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-eicosatetraenoic acids (5,12 diHETEs) 5-hydroperoxy and hydroxyeicosatetraenoic acids (5-HPETE and 5-HETE) as well as a novel metabolite 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The crude 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 microM. Activity was also stimulated by ATP (EC50 = 160 microM). The cytosolic 5-lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 microM (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 of several hydroperoxides, i.e. 5-,8-,9- or 15-HPETE (EC50 ca. 1 microM), 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.
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 / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D015289:Leukotrienes; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D051379:Mice
D001095:Arachidonic Acids; D015289:Leukotrienes; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D000255:Adenosine Triphosphate; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D005978:Glutathione; D002118:Calcium