Kinetic studies on the inactivation of 5-lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid

The oxygenation of arachidonic acid (AA) by guinea-pig neutrophil 5-lipoxygenase terminates prematurely at a substrate utilization of only 50%. In the presence of dithiothreitol (DTT), reaction progress continues longer but still terminates prematurely, at about 70% substrate turnover. The addition of more substrate during the first 60 seconds of the initial reaction resulted in continued product formation. However, at times after 120 seconds, the addition of more AA could not produce additional product formation. Together, these results indicate a time-dependent ( ), irreversible loss of enzyme activity. To determine if the product 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) mediates the inactivation, it was tested for its ability to irreversibly inhibit the enzyme and found to inactivate 5-lipoxygenase with K_i = 0.05 ± 0.01 μM and k_i = 1.4 ± 0.4 min⁻. DTT changed the apparent affinity of 5-HPETE (K_i = 0.33 ± 0.09 μM) but had no effect on the rate of inactivation (k_i = 1.26 ± 0.62 min⁻¹). In contrast, the hydroxy derivative of 5-HPETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), is a reversible, time-independent inhibitor with _K = 6.3 ± 0.9 μM regardless of DTT. The ability of thiols to protect 5-lipoxygenase from production inactivation is due, at least in part, to a non-enzymatic reaction between DTT and 5-HPETE that converts the hydroperoxy acid to a material that can no longer inactivate the enzyme.     

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.     

Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase

The kinetic mechanism of guinea pig neutrophil 5-lipoxygenase was investigated using a continuous spectrophotometric assay that monitors product diene formation at 236 nm due to substrate oxygenation. Progress curves for reactions with both arachidonic acid and eicosapentaenoic acid are characterized by 1-3-min lag phases in the attainment of steady-state velocities and product inhibition, as indicated by the total cessation of the reaction prior to complete depletion of substrate. The dependence of the steady-state velocity on arachidonic acid concentration appears to follow Michaelis-Menten kinetics, with Vmax = 4.2 +/- 0.4 nmol of 5-hydroxy-6,8,11,14-eicosatetraenoic acid/min/mg of protein and Ks = 25 +/- 4 microM. The addition of Ca2+ results in an overall activation: lag phases are shortened to 10-20 s, Vmax increases to 24 +/- 2 nmol/min/mg of protein, and Ks decreases to 7.7 +/- 1.7 microM; and a change in a mechanism to one involving substrate inhibition (Kss = 13 +/- 1 microM). The observed activation by Ca2+ has a half-maximal response at around 30 microM. In the presence of Ca2+, ATP causes an increase in Vmax to 30 +/- 4 nmol/min/mg of protein without changing Ks or Kss and a reduction of the lag to less than 5 s. The half-maximal response for ATP is 31 +/- 7 microM. Oxygenation of eicosapentaenoic acid in the presence of Ca2+ and ATP occurs with similar kinetics, except for significantly less substrate inhibition: Vmax = 31 +/- 6 nmol/min/mg of protein, Ks = 7 +/- 1 microM, and Kss = 33 +/- 2 microM. This is the first report suggesting a kinetic mechanism for 5-lipoxygenase, which accounts for substrate inhibition, regulation by Ca2+, and ATP and substrate specificity.     

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.     

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.     

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.     

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%.     

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.     

Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.

Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.     

Metabolism of 5-hydroxyicosatetraenoate by human neutrophils: production of a novel omega-oxidized derivative

Human neutrophils incorporated 5-hydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5-HETE) into cellular triglyceride and phospholipid. They also metabolized 5-HETE into a novel, extracellularly released derivative, 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5,20-diHETE). 5,20-diHETE formation predominated at higher substrate concentrations and longer incubation intervals. In the absence of added 5-HETE, 1 X 10(8) neutrophils stimulated with 20 microM ionophore A23187 produced up to 243 ng of 5,20-diHETE, indicating that both endogenously formed and exogenously added substrate could be oxidized at carbon 20. 5,20-diHETE was about 10- to 100-fold weaker than 5-HETE in enhancing human neutrophil degranulation responses to platelet-activating factor. omega-Oxidation appears to be a general enzymatic mechanism for inactivation of arachidonic acid metabolites.     

Use of simplified substrates for the study of 5-lipoxygenase from RBL-1 cells

5,8,14-eicosatrienoic (5,8,14-ETA) and 5,8-eicosadienoic (5,8-EDA) acids are converted by the 5-lipoxygenase from RBL-1 cells into 5-hydroperoxy-6,8,14-eicosatrienoic (5-OOH-ETA) and 5-hydroperoxy-6,8-eicosadienoic (5-OOH-EDA) acids, respectively. These hydroperoxy fatty acids, unlike 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), are not further processed into leukotrienes by the leukotriene A4 synthase activity of 5-lipoxygenase. 5,8,14-ETA was used to establish the saturation kinetics of 5-lipoxygenase in the 100,000g supernatant from RBL-1 cells. The study was performed by measuring the rate of product formation at optimal concentrations of the cofactors, calcium and ATP. Kinetics performed at various concentrations of supernatant did not follow the Michaelis-Menten equation. This aspect is discussed in relation to the presence of hydroperoxide-reducing system(s) in the supernatant. 5,8,14-ETA and 5,8-EDA turnover rates were also compared.     

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.     

Purification and Properties of a New l-Fucose-specific Lectin Produced by Streptomyces No. 100–2

The inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamide derivatives on human 5-lipoxygenase (5-LO), a key enzyme in arachidonic acid cascades, were examined using 5-LO produced by Escherichia coli. Some of the tested compounds inhibited the conversion of arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), and in particular the N-phenylbutyl derivative was about 30 times more active (IC₅₀ = 35 μm) than caffeic acid (IC₅₀ = 1000 μm), a known selective 5-LO inhibitor.     

Biosynthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid from 5-hydroperoxyeicosatetraenoic acid in the murine macrophage

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a metabolite of arachidonic acid shown to possess important biological activities within different cell types. In the neutrophil, a specific NADP(+)-dependent dehydrogenase utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in the murine macrophage. The macrophage was not able to convert 5-HETE into 5-oxo-ETE even when preincubated with phorbol ester or with other lipid hydroperoxides. The factor responsible for the conversion of 5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic fraction of the macrophage, with an approximate molecular weight of 50,000-60,000, as assessed by size exclusion chromatography. Formation of 5-oxo-ETE was rapid and the catalytic protein was found to have an apparent K(m) of 5.3 microM for the eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the murine macrophage was consistent with reduction of a 5-hydroperoxy group to an intermediate alkoxy radical that could be subsequently oxidized to the 5-oxo product. Such a mechanism would enable racemic 5-HpETE, derived from free radical oxidation of arachidonic acid, to be efficiently converted into this potent chemotactic eicosanoid.     

Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells

Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).     

Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation.

The characteristics of hydroperoxide activation of 5-lipoxygenase were examined in the high speed supernatant fraction prepared from rat polymorphonuclear leukocytes. Stimulation of 5-lipoxygenase activity by the 5-hydroperoxyeicosatetraenoic acid (5-HPETE) reaction product was strongly dependent on the presence of thiol compounds. Various reducing agents such as mercaptoethanol and glutathione (0.5-2 mM) inhibited the reaction and increased the concentrations of 5-HPETE (1-10 microM) necessary to achieve maximal arachidonic acid oxidation. The requirement for 5-HPETE was not specific and could be replaced by H2O2 (10 microM) but not by the 5-hydroxyeicosatetraenoic acid (5-HETE) analogue. Furthermore, gel filtration chromatography of the soluble extract from leukocytes resolved different fractions which can increase the hydroperoxide dependence or fully replace the stimulation by 5-HPETE. Maximal activity of the 5-HPETE-stimulated reaction required Ca2+ ions (0.2-1 mM) and ATP with the elimination of the HPETE requirement at high ATP concentrations (2-4 mM). In addition, NADPH (1-2 mM), FAD (1 mM), Fe2+ ions (20-100 microM) and chelated Fe3+ (0.1 mM-EDTA/0.1 mM-FeCl3) all markedly increased product formation by 5-lipoxygenase whereas NADH (1 mM) was inhibitory and Fe3+ (20-100 microM) alone had no effect on the reaction. The stimulation by Fe2+ ions and NADPH was also observed under various conditions which increase the hydroperoxide dependence such as pretreatment of the enzyme preparation with glutathione peroxidase or chemical reduction with 0.015% NaBH4. These results provide evidence for an hydroperoxide activation of 5-lipoxygenase which is not product-specific and is modulated by thiol levels and several soluble components of the leukocytes. They also indicate that stimulation of 5-lipoxygenase activity can contribute to increase lipid peroxidation in iron and nucleotide-promoted reactions.     

Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.     

Effects of stilbene derivatives on arachidonate metabolism in leukocytes

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.     

Modulation of the 5-lipoxygenase activity of MC-9 mast cells: Activation by hydroperoxides

In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in the 100,000 xg supernatant from sonicates of cells of an IL-3 dependent murine mast cell clone, MC-9 were determined. Principal products from exogenous ¹⁴C-arachidonic acid were identified as leukotriene B₄, diastereomeric 5,12-dihydroxy-eicossatetraenoic acids (5.12 diHETEs) 5-hydroperoxy and hydroxyeicosatetraenoic acids (5-HPETE and 5-HEYE) as well as a novel metabolite 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added Ca⁺⁺. The effective Ca⁺⁺ concentration for 50 per cent activation (EC₅₀) was 3 uM. Activity was also stimulated by ATP (EC₅₀ = 160 uM). The cytosolic 5-lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 uM (ca. 20 nmole/mg/4 min). The lipoxygenase activity exhibited apparent lag phase kinetics which were more pronounced at low protein concentrations (0.3 mg/ml). In addition, the lag phase was greatly accentuated by the addition of a hydroperoxide scavenging system consisting of glutathione (1 mM) plus glutathione peroxidase (0.4 unit/ml). In contrast, addition of any several hydroperoxides, i.e. 5-,8-,9- or 15-HPETE (EC₅₀ ca. 1 uM), but not the corresponding alcohols (5-HETE and 15-HETE), shortened the lag phase. These results show that the 5-lipoxygenase requires hydroperoxide for activation and that cellular level of hydroperoxides may be an important factor regulating leukotriene synthesis.     

5-lipoxygenase from rat PMN lysate

The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA4, particularly LTB4. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5-lipoxygenase activity is stimulated by Ca++ and ATP.