Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids.

Abstract

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-hydroper-o~y-6,8,l1,14-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, 6truns-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 B-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,lEiS)-dihydroperoxy acids, respectively.Maximal velocities of the reactions with these hydroperoxy acids as compared with that of arachidonic acid (loo%, 0.6 pmo1/3 min/mg of protein) were as follows: 5-hydroperoxy acid, 3.5%, 12-hydroperoxy acid, 22%, and 15-hydroperoxy acid, 30%.