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

以花生四烯酸为原料，经环氧合酶和脂氧合酶催化合成前列腺素、血栓素和白三烯等生物活性化合物。本研究旨在从细胞和分子水平研究这些酶的生理和病理动力学。以下是我们为研究目的而采取的方法。(1)酶的催化性质：对人血小板12-脂氧合酶进行免疫亲和纯化，并研究其底物特异性。(2)过氧化物酶联免疫分析法筛选酶分布：猪垂体前叶组织中检测到相当数量的12-脂氧合酶。(3)免疫组织化学方法筛选酶的分布：猪组织中，5-和12-脂氧合酶定位于粒细胞，而不定位于淋巴细胞。此外，肺和胰腺中存在5-脂氧合酶，垂体中存在12-脂氧合酶。(4)环氧合酶代谢物的检测：制备了抗11-脱氢血栓素B2的单抗。并用于其免疫亲和纯化和免疫分析。(5)酶的生理学和病理学动态：用抗环氧合酶抗体进行Western印迹和用环氧合酶基因的Northern印迹证实EGF、转化生长因子和肾上腺素对小鼠成骨细胞环氧合酶的诱导作用。(6)基因克隆：克隆了猪白细胞和人红白血病细胞12-脂氧合酶和人血小板环氧合酶的cDNAs，并根据这些cDNA的核苷酸序列推测了这些酶的一级结构。

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.