Development by Gene Engineering of Kits for Screening the Enzymes of the Arachidonate Cascade

点击翻译按钮获取中文摘要

A variety of bioactive compounds are produced in a metabolic pathway referred to as the arachidonate cascade. Several key enzymes of the cascade must be studied to elucidate the physiological and pathological roles of these compounds. These enzymes are also the targets in the development of various drugs. The purpose of this research project is the development of the assay kits for the arachidonate cascade enzymes by gene engineering techniques.1) Cloning of cDNAs for the arachidonate cascade enzymes: In addition to previous cDNA clonings for 5- and 12-lipoxygenases, cyclooxygenase and leukotriene A hydrolase, we cloned cDNAs for human platelet and vascular endothelial cyclooxygenases and for human platelet thromboxane A synthase.2) Expression of the enzyme cDNAs: 5-Lipoxygenase cDNA was expressed in E. coli and yeast, 12-lipoxygenase in E. coli, thromboxane A synthase in insect cells and leukotriene A hydrolase in E. coli.3) Quantitative assay of the enzyme mRNA: By the use of 12-lipoxygenase cDNA as a probe, the 12-lipoxygenase mRNA in human platelets was PCR-amplified and quantitated with a cRNA as an internal standard. The assay was applied to leukemia patients with reduced 12-lipoxygenase activity.4) Enzyme immunoassays for the arachidonate cascade enzymes: A peroxidase-linked immunoassay was developed for human platelet 12-lipoxygenase and applied to the leukemia patients with 12-lipoxygenase deficiency. An enzyme immunoassay was also set up for leukotriene A hydrolase.