ISOLATION AND PURIFICATION OF PROSTACYCLIN SYNTHASE

PROSTACYCLIN SYNTHASE (PGI2 SYNTHASE) CATALYZES AN INTRAMOLECULAR REDOX REACTION IN WHICH PROSTAGLANDIN ENDOPEROXIDE, PGH2, IS CONVERTED TO PROSTACYCLIN, PGI2. THE POTENT BIOLOGICAL ACTIVITIES OF PGI2 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. PHASE I RESEARCH WILL PURIFY PROSTACYCLIN SYNTHASE FROM RAM SEMINAL VESICLES (RSV), DETERMINE SUITABLE CONDITIONS FOR ENZYME ASSAY AND STORAGE, AND DETERMINE COFACTOR REQUIREMENTS OF THE PURIFIED PROTEIN. THE PARTIALLY PURIFIED ENZYME WILL THEN BE MARKETED. IN PHASE II RESEARCH, AFFINITY CHROMATOGRAPHY AND IMMUNOAFFINITY CHROMATOGRAPHY WILL BE EMPLOYED TO PURIFY THEENZYME TO HOMOGENEITY. THE PURIFIED ENZYME WILL BE USED FORTHE PRODUCTION OF POLYCLONAL ANTIBODIES AND AMINO ACID SEQUENCING. POLYCLONAL ANTIBODIES AND AN OLIGONUCLEOTIDE PROBE BASED ON A PARTIAL AMINO ACID SEQUENCE WILL BE PREPARED AND USED AS REAGENTS FOR THE IDENTIFICATION, ISOLATION, AND MOLECULAR CLONING OF THE PROSTACYCLIN SYNTHASE GENE.