Molecular pathophysiological studies on arachidonate lipoxygenases

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Cyclooxygenase is well known asan enzyme responsible for the biosynthesis of prostaglandins and thromboxanes, and 5-lipoxygenase initiates the leukotriene synthesis. There are two similar enzyme with still unknown biological functions, i.e., 12-lipoxygenase and 15 -lipoxygenase. Catalytic properties and transcriptional regulation of these enzymes were investigated in collaboration with two German research groups.1) Transcriptional regulation of 12-lipoxygenase : As examined by the luciferase assay, a negative regulatory region including the NFkB motif was found in the 5'-flanking region of the 12-lipoxygenase gene in human erythroleukemia cells. The negative control was abolished by a site-specific mutation of the NFkB motif. Probes including the NFkB region gave positive bands upon a gel-shift assay. The bands were supershifted by antibodies for NFkB p50, NFkB p65 and c-Rel, and were lost by a NFkB competitor DNA.Furthermore, the NFkB sequence was protected in DNase I footprinting. Th … More us, two kinds of heterodimer (p50 and p65 ; p50 and c-Rel) seemed to control the over-expression of the human 12-lipoxygenase gene.2) Suicide inactivation of 12-and 15-lipoxygenases : Leukocyte 12-lipoxygenase showed a typical "suicide inactivation" during its catalysis, whereas platelet 12-lipoxygenase reaction proceeded almost linearly. The mechanism of the 12-lipoxygenase inactivation was investigated. The leukocyte enzyme produced a small amount of 15-hydroperoxy acid from arachidonic acid in addition to 12-hydroperoxy acid. The former product was further converted to 14,15-epoxy acid. The platelet enzyme produced a much less amount of 15-hydroperoxy acid. Several lines of evidence suggested that the 14,15-epoxy acid was covalently incorporated into the enzyme protein and the leukocyte 12-lipoxygenase was suicide-inactivated. 15-Lipoxygenase is now under investigations.3) 12-Lipoxygenase of human uterine cervix : Nigam's group of Free University of Berlin had found that human uterine cervix had 12-lipoxygenase activity which was lower in cancer cells. Last November Natsuo Ueda visited Free University in Berlin and confirmed the 12-lipoxygenase activity in human uterine cervix. Recently Sravan kumar from Berlin visited our laboratory in Tokushima for collaboration. The uterine tissue was homogenized, and its differential centrifugation showed the presence of the enzyme both in the high-speed supernatant and in the particles. The enzyme activity in the supernatant was immunoprecipitated by the use of anti-human platelet 12-lipoxygenase antibody. Results of Western blotting was agreeable with this finding. The enzyme was almost inactie with linoleic acid. These findings indicated that the 12-lipoxygenase of human uterine cervix was of platelet-type. Less