5-Lipoxygenase and leukotriene synthesis: effects of calcium ions and of inhibitors.
5-Lipoxygenase and leukotriene (LT) A4 synthase, the first two enzymes in the pathway converting arachidonic acid to leukotrienes, can be co-purified. The Ca2+-activated conversion of arachidonic acid and of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to LTB4 have been compared, using cytosol from human leucocytes. The two activities showed identical responses to a number of inhibitors, suggesting that the two catalytic activities may involve the same active centre. The effects of Ca2+ ions were further investigated. With 5-HPETE as substrate, substantial synthesis of LTB4 was given in the absence of Ca2+, and the inhibitor sensitivity of this component was quite different from that of the Ca2+-activated component. This Ca2+-independent synthase activity was, however, very low in saponin-permeabilised washed leucocytes and it may therefore be not significant physiologically. With arachidonic acid as substrate at pH 7, the activity was highly Ca2+-dependent at a low substrate concentration (6.6 microM), but at a high concentration (132 microM) substantial activity was observed without Ca2+. This was also found when 5-lipoxygenase was assayed in cytosol from RBL cells. At pH 8-8.5, however, Ca2+ was required at both high and low concentrations of arachidonic acid. This suggests that Ca2+ is required for 5-lipoxygenase activity on arachidonate ions in solution but possibly not on protonated arachidonic acid or micelles.
Reynolds
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Biochemistry Department, Wellcome Research Laboratories, Beckenham, Kent, U.K.
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007975:Leukotriene B4 / Q000096:biosynthesis*; D015289:Leukotrienes / Q000378:metabolism
D001095:Arachidonic Acids; D015289:Leukotrienes; D007975:Leukotriene B4; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium