Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1α promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11,14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on α-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and γ-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 μmol/45 min per mg protein by nickel–nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B₄, degradation products of unstable leukotriene A₄, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A₄ from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.     

The 6R-oxygenase activity of arachidonate 5-lipoxygenase purified from porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes was incubated with (5S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid. In addition to degradation products of leukotriene A4 (6-trans-leukotriene B4 and its 12-epimer and others), (5S,6R)-dihydroperoxy-7,9,11,14-eicosatetraenoic acid was produced as a major product especially when the incubation was performed on ice rather than at room temperature. The amount of the (5S,6R)-dihydroperoxy acid was close to the total amount of leukotriene A4 degradation products. Under the anaerobic condition, production of the (5S,6R)-dihydroperoxy acid was markedly reduced. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid could be a substrate of the enzyme and was transformed predominantly to a compound identified as (5S)-hydroxy-(6R)-hydroperoxy-7,9-trans-11,14-cis-eicosatetraenoic acid at about 1-2% rate of arachidonate 5-oxygenation. These findings indicated that the purified 5-lipoxygenase exhibited a 6R-oxygenase activity with (5S)-hydroxy and (5S)-hydroperoxy acids as substrates. The 6R-oxygenase activity, like the leukotriene A synthase activity, was presumed to be an integral part of 5-lipoxygenase because it required calcium and ATP and was affected by selective 5-lipoxygenase inhibitors.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 5-lipoxygenase was purified to near homogeneity from the 105,000 X g supernatant of porcine leukocyte homogenate by immunoaffinity chromatography using a monoclonal anti-5-lipoxygenase antibody. Reaction of the purified enzyme with arachidonic acid produced predominantly 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid with concomitant formation of several more polar compounds in smaller amounts. These minor products were identified as the degradation products of leukotriene A4, namely, 6-trans-leukotriene B4 (epimeric at C-12) and an epimeric mixture of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids. These compounds were also produced by reaction of the enzyme with 5-hydroperoxy-eicosatetraenoic acid. Association of the 5-lipoxygenase and leukotriene A synthase activities was demonstrated by several experiments: heat inactivation of enzyme, effect of selective 5-lipoxygenase inhibitors, requirements of calcium ion and ATP, and self-catalyzed inactivation of enzyme. The enzyme was also active with 12- and 15-hydroperoxy-eicosatetraenoic acids producing (5S,12S)- and (5S,15S)-dihydroperoxy acids, respectively. Maximal velocities of the reactions with these hydroperoxy acids as compared with that of arachidonic acid (100%, 0.6 mumol/3 min/mg of protein) were as follows: 5-hydroperoxy acid, 3.5%, 12-hydroperoxy acid, 22%, and 15-hydroperoxy acid, 30%.     

Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties

Arachidonic acid 15-lipoxygenase was purified from rabbit peritoneal polymorphonuclear leukocytes. The enzyme was recovered in the cytosol fraction after sonication and purified about 250-fold by acetone precipitation, column chromatography on CM52, Sephadex G-150, and hydroxyapatite. The enzyme catalyzed the conversion of arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which then decomposed to a mixture of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), 15-keto-5,8,11,13-eicosatetraenoic acid, 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid. The enzyme was specific for oxygenation at carbon 15 of arachidonic acid. The apparent molecular weight of the enzyme was about 61,000 as measured by Sephadex G-150 gel filtration chromatography. The enzyme was sensitive to sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid. The enzyme activity was inhibited by eicosatetraynoic acid (ETYA) or 3-amino-1-(m-(trifluoromethyl)-phenyl)2-pyrazoline (BW755C), but not by indomethacin up to 200 micrograms/ml.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Localization of Arachidonate 12-Lipoxygenase in Canine Brain Tissues

The cytosol fraction from a thoroughly irrigated canine cerebrum was subjected to immunoaffinity chromatography using a monoclonal antibody against porcine leukocyte 12-lipoxygenase. Arachidonate 12-lipoxygenase eluted from the column with some retardation. The enzyme, with a specific activity of 9 nmol/min/mg of protein, converted arachidonic acid to 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid. The enzyme was active not only with arachidonic acid, but also with linoleic and alpha-linolenic acids. In contrast, 12-lipoxygenase of canine platelets was almost inactive with linoleic and alpha-linolenic acids, and the platelet enzyme was also distinguished from the cerebral enzyme in terms of reactivity with the anti-12-lipoxygenase antibody. 12-Lipoxygenase activity was also detected in the cytosol fractions of other parts of canine brain: basal ganglia, hippocampus, cerebellum, olfactory bulb, and medulla oblongata.     

