Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets

• Published in: Biochemical journal Vol. 202; no. 3; pp. 771 - 776
• Main Authors: Chang, W C, Nakao, J, Orimo, H, Murota, S
• Format: Journal Article
• Language: English
• Published: England 15-03-1982
• Subjects: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids - metabolism; Blood Platelets - drug effects; Blood Platelets - enzymology; Disulfides - pharmacology; Glutathione - pharmacology; In Vitro Techniques; Leukotrienes; Lipoxygenase - blood; Lipoxygenase Inhibitors; Male; Metabolism, Regulation and Control Processes; Oxidation-Reduction; Peroxides - metabolism; Rats; Rats, Inbred Strains; Sulfhydryl Compounds - pharmacology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2020771

Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity.