle underlying this method is described in (10Dong L. Zou H. Yuan C. Hong Y.H. Kuklev D.V. Smith W.L. Different fatty acids compete with arachidonic acid for binding to the allosteric or catalytic subunits of cyclooxygenases to regulate prostanoid synthesis.J. Biol. Chem. 2016; 291: 4069-4078Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Agents that displace [1-14C]AA from Eallo of huPGHS-2 cause the disappearance of [1-14C]AA, which, following its displacement from Eallo, is converted by Ecat to an oxygenated product. A reaction was performed using a standard COX assay mixture that included 20 μg of 2-AG ether as the substrate and sufficient huPGHS-2 to consume approximately 80% of the substrate during a 2 min incubation. Immediately afterwards, a volume of 100 mM SnCl2 in methanol was added to the sample such that the final SnCl2 concentration was 1 mM. The sample was vortexed and incubated at room temperature, then extracted with ethyl acetate, dried under N2, and kept in a sealed tube until analyzed. Control reactions were performed with assay buffer alone, with assay buffer that included heme and phenol, with huPGHS-2 in buffer alone, with huPGHS-2 in buffer that included heme and phenol, with 2-AG ether in buffer alone, with 2-AG ether in buffer containing heme and phenol, and with 2-AG ether plus huPGHS-2 in buffer alone. Acetylation of the extracted reaction products was performed with a 1:1 mixture of acetic anhydride and pyridine for 20 min at 100°C. The reaction mixture was dried under nitrogen, dissolved in methanol, and then diluted with water to a final methanol concentration of less than 15%. Solid phase extraction and reversed phase chromatography were performed essentially as previously described (16Suram S. Gangelhoff T.A. Taylor P.R. Rosas M. Brown G.D. Bonventre J.V. Akira S. Uematsu S. Williams D.L. Murphy R.C. et al.Pathways regulating cytosolic phospholipase A2 activation and eicosanoid production in macrophages by Candida albicans.J. Biol. Chem. 2010; 285: 30676-30685Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The LC effluent was directly interfaced into the electrospray ionization source of a triple quadrupole mass spectrometer (Sciex API 5500; PE-Sciex, Thornhill, ON, Canada) where mass spectrometric analyses were performed in the positive ion mode (m/z 250–800) or as MS2 product ions using nitrogen as collision gas at a collision voltage of 25 V. TMS ether derivatives were prepared from HPLC-purified metabolites and analyzed by electron ionization by capillary GC/MS, as previously described (17Wheelan P. Zirrolli J.A. Murphy R.C. Analysis of hydroxy fatty acids as pentafluorobenzyl ester, trimethylsilyl ether derivatives by electron ionization gas chromatography/mass spectrometry.J. Am. Soc. Mass Spectrom. 1995; 6: 40-51Crossref PubMed Scopus (33) Google Scholar), using a Finnigan DSQ GC-MS system (Thermo Finnigan, Thousand Oaks, CA) with a ZB-l column (30 m, 0.25 mm inner diameter 0.25 mm film thickness; Phenomenex). The gas chromatograph was programmed from 150 to 270°C at 30°C/min, 270 to 315°C at 10°C/min, and finally held at 315°C for 6 min. The injector was maintained at 230°C, the transfer line was maintained at 290°C, and the ion source at 200°C. Student's t-tests were performed in Microsoft Excel. If the experiments had the same numbers of repetitions, probabilities were calculated with a Student's paired t-test, with a two-tailed distribution. If the experiments had different numbers of repetitions, probabilities were calculated with a Student's unequal variance t-test, with a two-tailed distribution. When 2-AG ether (75 nmoles) was incubated with an excess of huPGHS-2 (240 nmoles) at 37°C in a standard COX assay mixture for 2 min, 112 nmoles of O2 were consumed. We assumed that the excess enzyme led to complete conversion of the substrate and that either one or two O2 molecules were incorporated into the 2-AG ether substrate. These assumptions were corroborated by the mass spectrometric results described below. Accordingly we calculated that 1.49 mol O2 were incorporated per mole of 2-AG ether, indicating that about 70% of the products were bis-oxygenated (i.e., 2-PGH2-glycerol ether) and 30% were mono-oxygenated [i.e., 2-(hydroxy-eicosatetraenoyl)-glycerol ether(s)]. Mass spectrometric studies using LC-MS and LC-MS/MS were performed to directly characterize the structural products obtained following the action of huPGHS-2 on 2-AG ether (Fig. 1). In order to impart favorable mass spectrometric characteristics, the products extracted from the reaction mixture were first reduced with SnCl2 and then derivatized by acetylation. RP-HPLC was able to separate two less lipophilic products from the starting 2-AG ether that were not present in control incubations with huPGHS-2 when no phenol or heme was present (Fig. 1A). These are labeled peak A and the more lipophilic peak B. Because these were presumed to be acetylated