ISOLATION/PURIFICATION MAMMALIAN PROSTACYCLIN SYNTHASE

前列环素合酶催化分子内氧化还原反应，其中 前列腺素内过氧化物PGH2转化为前列环素。 的 前列环素的强有力的生物活性表明了其重要作用， 前列环素合酶在血栓形成的病理生理学中的作用， 心血管疾病 纯化的 因此，哺乳动物前列环素合酶的制备应该 促进基础和药物研究，包括 前列环素合酶基因调控因子 表达，开发用于治疗 心血管疾病和 制定新的战略， 血栓栓塞发作的预防和管理。 在第二阶段，我们将纯化前列环素合酶， 关于ram seminal 使用各种方法，包括离子， 交换层析，亲和层析，等电聚焦 和凝胶渗透色谱法。 纯化的蛋白质将用于 产生多聚抗体和单克隆抗体。 多克隆抗体 将与一个GT11表达文库结合使用， 隔离 前列环素合酶特异性cDNA序列。 这些 然后将cDNA序列用于产生DNA和RNA探针。 将尝试使用开发的单克隆抗体， 大规模单步纯化方法。 纯化的酶， 抗体和核酸探针将在牛津市售 生物医学研究。

Prostacyclin synthase catalyzes an intramolecular redox reaction in which prostaglandin endoperoxide, PGH2, is converted to prostacyclin. The potent biological activities of prostacyclin indicate an important role for prostacyclin synthase in the pathophysiology of thrombosis and cardiovascular diseases. Commercial availability of a purified preparation of mammalian prostacyclin synthase should therefore facilitate basic and pharmaceutical research, including studies of factors involved in the regulation of prostacyclin synthase gene expression, development of novel therapeutic agents for the treatment of cardiovascular diseases, and development of new strategies for the prevention and management of thromboembolic episodes. In Phase II, we will purify prostacyclin synthase from ram seminal vesicles to homogeneity using a variety of methodologies including ion- exchange chromatography, affinity chromatography, isoelectric focusing and gel permeation chromatography. The purified protein will be used to raise both poly- and monoclonal antibodies. The polyclonal antibodies will be used in conjunction with a lambda-gt11 expression library to isolate cDNA sequences specific for prostacyclin synthase. These cDNA sequences will then be used to produce DNA and RNA probes. Attempts will be made to use the monoclonal antibodies developed in a large scale, single step purification procedure. The purified enzyme, the antibodies and the nucleic acid probes will be marketed Oxford Biomedical Research.