When arachidonic acid is used as a substrate the platelet

When arachidonic PD 151746 is used as a substrate, the platelet-type 12S-lipoxygenase produces predominantly the 12S-hydroperoxy derivative. In contrast, the leukocyte-type 12S-lipoxygenases generate substantial amounts of the 15-lipoxygenase product in addition to the 12S-hydroperoxy derivative. The ratio of 12- to 15-lipoxygenase products varies depending on the enzymes: ∼3:1 for the murine leukocyte-type 12-lipoxygenase, ∼6:1 for the rat brain 12-lipoxygenase, ∼9:1 for the porcine leukocyte 12-lipoxygenase and ∼11:1 for the bovine tracheal 12-lipoxygenase [6]. With regards to amino acid residues that determine this positional specificity, it has been proposed that the overall size and shape of the arachidonic acid-binding pocket is most important [43]. X-ray crystallographic studies show a U-shaped active site cavity lined with side chains from Phe-353, Met-419, Ile-418, and Ile-593 of the reticulocyte 15-lipoxygenase [43] that are in positions corresponding to those of the leukocyte-type 12-lipoxygenase. The active site cavity of the 12-lipoxygenase is predicted to be slightly larger (6%) than that of the 15-lipoxygenase [40]. In fact, a double mutation of Ile-418 and Met-419 of human 15-lipoxygenase to residues with smaller side chain (Val) yields an enzyme that performs 12-lipoxygenation [44]; conversely, porcine leukocyte 12-lipoxygenase can be converted to a 15-lipoxygenase by mutation of two amino acids [42]. Nonetheless, there are a number of differences in the amino acids that define the specificity of lipoxygenase reactions between enzymes from different species [45]. The leukocyte-type 12S-lipoxygenase exhibits a rather broad substrate specificity. As shown in Table 2, the porcine and murine leukocyte-type 12S-lipoxygenases oxygenate not only various C-18 and C-20 free fatty acids but also complex substrates such as phospholipids and cholesterol esters of biomembranes and low density lipoproteins (LDL) [5], [6], [46]. This is in contrast to the platelet-type 12S-lipoxygenase which solely oxygenates arachidonic acid, and is almost inactive with C-18 and esterified polyenoic fatty acids [5], [10]. The epidermal 12S-lipoxygenase exhibits no or very low reactivity towards free arachidonic and linoleic acids, but metabolizes the corresponding fatty acid methyl esters [47]. Porcine leukocyte 12S-lipoxygenase has recently been reported to oxygenate 2-arachidonylglycerol, an endocannabinoid, but the platelet-type 12S-lipoxygenase is inactive with this compound [48]. The enzyme reaction catalyzed by the platelet and epidermal 12S-lipoxygenases proceeds almost linearly over 30min, while that of the leukocyte-type enzyme ceases within a few minutes due to suicide inactivation of the enzyme [4], [5], [47]. Self-inactivation of porcine leukocyte 12S-lipoxygenase is associated with a rapid and stoichiometric incorporation of 15-HPETE into the enzyme [49]. As described above, the 15-HPETE is generated as a minor product of the leukocyte-type 12S-lipoxygenase, and is then transformed to 14,15-leukotriene A4 which presumably reacts irreversibly with the enzyme protein. Covalent binding of the hydroperoxy product is not observed with the platelet-type enzyme [49]. It has recently been reported that mouse epidermal 12-lipoxygenase is inactivated by an antipsoriatic drug, anthralin, by the mechanism involving an active oxygen species formed during the auto-oxidation of the drug [50]. In an attempt to identify potential regulators of the 12S-lipoxygenase, specific interacting proteins such as type II keratin 5 and lamin A were isolated from human epidermoid carcinoma A431 cells by the yeast two-hybrid system [51]. Important roles of albumin are postulated in regulating the availability of substrates for platelet 12S-lipoxygenase and the metabolism of 12S-HETE [52], [53]. Namely, the in vitro metabolism of 12S-HETE by human leukocytes is effectively inhibited by the addition of physiological concentrations of albumin, probably by sequestration of the compound, suggesting that 12S-HETE is a long-lived substance in the circulation [52]. In accordance with these findings, putative receptors for 12S-HETE are found in human epidermal Langerhans cells [54] and Lewis lung carcinoma cells [55]. The latter binding sites have a cytosolic–nuclear localization and contain the heat shock proteins as components of a high molecular weight cytosolic-binding complex. The 50-kDa 12S-HETE-binding protein interacts as a homodimer with steroid receptor coactivator-1 (SRC-1) in the presence of 12S-HETE in Lewis lung carcinoma cells [56].