The importance of hydroperoxide activation for the detection and assay of mammalian 5-lipoxygenase.
Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1-5 microM) during the reaction which effected a 10-20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.
D001091:Arachidonate Lipoxygenases; D001095:Arachidonic Acids / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D015289:Leukotrienes; D008054:Lipid Peroxides / Q000494:pharmacology; D008084:Lipoxygenase / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001095:Arachidonic Acids; D015289:Leukotrienes; D008054:Lipid Peroxides; D013439:Sulfhydryl Reagents; C021201:12-HPETE; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; C025348:arachidonic acid 5-hydroperoxide; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase