Molecular pathophysiological studies on arachidonate lipoxygenases

环氧合酶是一种众所周知的酶，负责前列腺素和凝血酶的生物合成，5-脂氧合酶启动白三烯的合成。有两种类似的酶，生物学功能尚不清楚，即12-脂氧合酶和15 -脂氧合酶。这些酶的催化特性和转录调控是与两个德国研究小组合作研究的。1) 12-脂氧合酶的转录调控：通过荧光素酶检测，在人红细胞12-脂氧合酶基因的5'侧区发现了一个包含NFkB基序的负调控区。阴性对照被NFkB基序的位点特异性突变所消除。包括NFkB区域的探针在凝胶移位试验中呈现阳性带。这些条带被NFkB p50、NFkB p65和c-Rel抗体超移，并被NFkB竞争对手DNA丢失。此外，NFkB序列在DNase I足迹中受到保护。另外，两种异源二聚体（p50和p65， p50和c-Rel）似乎控制了人类12-脂氧合酶基因的过表达。2) 12-和15-脂氧合酶的自杀式失活：白细胞12-脂氧合酶在催化过程中表现出典型的“自杀式失活”，而血小板12-脂氧合酶的催化过程几乎呈线性进行。探讨了12-脂氧合酶失活的机理。白细胞酶从花生四烯酸中除产生12-氢过氧酸外，还产生少量的15-氢过氧酸。前产物进一步转化为14,15-环氧酸。血小板酶产生的15-羟基过氧酸的量要少得多。几条线索的证据表明，14,15-环氧酸共价结合到酶蛋白和白细胞12-脂氧合酶自杀失活。15-脂氧合酶目前正在研究中。3)人子宫颈的12-脂氧合酶：柏林自由大学Nigam课题组发现，人子宫颈的12-脂氧合酶活性在癌细胞中较低。去年11月，Natsuo Ueda访问了柏林自由大学，证实了人类子宫颈中的12-脂氧合酶活性。最近，来自柏林的Sravan kumar访问了我们在德岛的实验室进行合作。子宫组织匀浆，差速离心显示高速上清和颗粒中均存在酶。用抗人血小板12-脂氧合酶抗体对上清酶活性进行免疫沉淀。Western blotting的结果与这一发现一致。该酶对亚油酸几乎无活性。这些结果表明，人子宫颈的12-脂氧合酶为血小板型。少

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