flavin-adenine-dinucleotide and arachidonic-acid-5-hydroperoxide

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.

Topics: Adenosine Triphosphate; Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcium; Enzyme Activation; Ferric Compounds; Ferrous Compounds; Flavin-Adenine Dinucleotide; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxidation; Lipoxygenase Inhibitors; Mercaptoethanol; NAD; NADP; Rats

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cell Compartmentation; Enzyme Activation; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Leukotrienes; Male; NAD; NADP; Neutrophils; Rats