Isolation and molecular cloning of prostacyclin synthase from bovine endothelial cells.
Prostacyclin synthase catalyzes the conversion of prostaglandin H2 to prostacyclin, which is a powerful vasodilator and the most potent natural occurring inhibitor of platelet aggregation. In the present study, we determined the amino acid sequence of bovine prostacyclin synthase by combined protein chemical and molecular cloning techniques. The enzyme was purified and characterized from bovine aorta microsomes, and the partial amino acid sequences were determined with the native enzyme and endoproteinase Lys-C-cleaved peptides. Using primers synthesized according to the amino acid sequences, cDNA coding for prostacyclin synthase was amplified by polymerase chain reaction with bovine endothelial cell poly(A)+ RNA and cloned into pBluescript II. Nucleotide sequence analyses of the cloned cDNA inserts revealed that cDNA for this enzyme contained a 1500-base pair open reading frame coding for a 500-amino acid polypeptide with a M(r) of 56,628. COS-7 cells transfected with an expression plasmid harboring this cDNA clone expressed prostacyclin synthase activity. The primary structure of the enzyme showed structural characteristics of cytochrome P450 and exhibited a 32% identity to that of human cholesterol 7 alpha-hydroxylase. However, the identity between the amino acid sequences of bovine prostacyclin synthase and human thromboxane synthase was only 16%, and no P450 showed an identity higher than 40%, suggesting that prostacyclin synthase represents a new family in the P450 superfamily. RNA blot analysis indicated that the mRNA for prostacyclin synthase from bovine endothelial cells showed a size of approximately 2.7 kilobases and that the mRNA level increased about 3-fold by treatment of tumor necrosis factor-alpha.
Hara
S
S
Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka, Japan.
Miyata
A
A
Yokoyama
C
C
Inoue
H
H
Brugger
R
R
Lottspeich
F
F
Ullrich
V
V
Tanabe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics* / Q000302:isolation & purification; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018076:DNA, Complementary; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase