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

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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.