Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids.
Arachidonate 5-lipoxygenase was purified to near homogeneity from the 105,000 X g supernatant of porcine leukocyte homogenate by immunoaffinity chromatography using a monoclonal anti-5-lipoxygenase antibody. Reaction of the purified enzyme with arachidonic acid produced predominantly 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid with concomitant formation of several more polar compounds in smaller amounts. These minor products were identified as the degradation products of leukotriene A4, namely, 6-trans-leukotriene B4 (epimeric at C-12) and an epimeric mixture of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids. These compounds were also produced by reaction of the enzyme with 5-hydroperoxy-eicosatetraenoic acid. Association of the 5-lipoxygenase and leukotriene A synthase activities was demonstrated by several experiments: heat inactivation of enzyme, effect of selective 5-lipoxygenase inhibitors, requirements of calcium ion and ATP, and self-catalyzed inactivation of enzyme. The enzyme was also active with 12- and 15-hydroperoxy-eicosatetraenoic acids producing (5S,12S)- and (5S,15S)-dihydroperoxy acids, respectively. Maximal velocities of the reactions with these hydroperoxy acids as compared with that of arachidonic acid (100%, 0.6 mumol/3 min/mg of protein) were as follows: 5-hydroperoxy acid, 3.5%, 12-hydroperoxy acid, 22%, and 15-hydroperoxy acid, 30%.
D000818:Animals; D001091:Arachidonate Lipoxygenases; D001095:Arachidonic Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D015289:Leukotrienes; D008054:Lipid Peroxides / Q000378:metabolism*; D008084:Lipoxygenase / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D011485:Protein Binding; D013379:Substrate Specificity; D013552:Swine
D001095:Arachidonic Acids; D015289:Leukotrienes; D008054:Lipid Peroxides; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase