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.
Riendeau
D
D
Merck Frosst Centre for Therapeutic Research, Dorval, Québec, Canada.
Denis
D
D
Choo
L Y
LY
Nathaniel
D J
DJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D015289:Leukotrienes / Q000494:pharmacology; D015227:Lipid Peroxidation; D016859:Lipoxygenase Inhibitors; D008623:Mercaptoethanol / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D051381:Rats
D001095:Arachidonic Acids; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D016718:Arachidonic Acid; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D009249:NADP; D008623:Mercaptoethanol; C025348:arachidonic acid 5-hydroperoxide; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium