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• W2022077377 abstract "The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor α agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor α agonists. The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor α agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor α agonists. arachidonylglycerol cyclooxygenase anandamide lipoxygenase hydroxyeicosatetraenoic acid hydroperoxyeicosatetraenoic acid glyceryl ester mass spectrometry liquid chromatography reversed phase high performance liquid chromatography peroxisome proliferator-activated receptor arachidonic acid normal human epidermal keratinocyte Hepes-buffered saline solution In 1995, 2-arachidonylglycerol (2-AG)1 was isolated from rat brain and canine gut and shown to bind both the central and peripheral cannabinoid receptors (1Mechoulam R. Ben-Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2374) Google Scholar, 2Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Crossref PubMed Scopus (1848) Google Scholar). Subsequently, 2-AG was shown to be present in vivo at levels several orders of magnitude higher than the other known endocannabinoid, AEA (2Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Crossref PubMed Scopus (1848) Google Scholar, 3Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Crossref PubMed Scopus (1260) Google Scholar, 4Bisogno T. Berrendero F. Ambrosino G. Cebeira M. Ramos J.A. Fernandez-Ruiz J.J. Di Marzo V. Biochem. Biophys. Res. Commun. 1999; 256: 377-380Crossref PubMed Scopus (275) Google Scholar). Accumulating evidence supports assertions that 2-AG serves as a physiologically relevant cannabinoid receptor ligand, occupying a central role within the endogenous cannabinoid system (5Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 6Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Therefore, the identification and characterization of enzymes capable of metabolizing this lipid mediator should aid in the elucidation of mechanisms by which cannabinoid tone is modulated in vivo. We are particularly interested in the role of fatty acid oxygenases, such as cyclooxygenases (COXs) and lipoxygenases (LOXs), in 2-AG metabolism and have previously shown that 2-AG is an excellent substrate for COX-2 and leukocyte-type 12-LOX (7Kozak K.R. Rowlinson S.W. Marnett L.J. J. Biol. Chem. 2000; 275: 33744-33749Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 8Moody J.S. Kozak K.R., Ji, C Marnett L.J. Biochemistry. 2001; 40: 861-866Crossref PubMed Scopus (74) Google Scholar). LOXs are a diverse family of nonheme ferroproteins that catalyze the hydroperoxidation of polyunsaturated fatty acids both regio- and stereospecifically (9Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar, 10Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (238) Google Scholar, 11Gaffney B.J. Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 431-459Crossref PubMed Scopus (79) Google Scholar, 12Gardner H.W. Biochim. Biophys. Acta. 1991; 1084: 221-239Crossref PubMed Scopus (514) Google Scholar, 13Kuhn H. Schewe T. Rapoport S.M. Adv. Enzymol. Relat. Areas Mol. Biol. 1986; 58: 273-311PubMed Google Scholar, 14Yamamoto S. Biochim. Biophys. Acta. 1992; 1128: 117-131Crossref PubMed Scopus (569) Google Scholar). Six LOXs have been identified in humans: platelet-type 12-LOX, 12(R)-LOX, 15-LOX-1, 15-LOX-2, e-LOX-3, and 5-LOX (9Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar, 15Krieg P. Marks F. Furstenberger G. Genomics. 2001; 73: 323-330Crossref PubMed Scopus (61) Google Scholar). The ability of leukocyte 12-LOX, but not platelet 12-LOX, to oxidize 2-AG and the ability of some lipoxygenases to oxidize the endocannabinoid AEA prompted us to evaluate additional possible lipoxygenase metabolic pathways for 2-AG (8Moody J.S. Kozak K.R., Ji, C Marnett L.J. Biochemistry. 