ISOLATION AND MOLECULAR-CLONING OF PROSTACYCLIN SYNTHASE FROM BOVINE ENDOTHELIAL-CELLS

Prostacyclin synthase catalyzes the conversion of prostaglandin H-2 to prostacyclin, which is a powerful vasodilator and the most potent nat ural occurring in hibitor of platelet aggregation. In the present stud y, we determined the amino acid sequence of bovine prostacyclin syntha se by combined protein chemical and molecular cloning techniques. The enzyme was purified and characterized from bovine aorta microsomes, an d the partial amino acid sequences were determined with the native enz yme and endoproteinase Lys-C-cleaved peptides. Using primers synthesiz ed according to the amino acid sequences, cDNA coding for prostacyclin synthase was amplified by polymerase chain reaction with bovine endot helial cell poly(A)(+) RNA and cloned into pBluescript II. Nucleotide sequence analyses of the cloned cDNA inserts revealed that cDNA for th is enzyme contained a 1500-base pair open reading frame coding for a 5 00-amino acid polypeptide with a M(r) of 56,628. COS-7 cells transfect ed with an expression plasmid harboring this cDNA clone expressed pros tacyclin synthase activity. The primary structure of the enzyme showed structural characteristics of cytochrome P450 and exhibited a 32% ide ntity to that of human cholesterol 7 alpha-hydroxylase. However, the i dentity between the amino acid sequences of bovine prostacyclin syntha se and human thromboxane synthase was only 16%, and no P450 showed an identity higher than 40%, suggesting that prostacyclin synthase repres ents a new family in the P450 superfamily. RNA blot analysis indicated that the mRNA for prostacyclin synthase from bovine endothe lial cell s showed a size of approximately 2.7 kilobases and that the mRNA level increased about 3-fold by treat ment of tumor necrosis factor-alpha.