Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin.
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Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes. (+info)
Characterization of the human 5-lipoxygenase gene.
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The gene for human 5-lipoxygenase has been isolated from three different bacteriophage genomic libraries and a genomic cosmid library. The gene spans greater than 82 kilobases and consists of 14 exons. The size range for the exons is 82-613 base pairs, whereas that for the introns is approximately 200 bp to greater than 26 kb. A major site of transcription initiation in leukocytes was mapped to a thymidine residue 65 base pairs upstream of the ATG initiation codon by nuclease S1 protection and primer extension experiments. Other potential minor initiation sites were found. The putative promoter region contains no TATA and CCAAT sequences in the expected positions upstream of the major transcription initiation site but contains multiple GC boxes within a (G + C)-rich region, as does the immediate 5' region of the first intron. Characteristics common to the 5' end of the human 5-lipoxygenase gene and the promoter regions of the housekeeping genes raise important questions concerning the regulation of 5-lipoxygenase gene expression. (+info)
Endogenous epoxyeicosatrienoic acids. Cytochrome P-450 controlled stereoselectivity of the hepatic arachidonic acid epoxygenase.
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Chiral analysis of the endogenous rat liver epoxyeicosatrienoic acids shows the biosynthesis of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids in a 4:1, 2:1, and 3:1 ratio of antipodes, respectively. Animal treatment with phenobarbital results in a 3.7-fold increase in microsomal cytochrome P-450 concentration and a concomitant, regioselective 6.8- and 3.4-fold increase in the liver concentration of 8,9- and 14,15-epoxyeicosatrienoic acids, respectively. Phenobarbital induces the in vivo synthesis of both regioisomers as nearly optically pure enantiomers. These results demonstrate the enzymatic origin of the epoxyeicosatrienoic acids present in rat liver and document a novel metabolic function for cytochrome P-450 in the regio- and enatioselective epoxygenation of endogenous pools of arachidonic acid. (+info)
Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system.
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Human 5-lipoxygenase (EC 1.13.11.34), the key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus (3B6) carrying the human 5-lipoxygenase coding sequence downstream of the strong polyhedrin protein promoter was isolated. Approximately 48 hr after infection of Spodoptera frugiperda cells with the recombinant baculovirus, maximal intracellular enzyme activity and protein levels were detected. The recombinant 5-lipoxygenase in 10,000 x g supernatant fractions was able to synthesize large amounts of 5-hydroperoxy-6,8,10,14-icosatetraenoic acid, together with smaller amounts of the nonenzymatic hydrolysis products of leukotriene A4, and exhibited a dependence on Ca2+ and ATP for maximal activity. Immunoblot analysis of supernatant proteins from human leukocytes and recombinant virus-infected cells indicated the presence of indistinguishable approximately 80-kDa bands. However, 5-lipoxygenase protein in recombinant-infected cells was found to be present in amounts 50-200 times that present in leukocytes on a per-cell basis. Histidine-362 and histidine-372, potential iron-atom ligands within a putative iron-binding domain, were changed to serine residues. Recombinant baculoviruses carrying the mutations were isolated and used to infect insect cells. Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity was not substantially altered, suggesting the nonessential nature of these histidines in binding iron at the putative ferric catalytic site. (+info)
Expression of human 5-lipoxygenase cDNA in Escherichia coli.
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A cDNA for human 5-lipoxygenase (5LO) was inserted into the vector pKC (constructed from pKK223-3 by replacing its replication origin with that of pUC18) and expressed in Escherichia coli. The enzyme expressed was purified to homogeneity from the cellular soluble fraction. The purified enzyme showed both 5LO and leukotriene A4 synthase activities, which were stimulated by Ca2+ and ATP. Its molecular mass (78 kDa) and NH2-terminal sequence were identical with those of 5LO purified from human leukocytes. The availability of the expression system will facilitate further studies on its regulation and the reaction mechanism of the enzyme. (+info)
Species difference of 5-lipoxygenase derived from polymorphonuclear leukocytes on sensitivity to drugs.