2001; 40: 861-866Crossref PubMed Scopus (74) Google Scholar, 16Ueda N. Yamamoto K. Kurahashi Y. Yamamoto S. Ogawa M. Matsuki N. Kudo I. Shinkai H. Shirakawa E. Tokunaga T. Adv. Prostaglandin Thromboxane Leukotriene Res. 1995; 23: 163-165PubMed Google Scholar, 17Ueda N. Yamamoto K. Yamamoto S. Tokunaga T. Shirakawa E. Shinkai H. Ogawa M. Sato T. Kudo I. Inoue K. et al.Biochim. Biophys. Acta. 1995; 1254: 127-134Crossref PubMed Scopus (161) Google Scholar, 18Edgemond W.S. Hillard C.J. Falck J.R. Kearn C.S. Campbell W.B. Mol. Pharmacol. 1998; 54: 180-188Crossref PubMed Scopus (115) Google Scholar, 19Hampson A.J. Hill W.A. Zan-Phillips M. Makriyannis A. Leung E. Eglen R.M. Bornheim L.M. Biochim. Biophys. Acta. 1995; 1259: 173-179Crossref PubMed Scopus (117) Google Scholar, 20van Zadelhoff G. Veldink G.A. Vliegenthart J.F. Biochem. Biophys. Res. Commun. 1998; 248: 33-38Crossref PubMed Scopus (35) Google Scholar). In the present study, we investigated the ability of two plant and four animal LOXs to catalyze the hydroperoxidation of 2-AG. 5-LOX catalyzes the hydroperoxidation of arachidonic acid providing 5-hydroperoxyeicosatetraenoic acid (HpETE), the precursor to the leukotrienes. The possibility that 5-LOX might oxygenate 2-AG to generate 5-HpETE glyceryl ester (HpETE-G) and, subsequently, leukotriene glyceryl esters in a manner similar to the ability of cyclooxygenase-2 to generate prostaglandin glycerol esters was investigated using two 5-LOX enzymes. The endocannabinoid oxygenase activities of four 15-LOX enzymes were also rigorously characterized. Lipoxygenase metabolism of 2-AG in an eukaryotic cellular environment was examined in two distinct cell systems. The results suggest that 15-LOX enzymes, but not 5-LOX enzymes, may play a role in endogenous cannabinoid signaling and should be included in the growing family of oxygenases capable of 2-AG metabolism. Various oxygenated derivatives of arachidonic and linoleic acids have been found to be ligands for peroxisome proliferator-activated receptors (PPARs) (21Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. Witztum J.L. Funk C.D. Conrad D. Glass C.K. Nature. 1999; 400: 378-382Crossref PubMed Scopus (781) Google Scholar, 22Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. J. Biol. Chem. 1995; 270: 23975-23983Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). Of particular interest, 15-HETE is reported to be a ligand for the PPARγ receptor (21Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. Witztum J.L. Funk C.D. Conrad D. Glass C.K. Nature. 1999; 400: 378-382Crossref PubMed Scopus (781) Google Scholar, 23Shappell S.B. Gupta R.A. Manning S. Whitehead R. Boeglin W.E. Schneider C. Case T. Price J. Jack G.S. Wheeler T.M. Matusik R.J. Brash A.R. Dubois R.N. Cancer Res. 2001; 61: 497-503PubMed Google Scholar). Characterization of the ability of 15-HETE-G to transactivate various PPAR receptors revealed that it is a specific agonist for the PPARα receptor. This is the first reported biological activity of a glyceryl eicosanoid and suggests that fatty acid oxygenase metabolism of 2-AG might represent a pathway for the generation of ligands for nuclear receptors. Arachidonic acid and arachidonyl ethyl ester were purchased from Nu-Chek Prep (Elysian, MN). Arachidonylglycerols (1- and 2-), HETEs (15(R)-, 15(S)-, and (±)-15-HETE), soybean 15-LOX (P1), potato 5-LOX, human 5-LOX, and 15-LOX (rabbit reticulocyte) polyclonal antiserum were purchased from Cayman Chemical (Ann Arbor, MI). Soybean 15-LOX and potato 5-LOX were obtained at ≥98% purity. Human, recombinant 5-LOX was obtained as a 16,000 × g supernatant from baculovirus-infectedSf21 cells overexpressing the enzyme. Human 15-LOX-2 polyclonal antiserum was generated and characterized as previously described (24Shappell S.B. Boeglin W.E. Olson S.J. Kasper S. Brash A.R. Am. J. Pathol. 1999; 155: 235-245Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Rabbit reticulocyte 15-LOX was obtained from Calbiochemas a partially purified preparation from rabbit reticulocytes generated essentially as previously described (25Schewe T. Wiesner R. Rapoport S.M. Methods Enzymol. 1981; 71: 430-441Crossref PubMed Scopus (62) Google Scholar).l-α-Phosphatidylcholine (egg) was purchased from Avanti Polar Lipids. All other chemicals and solvents were purchased from Aldrich unless otherwise noted. Restriction enzymes were obtained from New England Biolabs (Beverly, MA). The vectors pcDNA3 and pCR2.1 were purchased from Invitrogen and pET3a was obtained from Stratagene (La Jolla, CA). The Bac-to-Bac baculovirus expression system, including the pFastBac HT vector, was purchased from Invitrogen. The cDNA for wild-type human 15-LOX-1 in a pcDNA3 vector was a generous gift of Professor C. D. Funk (University of Pennsylvania, Philadelphia, PA). The cDNA for wild-type human 15-LOX-2 was subcloned into the pcDNA3 vector as previously described (26Brash A.R. Boeglin W.E. Chang M.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6148-6152Crossref PubMed Scopus (360) Google Scholar). The baculovirus vector containing hexahistidine-tagged human 15-LOX-2 was prepared by subcloning human 15-LOX-2 into pFastBac HT usingXbaI and BamHI. The pET3a vector containing hexahistidine-tagged human 15-LOX-2 was prepared by PCR amplification of 15-LOX-2 in pcDNA3 using an upstream primer encoding six histidines. The PCR product was cloned into pCR2.1 and amplified. The 15-LOX-2 fragment was then subcloned into the pET3a vector usingBamHI and EcoRI. All vectors were sequenced to ensure no mutations had been incorporated. COS-7 cells were obtained from American Type Culture Collection (Rockville, MD). Undifferentiated human keratinocytes were a generous gift from Professor D. S. Keeney (Veterans Administration Hospital, Nashville, TN). LipofectAMINE was purchased from Invitrogen. Immobilon-P transfer membranes were obtained from Millipore (Bedford, MA). Enhanced chemiluminescence reagent was purchased from Amersham Biosciences. Eicosa-5,8,11,14-tetraenoic acid 2-hydroxyethyl ester and eicosa-5,8,11,14-tetraenoic acid 2-methoxyethyl ester were prepared as described (8Moody J.S. Kozak K.R., Ji, C Marnett L.J. Biochemistry. 2001; 40: 861-866Crossref PubMed Scopus (74) Google Scholar). 11- and 15-HETE-G were generated by incubating purified murine COX-2 with 2-AG, and 12-HETE-G was obtained by incubating purified leukocyte 12-lipoxygenase with 2-AG as described (8Moody J.S. Kozak K.R., Ji, C Marnett L.J. Biochemistry. 2001; 40: 861-866Crossref PubMed Scopus (74) Google Scholar, 27Kozak K.R. Prusakiewicz J.J. Rowlinson S.W. Schneider C. Marnett L.J. J. Biol. Chem. 2001; 276: 30072-30077Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Large scale 15-HETE-G synthesis was effected enzymatically using purified soybean 15-LOX. To 300 ml of 100 mmNa2B4O7 containing 10 mm sodium deoxycholate (pH 9.0) was added ∼15 mg of purified soybean 15-LOX. The solution was stirred to homogeneity at room temperature, followed by the addition of 2-AG (10 mg, 26 μmol). A reaction mixture aliquot was followed by UV spectroscopy (240 nm) until no further increase in absorbance was detected (≈5 min). The reaction was quenched with 300 ml of EtOAc containing 25 mg of triphenylphosphine to reduce 15-HpETE-G to 15-HETE-G. Organics were removed and the aqueous layer was re-extracted with 300 ml of EtOAc. The combined organics were washed with saturated NaHCO3 and H2O, dried (MgSO4), filtered, and concentratedin vacuo. The resultant residue was resuspended in EtOAc, filtered over glass wool, and purified by silica gel chromatography (EtOAc:hexanes, 1:3 then 1:1; fractions containing the desired product were concentrated and rechromatographed using CHCl3 then CHCl3:MeOH, 98:2) to provide the desired ester as a colorless oil (6.1 mg, 58%). RF = 0.44 (EtOAc:hexanes, 7:3); UV (MeCN) λmax 196, 238 nm;1H NMR (CDCl3) δ 6.50–6.57 (dd, 1H,J = 11.1, 15.1 Hz, CH), 5.98–6.03 (t, 1H,J = 10.9 Hz, CH), 5.68–5.74 (dd, 1H, J= 6.6, 15.1 Hz, CH), 5.33–5.45 (m, 5H, 5× CH), 4.10–4.26 (m, 2H, CH2), 3.89–3.95 (m, 1H, CH), 3.81–3.83 (t, 1H,J = 5.1 Hz, CH), 3.66–3.75 (m, 1H, CH), 3.56–3.62 (m, 1H, CH), 2.95–3.02 (m, 2H, CH2), 2.78–2.83 (m, 2H, CH2), 2.31–2.42 (m, 2H, CH2), 2.10–2.16 (m, 2H, CH2), 1.68–1.76 (m, 2H, CH2), 1.51–1.59 (m, 2H, CH2), 1.15–1.41 (m, 6H, 3CH2), 0.85–0.91 (t, 3H, J = 6.7 Hz, CH3); ESI-MSm/z calculated for C23H38O4 (M + Na+), 417.3; found, 417.3. Hexahistidine-tagged human 15-LOX-2 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions and in Escherichia coli from a pET3a vector as previously described (28Tijet N. Waspi U. Gaskin D.J. Hunziker P. Muller B.L. Vulfson E.N. Slusarenko A. Brash A.R. Whitehead I.M. Lipids. 2000; 35: 709-720Crossref PubMed Scopus (69) Google Scholar). The lipoxygenase product was then purified on nickel-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) according to the manufacturer's instructions. Fractions containing 15-LOX-2 were pooled and dialyzed against phosphate-buffered saline containing 5% glycerol to remove imidazole. E. coli-expressed human 15-LOX-2 was further purified by anion exchange chromatography. Lipoxygenase activity, except in the case of human 5-LOX, was detected by monitoring the absorbance of the conjugated diene product at 236 nm as previously described (8Moody J.S. Kozak K.R., Ji, C Marnett L.J. Biochemistry. 2001; 40: 861-866Crossref PubMed Scopus (74) Google Scholar). Briefly, UV assays were monitored using a Hewlett-Packard 8452A diode array spectrophotometer equipped with a water-jacketed cuvette. The enzyme reactions included reaction buffer (50 mm Tris-Cl, 0.03% Tween 20, pH 7.4), arachidonic acid or arachidonyl ester, and enzyme. The reaction temperature was 30 °C, and the final reaction volume was 1 ml. Potato 5-LOX reactions were conducted at 30 °C and included arachidonic acid or 2-AG (10 μm) and enzyme (200 units) in 1 ml of buffer (50 mm Tris-Cl, pH 7.4). The commercially obtained, human 5-LOX preparation displayed considerable background absorbance at 236 nm, preventing spectrophotometric quantitation of enzyme activity. Consequently, human 5-LOX activity was assessed by measuring oxygen consumption according to the manufacturer's instructions. Briefly, oxygen consumption was measured with a Gilson model 5/6 oxygraph (Gilson Medical Electronics, Middleton, WI) equipped with a Clark electrode and a thermostated cuvette (37 °C). Enzyme aliquots (10 units) were added to 50 mm Tris-Cl, 2 mm CaCl2, and 1 mm ATP, pH 7.4, containing either 0.03% Tween 20 or 0.015% Tween 20 and 100 μg/ml phosphatidylcholine in a final volume of 1 ml. Oxygen uptake was initiated by the addition of 100 μm arachidonic acid or 2-AG. Enzyme kinetics were assessed using the computer program Enzyme Kinetics 1.5 (Trinity Software, Campton, NH). Kinetic values were determined using nonlinear regression analysis. Velocity data were obtained by taking the slope of the reaction curve at the point of maximal reaction velocity. Because of the characteristic lag phase of lipoxygenases in some reactions, this rate was not necessarily the initial rate. HpETE-G regiochemistry was established by mass spectrometry (MS). Incubations of enzyme and 2-AG in 25 mm Tris, 0.015% Tween 20, pH 7.4 (37 °C, 10 min), were extracted with EtOAc and dried under argon. The residue was redissolved in 1:1 MeCN:H2O and infused into the mass spectrometer. Regiochemistry was established by diagnostic, collision-induced hydroperoxide cleavage. HpETE-Gs were reduced with triphenylphosphine to the corresponding HETE esters. Saponification with 1 n NaOH followed by RP-HPLC (Supelcosil LC-18, 250 × 4.6 mm, 5 μm, 80:20:0.01 MeOH:H2O:HOAc, 1.4 ml/min) provided purified HETEs for chiral analysis and confirmed regiochemical assignment established by MS. Following methylation with diazomethane, HETE methyl esters were analyzed by chiral-phase HPLC (Chiralpak AD, 250 × 4.6 mm, 1.5 ml/min, hexanes:EtOH, 100:1) (29Schneider C. Boeglin W.E. Brash A.R. Anal. Biochem. 2000; 287: 186-189Crossref PubMed Scopus (47) Google Scholar). Enantiomerically pure HETE methyl ester standards were well resolved on the chiral column. LC effluents were routinely monitored by UV at 235 nm. COS-7 cells were maintained at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. Human 15-LOX cDNAs in pcDNA3 or vector without insert were transfected into COS-7 cells using LipofectAMINE according to the manufacturer's instructions. Following transfection, medium was replaced with HBSS and cells were treated with 2-AG (20 μm) or Me2SO vehicle for 30 min (15-LOX-1) or 45 min (15-LOX-2) at 37 °C. After treatment, HBSS was removed and extracted twice with an equal volume of 2:1 CHCl3:MeOH. The organic extract was dried under argon, and the residue was analyzed by LC/MS. Cells were harvested for immunoblotting by scraping, washed twice with phosphate-buffered saline, and lysed in 50 mm Tris-HCl, pH 7.5, containing 150 mm NaCl, 0.1% Triton X-100, 0.1% Nonidet P-40, 4 mm EDTA, 50 mm NaF, 0.1 mmNa3VO4, 1 mm dithiothreitol, and protease inhibitors for 30 min at 4 °C. Cell lysates were cleared by centrifugation at 15,000 × g for 15 min, and the resulting supernatant was collected. Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis (8%) and then electrophoretically transferred to Immobilon-P transfer membrane. The membrane was probed with goat polyclonal antiserum to rabbit reticulocyte 15-LOX (cross-reactive with human 15-LOX-1) or rabbit polyclonal antiserum to human 15-LOX-2, followed by anti-goat or anti-rabbit horseradish peroxidase conjugate. Immunoreactive bands were visualized by enhanced chemiluminescence. Undifferentiated human epidermal keratinocyte production of 15-HETE-G was evaluated by treating cells in HBSS (50–70% confluence) with vehicle or 2-AG (50 μm). Following incubation at 37 °C for 45 min, HBSS was removed and extracted twice with an equal volume of CHCl3:MeOH (2:1). The organic extract was dried under argon, and the residue was analyzed by LC/MS. Human epidermal keratinocyte expression of 15-LOX enzymes was examined by immunoblotting as described above. UAS-tk-luciferase, PPARδ-pCMX, pGAL4, PPARα-GAL4, PPARγ-GAL4, and PPARδ-GAL4 plasmids have been described previously (30Gupta R.A. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13275-13280Crossref PubMed Scopus (359) Google Scholar). NIH 3T3 cells (5.0 × 105cells/well using 24-well plates) were transfected using FuGENE 6 at a lipid:DNA ratio of 3:1. Cells were exposed to a mix containing 300 ng/ml UAS-tk-luciferase (GAL4 reporter plasmid expressing firefly luciferase), 300 ng/ml indicated PPAR-GAL4 plasmid, and 1.0 ng/ml pRL-SV40 (control plasmid expressing Renillaluciferase) in Opti-MEM (Invitrogen). The transfection mix was replaced after 4–5 h with 10% charcoal-stripped fetal bovine serum-containing medium supplemented with either 0.1% vehicle or the indicated compound. After 24–36 h, cells were harvested in 1× luciferase lysis buffer. Relative light units from firefly luciferase activity were determined using a luminometer (MGM Instruments, Hamden, CT) and normalized to the relative light units from Renillaluciferase using the dual luciferase kit (Promega, Madison, WI). Human recombinant 5-LOX and purified potato 5-LOX were used to assess the ability of 5-LOXs to oxygenate 2-AG. Potato 5-LOX was incubated with substrate (10 μm arachidonate or 2-AG) in 50 mm Tris-HCl, pH 7.4, and the reaction mixture was monitored by spectrophotometry. A marked increase in absorbance at 236 nm demonstrated that the enzyme was catalytically active with arachidonic acid as substrate. However, no change in absorbance was detected upon incubation with 2-AG (data not shown). To evaluate the ability of human 5-LOX to metabolize 2-AG, enzymatic hydroperoxidation was monitored by oxygen uptake. When human 5-LOX was incubated with substrate (100 μm arachidonate or 2-AG) in 50 mm Tris-Cl, 0.03% Tween 20, pH 7.4, containing 2 mm CaCl2 and 1 mm ATP, robust oxygen uptake was detected with arachidonic acid but not with 2-AG (Fig. 1). The maximal rate of 2-AG oxygenation catalyzed by 5-LOX was 6.6 ± 0.9% of that observed with arachidonic acid (mean ± S.E., n = 3). In addition, direct liquid infusion MS of organic extracts of human 5-LOX incubations with 2-AG failed to reveal a mass ion consistent with a HpETE, HETE, dihydroxyeicosatetraenoic acid, or leukotriene A4 glycerol ester (data not shown). The inclusion of phosphatidylcholine in human 5-LOX assays has been shown, under some conditions, to enhance enzyme activity (31Reddy K.V. Hammarberg T. Radmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) Google Scholar). To confirm our findings with human 5-LOX, we repeated activity assays with arachidonic acid and 2-AG using a phospholipid-containing buffer (50 mm Tris-Cl, 0.015% Tween 20, 100 μg/ml phosphatidylcholine, pH 7.4, containing 2 mmCaCl2 and 1 mm ATP). Similar results were obtained in the absence or presence of exogenous phosphatidylcholine (data not shown). Purified soybean 15-LOX was incubated with substrate (50 μm arachidonate or 2-AG) in 50 mm Tris-HCl, 0.03% Tween 20, pH 7.4, and the reaction course was followed spectrophotometrically. Increases in absorbance at 236 nm demonstrated that soybean 15-LOX converted both arachidonic acid and 2-AG into a conjugated diene (Fig.2, A and B). Although the maximal rate of arachidonic acid oxygenation was markedly higher than that observed with 2-AG, total product formation after 5 min was only slightly lower with the endocannabinoid substrate (∼80%). To assess the substrate structural requirements for soybean 15-LOX oxygenation, arachidonic acid and a series of related arachidonyl esters (50 μm) were evaluated as substrates. The maximal rate of 2-AG oxygenation by soybean 15-LOX under these screening conditions was only 15% of that observed with arachidonic acid. However, 2-AG proved to be the preferred arachidonyl ester substrate (Fig. 3 A). Having established that soybean 15-LOX oxygenates 2-AG, we determined the steady-state kinetic values for both 2-AG and arachidonate metabolism (Table I). The enzyme displayed K m values in the low micromolar range for both substrates (7 ± 2 μm for 2-AGversus 13 ± 4 μm for arachidonic acid). Consistent with the initial observations of more rapid oxygenation of arachidonic acid by soybean 15-LOX, the V maxvalue with the free acid substrate was ∼9-fold higher than that observed with 2-AG, yielding a 4–5-fold higherV max/K m ratio for arachidonic acid (Table I). Soybean 15-LOX therefore appears capable of more rapidly metabolizing arachidonic acid, but the affinities of the enzyme toward both substrates and total product synthesis appear similar.Table ISteady-state kinetic values of soybean 15-LOXKinetic valueAA2-AG K M (μm)13 ± 47 ± 2 V max (nmol s−1 mg−1)76 ± 98.9 ± 0.8 V max/K M5.81.3Kinetic values were determined using the UV assay as described under “Experimental Procedures.” The maximum rates of reaction were obtained at least in triplicate with substrate concentrations varying from 5 to 60 μm. Substrate stocks were prepared in acetonitrile and diluted 500-fold to yield final concentrations. Values given are the mean ± S.E. Open table in a new tab Kinetic values were determined using the UV assay as described under “Experimental Procedures.” The maximum rates of reaction were obtained at least in triplicate with substrate concentrations varying from 5 to 60 μm. Substrate stocks were prepared in acetonitrile and diluted 500-fold to yield final concentrations. Values given are the mean ± S.E. Characterization of the product of 2-AG metabolism by soybean 15-LOX was achieved by chromatography, UV spectroscopy, and MS. As described above, incubations of 2-AG with soybean 15-LOX resulted in an increase in absorbance at 236 nm, suggesting the formation of a conjugated diene. Direct infusion of the organic extract of 2-AG/soybean 15-LOX incubations into the mass spectrometer revealed a single predominant product with a mass-to-charge ratio of 433 consistent with a HpETE-G sodium adduct (Fig.4 A). Collision-induced dissociation of this metabolite produced the expected hydroperoxide cleavage and established the C-15 regiochemistry of 2-AG oxygenation by 15-LOX (Fig. 4 B). In addition to the major ion atm/z 433, minor ions were detected with mass-to-charge ratios of 449 and 465. The former (m/z 449) represents the potassium adduct of the HpETE-G species. The latter (m/z 465) is consistent with a sodium adduct of abis-dioxygenation product (M + 2O2 + Na+). Both soybean and rabbit reticulocyte 15-LOX enzymes have been reported to carry out bis-dioxygenations of arachidonic acid under some conditions, and thus the appearance of an ion at m/z 465 is not surprising (32Van Os C.P. Rijke-Schilder G.P. Van Halbeek H. Verhagen J. Vliegenthart J.F. Biochim. Biophys. Acta. 1981; 663: 177-193Crossref PubMed Scopus (153) Google Scholar, 33Bryant R.W. Schewe T. Rapoport S.M. Bailey J.M. J. Biol. Chem. 1985; 260: 3548-3555Abstract Full Text PDF PubMed Google Scholar). However, under the conditions employed, this species represented only a minor product and unambiguous characterization was not pursued. Reduction of the HpETE-G product of 2-AG oxygenation by 15-LOX with triphenylphosphine followed by saponification afforded a product that co-eluted on RP-HPLC with a 15-HETE standard, confirming the regiochemical assignment provided by MS. RP-HPLC-purified HETEs were methylated with diazomethane and analyzed by chiral chromatography to establish the stereochemistry of enzymatic hydroperoxidation. Soybean 15-LOX produced the expected 15(S) enantiomer almost exclusively (95.3 ± 0.2% S (mean ± S.E., n = 3)). Investigations into mammalian 15-LOX metabolism of 2-AG began with partially purified rabbit reticulocyte 15-LOX (15-LOX-1). As with the soybean enzyme, rabbit reticulocyte 15-LOX catalyzed the formation of conjugated diene products when incubated with 50 μm arachidonate or 2-AG (Fig. 2, C andD). The maximal rate of arachidonic acid oxygenation was higher than that observed with 2-AG. However, the diffe" @default.
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• W2022077377 title "15-Lipoxygenase Metabolism of 2-Arachidonylglycerol" @default.
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