15-lipoxygenation of leukotriene A4 -: Studies of 12-and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) Aa is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 13- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100 000 x g supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100 000 x g supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07. 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios < 0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K-m for LTA(4) (9-27 mu M), as compared to the other lipoxygenases tested (58-83 mu M). The recombinant human 12-lipoxygenase demonstrated the lowest K-m value for LTA(4) (9 mu M) whereas the porcine leukocyte 12-lipoxygenase had the highest V-max. The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB4 isomers were isolated. Production of LXB4 was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC50 1 and 50 mu M, respectively) in the human platelet cytosolic fraction. (C) 2000 Elsevier Science B.V. All rights reserved.