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Effects of 12 compounds including 5-lipoxygenase (5-LO) inhibitors and antiallergic drugs on the activity of 5-LO from the polymorphonuclear leukocytes (PMN) of guinea pigs, rats, rabbits, monkeys and humans were examined. The 5-LO activity was inhibited by the drugs according to the following orders of efficacy: Guinea pig 5-LO, 5-HDHDMF greater than TZI-2721 greater than NDGA greater than AA861 greater than FPL55712 greater than isoproterenol; rat 5-LO, 5-HDHDMF greater than AA861 greater than TZI-2721 greater than NDGA greater than FPL55712 greater than KP-136 greater than MCI-826 greater than benoxaprofen; and rabbit 5-LO, 5-HDHDMF greater than TZI-2721 greater than NDGA greater than AA861 greater than FPL55712 greater than KP-136 greater than MCI-826. The 5-LO activity from the rhesus monkey was dose-dependently inhibited by only 3 compounds, 5-HDHDMF greater than NDGA greater than TZI-2721, in this order, but the other compounds, except for AA861, did not show any effect on this activity. In humans, 5-LO activity was inhibited in the following order: TZI-2721 greater than 5-HDHDMF greater than NDGA greater than AA861 greater than FPL55712. From these results, it was strongly suggested that there is a species difference of this enzyme in its sensitivity to drugs. (+info)
Calcium induces membrane translocation of 12-lipoxygenase in rat platelets.
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Translocation of soluble 12-lipoxygenase to membranes was examined in rat platelets. Preincubation of platelet homogenates with 0.1-10 microM Ca2+ resulted in an increase in 12-lipoxygenase activity of the particulate fraction with a concomitant decrease in that of the soluble fraction. Kinetic parameters of 12-lipoxygenase of the soluble and membrane fractions were not changes in the presence of 10 microM Ca2+. Ca2+-induced association of 12-lipoxygenase to the particulate fraction was dependent on the amounts of platelet-soluble and membrane fractions but not on the incubation temperature. 12-Lipoxygenase activity associated with the particulate fraction was completely dissociated by reducing the concentration of Ca2+ to 10 nM. Ca2+-induced association of the enzyme also occurred in the boiled- and trypsin-treated membranes but was significantly reduced in the phospholipase A2-treated membranes. Soluble 12-lipoxygenase also associated to liposomes in a Ca2+-dependent manner. Pretreatment of platelets with thrombin (0.5-5 units/ml) significantly caused a translocation of soluble 12-lipoxygenase to particulate fraction; in the time course study, the translocation was observed at the thrombin pretreatment of 1, 5, and 10 min. These results suggest that stimulation of platelets is followed by the translocation of soluble 12-lipoxygenase to membranes, which is mediated by physiological concentration of Ca2+. (+info)
Studies on the metal-ion and lipoxygenase-catalysed breakdown of hydroperoxides using electron-spin-resonance spectroscopy.
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The breakdown of cumene hydroperoxide and peroxidized fatty acids by iron is shown, by use of the spin trap 5,5-dimethyl-l-pyrroline-N-oxide, to be sensitive to (a) the oxidation state of the metal and (b) the nature of the chelating ligands. The initial step in the Fe2+-catalysed breakdown is the production of an alkoxyl radical by one-electron reduction, and this type of radical has been successfully trapped from each substrate. Subsequent reactions of this alkoxyl species produce both carbon-centred and peroxyl radicals, depending on the concentrations of the reagents present. The use of the same spin trap in microsomal systems undergoing either NADPH-supported or Fe2+-induced peroxidation led to the detection of low concentrations of radical adducts, among which are signals that are believed to be due to lipid alkoxyl radicals. Reaction of polyunsaturated fatty acid hydroperoxides with both Fe2+ and lipoxygenase under anaerobic conditions gives rise to signals not only from the alkoxy-radical adduct, but also from a further species which is tentatively identified as being due to an acyl [RC(O).]-radical adduct; chemical studies lend support to this assignment. (+info)