Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities.
The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activity. Furthermore, the two enzymatic activities coeluted exactly on three different HPLC systems. Maximal activity of purified LTA4 synthase required the addition of three nondialyzable stimulatory factors, two of which were cytosolic and one of which was membrane-bound. These findings were identical for 5-lipoxygenase activity. When incubated with arachidonic acid, the purified 5-lipoxygenase converted approximately equal to 15% of its endogenously generated 5-hydroperoxyicosatetraenoic acid (5-HPETE) to LTA4. LTA4 production was more efficient when the enzyme utilized 5-HPETE generated from arachidonic acid than when 5-HPETE was exogenously supplied as substrate. These findings suggest that a single protein from human leukocytes possesses 5-lipoxygenase and LTA4 synthase activities and that the synthesis of LTA4 from 5-HPETE is controlled by the same complex multicomponent system that regulates the 5-lipoxygenase reaction.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001091:Arachidonate Lipoxygenases; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000096:biosynthesis* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D017572:Leukotriene A4; D015289:Leukotrienes; D008084:Lipoxygenase / Q000302:isolation & purification* / Q000378:metabolism
D001095:Arachidonic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D000255:Adenosine Triphosphate; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase; D002118:Calcium