The autoxidation of arachidonic acid: formation of the proposed SRS-A intermediate.

The air oxidation of 5,8,11,14-eicosatetraenoic [arachidonic] acid and its methyl ester is reported. A mixture of hydroperoxy arachidonic acid products was obtained from the oxidation and subsequent separation of the mixture by high pressure liquid chromatography led to pure hydroperoxides. One of these hydroperoxides, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, is a proposed intermediate in the biosynthesis of slow reacting substance of anaphylaxis.     

Arachidonic acid metabolism in isolated pancreatic islets. V. The enantiomeric composition of 12-hydroxy-5,8,10,14-eicosatetraenoic acid indicates synthesis by a 12-lipoxygenase rather than a monooxygenase

Recent evidence indicates that the arachidonate metabolite 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) or its precursor may act as a second messenger in stimulus-response coupling in a variety of cells including Aplysia neurons, adrenal glomerulosa cells, and pancreatic islets. The compound 12(S)-HETE is generated from the precursor 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12(S)-HPETE), which is a product of the 12-lipoxygenase enzyme. Some cells have recently been found to produce the enantiomer 12(R)-HETE, apparently via a cytochrome P-450 monooxygenase, and the biologic actions of 12(R)-HETE and 12(S)-HETE differ. We have examined the stereochemistry of 12-HETE from isolated pancreatic islets both radiochemically and by a new mass spectrometric method capable of quantitating subnanogram amounts of 12-HETE stereoisomers. Endogenous 12-HETE from islets was found to be exclusively the S-isomer. D-Glucose stimulated both insulin secretion and islet accumulation of 12(S)-HETE but not of 12(R)-HETE. Pharmacologic inhibition of islet 12-HETE biosynthesis also suppressed glucose-induced insulin secretion. These findings suggest that islet 12-HETE is a product of a 12-lipoxygenase rather than of a cytochrome P-450 monooxygenase and further implicate 12-lipoxygenase products in stimulus-secretion coupling.     

Resolution by DEAE-cellulose chromatography of the enzymatic steps in the transformation of arachidonic acid into 8, 11, 12- and 10, 11, 12-trihydroxy-eicosatrienoic acid by the rat lung

The 30-50% ammonium sulfate fraction of the high speed supernatant (100,000 xg) of a rat lung homogenate is capable of catalysing the conversion of arachidonic acid into 8,11,12- and 10,11, 12-trihydroxyeicosatrienoic acids. This enzyme preparation was resolved through DEAE cellulose chromatography into three stages which were assayed with precursors specific for each stage. Thus in the first stage arachidonic acid is converted by 12-lipoxygenase into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) detected as the corresponding 12-hydroxy product (12-HETE). 12-HPETE in turn is converted into 8-hydroxy-11,12-epoxy-5,9,14-eicosatrienoic acid and 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid. These epoxides are in turn selectively converted through an epoxide hydrase into the respective triols. While the first and third stages were carried out by distinct fractions from the DEAE columns, the second i.e. conversion of 12-HPETE into epoxides, was detected in all fractions as was the reduction of 12-HPETE into 12-HETE.     

Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid.

Prostaglandin A2 is a major constituent of the gorgonian Plexaura homomalla, and there is evidence that its biosynthesis involves a noncyclooxygenase pathway. The coral contains an 8(R)-lipoxygenase and an allene oxide synthase; from arachidonic acid, the sequential action of these enzymes gives an allene epoxide, the cyclization of which forms an analogue of prostaglandin A2 (PGA2) with no 15-hydroxyl group. In this study we examined the metabolic fate of 15-hydroxyeicosatetraenoic acid (15-HETE), which via analogous reactions could lead to PGA2. The 8(R)-lipoxygenase metabolized preferentially the 15(R) enantiomer of 15-HETE, and this reaction was stimulated fivefold by including 1 M NaCl in the incubation. Further enzymic steps were detected by comparing the metabolic profiles of the 8(R)-hydroperoxy-15(R)-hydroxy intermediate with that of its 8(S),15(S) enantiomer. Two main products were formed exclusively from the 8(R),15(R) enantiomer: an allene epoxide and the comparatively stable epoxide, 8,9-epoxy-10,15-dihydroxyeicosa-5,11,14-trienoic acid. Formation of the allene oxide was inferred from detection of its hydrolysis and cyclization products. It cyclized to give two isomers of PGA2 which have a "cis" arrangement of the side chains. The main hydrolysis product (8,15-dihydroxy-9-ketoeicosa-5,11,13-trienoic acid) was unstable and prone to oxygenation, giving 8,14,15-trihydroxy-9-ketoeicosa-5,10,12-trienoic acids after reduction of the 14-hydroperoxide. We conclude that metabolism of a 15-hydroxy eicosanoid is a potential route to the A series prostaglandins, although the low yield and lack of stereochemical control suggest that this is not the natural pathway of biosynthesis in P. homomalla. Unexpectedly, the major end products of the pathway are trihydroxy ketols and the single diastereomer of a stable epoxyalcohol.     