ether diglycerides, positive ion electrospray ionization as the ammonium adduct ion (NH4+) was employed. The observed [M+NH4]+ adduct ions were m/z 642 (peak A; Fig. 1B) and m/z 524 (peak B; Fig. 1C), while the signal for unreacted starting material was found to produce m/z 466 (peak C; Fig. 1D). Collisional activation of the starting material (peak C) yielded a major product ion at m/z 273 corresponding to cleavage of the arachidonyl chain at the ether bond (Fig. 1D). The spectrum of least lipophilic reaction product (acetyl derivative, ammonium ion adduct, m/z 642) (Fig. 1B) was consistent with the addition of 3-hydroxyl groups (analyzed as acetyl esters) and reduction of one double bond. This was consistent with a PGF2-like structure generated from a PGH2 endoperoxide intermediate formed by the COX activity of huPGHS-2 acting on the arachidonyl ether chain of 2-AG ether. The collisional activation of m/z 642 [M+NH4]+ yielded a very prominent product ion at m/z 445 (Fig. 1B), consistent with three neutral losses of acetic acid (60 Da each) and ammonia (NH3). The abundant ion at m/z 269 could then be understood as cleavage of the arachidonyl carbon-ether bond with positive charge retention on the 20-carbon alkyl leaving group via the mechanism suggested in supplementary Fig. 1. The corresponding ether-bond fragment ion was observed in the MS/MS spectra of peaks B and C, but each was 2 Da and 4 Da, respectively, higher in measured m/z because the alkyl carbocation generated by collisional activation (supplementary Fig. 1) has five and six rings or double bonds, respectively, compared with the arachidonyl carbocation generated from CID of 2-AG ether, which has only four rings or double bonds. Considering the presence of three hydroxyl groups and loss of a double bond, the data are consistent with the presence of a novel ether lipid having a PGF2 structural element, 2-O-(PGF2)-glycerol. The scale employed in these experiments was not sufficient for NMR analysis of this metabolite. The increase in the observed adduct molecular ion mass for peak B was 58 Da (Fig. 1C), consistent with one additional hydroxyl group along the arachidonyl carbon chain that had been converted to an acetate ester. Collisional activation of peak B yielded ions at m/z 447 [M+H-CH3COOH]+, m/z 387 (m/z 477-CH3COOH), and the most abundant product ion at m/z 271 (Fig. 1C). This latter ion corresponded to the most abundant product ion observed in the MS/MS spectrum of the starting material (that being m/z 273), but 2 Da lower, corresponding to cleavage of the ether bond in 2-AG ether and one additional double bond introduced by the presence of one acetoxy group that had been lost as acetic acid following collisional activation. The position of this hydroxyl group on the 20-carbon chain was determined by electron ionization MS as the TMS derivative (Fig. 2), and the ion at m/z 225 [CH3(CH2)4CH=CH-CH=CH-CH=O+-TMS] was consistent with an introduction of a hydroxyl group at C-11 of the arachidonoyl carbon chain and migration of the Δ11,12 double bond to Δ12,13. Thus, this metabolite was determined to be 2-O-(11-hydroxy-eicosatetraenyl)-glycerol. Overall, our mass spectrometric studies and measurements of O2 consumption indicate that 2-AG ether is converted by huPGHS-2 to two major products: 2-O-(11-hydroxy-eicosatetraenyl)-glycerol ether (25%) and 2-O-(PGH2)-glycerol ether (75%). These two products are the glycerol ether homologs of the 2-(11-hydroxy-eicosatetraenoyl)-glycerol and 2-PGH2-glycerol products that are formed in similar proportions upon incubation of 2-AG with muPGHS-2 (18Kozak K.R. Rowlinson S.W. Marnett L.J. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2.J. Biol. Chem. 2000; 275: 33744-33749Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). We determined the following Vmax and Km values for recombinant huPGHS-2 when comparing 2-AG ether, 2-AG, and AA as substrates: Vmax of 27 units/mg and Km = 4.6 μM for 2-AG ether (Fig. 3); Vmax of 30 units/mg and Km ∼7 μM for 2-AG (15Dong L. Sharma N.P. Jurban B.J. Smith W.L. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.J. Biol. Chem. 2013; 288: 28641-28655Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar); and Vmax of 43 units/mg and Km ∼10 μM for AA (15Dong L. Sharma N.P. Jurban B.J. Smith W.L. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer.J. Biol. Chem. 2013; 288: 28641-28655Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Thus, the catalytic efficiencies (Vmax/Km) with huPGHS-2 are similar when each substrate is tested individually. To estimate the relative affinities of Eallo versus Ecat of huPGHS-2 for 2-AG ether, we first examined the effects of the nsFA, PA, on the oxygenation of 2-AG ether (Fig. 3). Increasing the ratio of 2-AG ether to PA did not change the ratio of the rates with 2-AG ether alone versus 2-AG ether plus PA. This indicates that 2-AG at any of the concentrations used in the assays fails to compete with 25 μM PA for Eallo. PA binds to Eallo of huPGHS-2 with a Kd ∼7.5 μM, but binds only very weakly to Ecat (Kd >50 μM) (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). PA increased the Vmax, but did not change the Km, of huPGHS-2 for 2-AG ether. Because it increases the rate of 2-AG ether oxygenation, PA is not competing for Ecat, but rather must act via Eallo; moreover, 2-AG ether did not compete with PA for Eallo at the concentrations tested. This indicates that 2-AG ether binds significantly less tightly to Eallo than PA, and thus, less tightly to Eallo than Ecat. The Kd for PA binding to Eallo is ∼7.5 μM, which is below its critical micelle concentration [∼25 μM (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 9Yuan C. Sidhu R.S. Kuklev D.V. Kado Y. Wada M. Song I. Smith W.L. Cyclooxygenase allosterism, fatty acid-mediated cross-talk between monomers of cyclooxygenase homodimers.J. Biol. Chem. 2009; 284: 10046-10055Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar)], where PA effectively binds only Eallo. The 2-AG ether did not displace PA from Eallo when the ratio of 2-AG ether/PA was 1.0, indicating that the Kd for 2-AG ether binding is significantly greater than 7.5 μM and, thus, greater than the Kd of 2-AG ether for Ecat. The Kd for 2-AG ether binding to Ecat is the Km of huPGHS-2 for 2-AG [∼5 μM (Fig. 3)]. This situation is unlike what is observed with AA (7Dong L. Vecchio A.J. Sharma N.P. Jurban B.J. Malkowski M.G. Smith W.L. Human cyclooxygenase-2 Is a sequence homodimer that functions as a conformational heterodimer.J. Biol. Chem. 2011; 286: 19035-19046Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) or EPA (10Dong L. Zou H. Yuan C. Hong Y.H. Kuklev D.V. Smith W.L. Different fatty acids compete with arachidonic acid for binding to the allosteric or catalytic subunits of cyclooxygenases to regulate prostanoid synthesis.J. Biol. Chem. 2016; 291: 4069-4078Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) that bind Eallo 30 times more tightly than Ecat of huPGHS-2. The results in Table 1 provide further evidence that 2-AG ether binds more tightly to Ecat than Eallo of huPGHS-2. In contrast to what is observed with PA or with 5 μM AA itself, 2-AG ether, at an initial concentration of 5 μM, more than 25 times that of unreacted [1-14C]AA (0.18 μM), failed to displace [1-14C]AA from Eallo of huPGHS-2. Only at concentrations higher than 7.5 μM does 2-AG ether cause any significant displacement of [1-14C]AA from Eallo. These findings provide additional evidence that 2-AG ether fails to bind efficiently to Eallo of PGHS-2.TABLE 1RP-HPLC analysis of the displacement of [1-14C]AA from Eallo of huPGHS-2 by unlabeled AA, PA, or 2-AG etherFA AddedaFA was added 4 min after initiating the reaction.Unreacted [1-14C]AA RemainingbThe amount of unreacted [1-14C]AA remaining after 8 min (percent of starting radioactivity, average ± SD from two reactions).,cValue shown is average ± SD for replicate determinations from one experiment representative of three separate experiments with different preparations of enzyme.Control (no FA added)18 ± 0.085 μM AA6.6 ± 1.4dSignificantly different from the control value (no FA or other agent added at 4 min) in Student's t-test (P < 0.05).5 μM PA6.2 ± 1.2dSignificantly different from the control value (no FA or other agent added at 4 min) in Student's t-test (P < 0.05).2.5 μM 2-AG ether17 ± 0.225 μM 2-AG ether16 ± 0.337.5 μM 2-AG ether14 ± 0.0915 μM 2-AG ether11 ± 0.09dSignificantly different from the control value (no FA or other agent added at 4 min) in Student's t-test (P < 0.05).25 μM 2-AG ether7.9 ± 1.8dSignificantly different from the control value (no FA or other agent added at 4 min) in Student's t-test (P < 0.05).The [1-14C]AA (1 μM) was incubated with huPGHS-2 (1 μM) at 37°C for 4 min, then unlabeled AA, PA, or 2-AG ether was added, and the incubation continued for another 4 min. Reactions were stopped by adding ethyl acetate/acetic acid (20:1), and an aliquot of the organic phase was subjected to radio-RP-HPLC to separate the radioactive products and unreacted AA, as described in the Experimental Procedures. The results are shown as the percentage of total 14C label that remained in the RP-HPLC fraction co-eluting with unreacted AA; the data represent averages of replicate samples ± SD. The results are shown for a single experiment that was performed a total of three times with similar results using different enzyme preparations in each case and concentrations of 2-AG ether ranging from 2.5–50 μM.a FA was added 4 min after initiating the reaction.b The amount of unreacted [1-14C]AA remaining after 8 min (percent of starting radioactivity, average ± SD from two reactions).c Value show" @default.
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• W2335438718 title "Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2" @default.
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