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

花生四烯酸经环加氧酶和脂加氧酶合成了前列腺素、血栓素和白三烯等生物活性化合物。本研究项目旨在研究这些酶在细胞和分子水平上的生理和病理动态。以下是我们的研究方法。(1)酶的催化性能：对人血小板12-脂氧合酶进行免疫亲和纯化，并对其底物特异性进行研究。(2)过氧化物酶联免疫法筛选酶的分布：猪垂体前叶中检测到大量的12-脂氧合酶。(3)免疫组织化学方法筛选的酶分布：5-和12-脂氧合酶在猪组织中定位于粒细胞，而不在淋巴细胞中。此外，肺和胰腺酶中检测到5-脂氧合酶，垂体酶中检测到12-脂氧合酶。(4)环加氧酶代谢产物测定：制备抗11-脱氢血栓素B2单克隆抗体。并将其用于免疫亲和纯化和免疫测定。(5)酶的生理和病理动态：EGF、TGF或肾上腺素对小鼠成骨细胞的环氧化酶诱导作用通过抗环氧化酶抗体的Western blotting和环氧化酶cDNA的Northern blotting证实。(6) cDNA克隆：克隆了猪白细胞和人红细胞的12脂加氧酶和人血小板的环加氧酶的cDNA，并根据核苷酸序列推断出这些酶的一级结构。

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.