Synthesis of 8,9-leukotriene A4 by murine 8-lipoxygenase

Arachidonate 8-lipoxygenase was identified in phorbol ester induced mouse skin. We expressed the enzyme in an Escherichia coli system using pET-15b carrying an N-terminal histidine-tag sequence. The enzyme, purified by nickel-nitrilotriacetate affinity chromatography, showed specific activity of about 0.1 micromol/min/mg of protein with arachidonic acid as a substrate. When metabolites of arachidonic acid were reduced and analyzed by reverse-phase HPLC, 8-hydroxy derivative was a major product as measured by absorbance at 235 nm. In addition, three polar compounds (I, II, and III) were detected by measuring absorbance at 270 nm. These compounds were also produced when the enzyme was incubated with 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. Neither heat-inactivated enzyme nor mutated enzyme produced these compounds, suggesting that they are enzymatically generated. Ultraviolet spectra of these compounds showed typical triplet peaks around 270 nm, indicating that they have a triene structure. Molecular weight of these compounds was determined to be 336 by liquid chromatography-mass spectrometry, indicating that they carry two hydroxyl groups. Compounds I and III were generated even under anaerobic condition, indicating that oxygenation reaction was not required for their generation from 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid. By analogy to the reactions of 5-lipoxygenase pathway where leukotriene A4 is generated, it is suggested that 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid is converted by the 8-lipoxygenase to 8,9-epoxyeicosa-5,10,12,14-tetraenoic acid which degrades to compounds I and III by non-enzymatic reaction. In contrast, compound II was not generated under anaerobic condition, indicating that it was produced by oxygenation reaction. Taken together, 8-lipoxygenase catalyzes both dehydration reaction to yield 8,9-epoxy derivative and oxygenation reaction presumably at 15-position of 8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid.     

Oxidation of glutathione to its thiyl free radical metabolite by prostaglandin H synthase. A potential endogenous substrate for the hydroperoxidase

The oxidation of glutathione to a thiyl radical by prostaglandin H synthase was investigated. Ram seminal vesicle microsomes, in the presence of arachidonic acid, oxidized glutathione to its thiyl-free radical metabolite, which was detected by ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. Oxidation of glutathione was dependent on arachidonic acid and inhibited by indomethacin. Peroxides also supported oxidation, indicating that the oxidation was by prostaglandin hydroperoxidase. Glutathione served as a reducingcofactor for the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid at 1.5-2 times the nonenzymatic rate. Although purified prostaglandin H synthase in the presence of either H2O2 or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid oxidized glutathione to a thiyl radical, arachidonic acid did not support glutathione oxidation. Glutathione also inhibited cyclooxygenase activity as determined by measuring oxygen incorporation into arachidonic acid. Reverse-phase high pressure liquid chromatography analysis of the arachidonic acid metabolites indicated that the presence of glutathione in an incubation altered the metabolite profile. In the absence of the cofactor, the metabolites were PGD2, PGE2, and 15-hydroperoxy-PGE2 (where PG indicates prostaglandin), while in the presence of glutathione, the only metabolite was PGE2. These results indicate that glutathione not only serves as a cofactor for prostaglandin E isomerase but is also a reducing cofactor for prostaglandin H hydroperoxidase. Assuming that glutathione thiyl-free radical observed in the trapping experiments is involved in the enzymatic reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid, then a 1-electron donation from glutathione to prostaglandin hydroperoxidase is indicated.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.