ISOLATION/PURIFICATION MAMMALIAN PROSTACYCLIN SYNTHASE

前列环素合成酶催化分子内氧化还原反应

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.