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Study on the physiological and pathological dynamics of cyclooxygenase and lipoxygenase enzymes

环氧合酶和脂氧合酶的生理病理动力学研究

基本信息

批准号：
01440025
负责人：
YAMAMOTO Shozo
金额：
$ 15.04万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

Bioactive compounds like prostaglandin, thromboxane and leukotriene are synthesized from arachidonic acid by cyclooxygenase and lipoxygenase. This research project is to investigate the physiological and pathological dynamics of these enzymes at cellular and molecular levels. The followings were our approaches for this research-purpose. (1) Catalytic properties of the enzymes : 12-Lipoxygenase of human platelets was immunoaffinity-purified, and its substrate specificity was studied. (2) Enzyme distribution screened by peroxidaselinked immunoassay : 12-Lipoxygenase was detected in a considerable quantity in porcine anterior pituitary. (3) Enzyme distribution screened by immunohistochemical methods : Among porcine tissues 5- and 12-lipoxygenases were localized in granulocytes but not in lymphocytes. In addition, 5-lipoxygenase was found in lung and pancrease, and 12-lipoxygenase in pituitary. (4) Assay of cyclooxygenase metabolites : A monoclonal antibody against 11-dehydro-thromboxane B2 was raised. and utilized for its inununoaffinity purification and immunoassay. (5) Physiological anc pathological dynamics of enzymes : Cyclooxygenase induction in mouse osteoblastic cells by EGF, TGF or epinephrine was confirmed by Western blotting using anti-cyclooxygenase antibody and by Northern blotting using cyclooxygense cDNA. (6) cDNA cloning : We cloned cDNAs for 12-lipoxygenases of porcine leukocytes and human erythroleukemia cells and cyclooxygenase of human platelets, and the primary structures of these enzymes were deduced from the nucleotide sequences of these cDNAs.

花生四烯酸通过环氧合酶和脂氧合酶合成前列腺素、血栓烷和白三烯等生物活性化合物。本研究项目旨在从细胞和分子水平研究这些酶的生理和病理动力学。以下是我们的研究方法。(1)酶的催化性质：免疫亲和纯化人血小板12-脂氧合酶，并研究其底物特异性。(2)过氧化物酶联免疫法检测酶的分布：猪垂体前叶中有大量的12-脂氧合酶。(3)酶分布免疫组化方法筛选：在猪组织5-和12-脂氧合酶定位于粒细胞，但不是在淋巴细胞。此外，在肺和胰腺中发现了5-脂氧合酶，在垂体中发现了12-脂氧合酶。(4)环氧合酶代谢产物的测定：制备了抗11-脱氢血栓烷B2的单克隆抗体。并用于其免疫亲和纯化和免疫测定。(5)酶的生理和病理动力学：EGF、TGF或肾上腺素在小鼠成骨细胞中的环氧合酶诱导通过使用抗环氧合酶抗体的Western印迹和使用环氧合酶cDNA的北方印迹来证实。(6)cDNA克隆：我们克隆了猪白细胞和人红白血病细胞的12-脂氧合酶和人血小板的环氧合酶的cDNA，并从这些cDNA的核苷酸序列推导出这些酶的一级结构。