Properties of prostacyclin synthase.
Prostacyclin synthase (PGIS) was isolated from bovine aortic microsomes after detergent solubilisation following purification by DEAE-Sephacel, immobilized metal affinity, and hydroxy apatite chromatography. The homogenous protein exhibited spectral characteristics of a heme-thiolate protein (P450) like the enzyme purified earlier from porcine microsomes and had an apparent mass of 52 kDa on SDS/PAGE. Three peptides from an endoproteinase Lys-C digest were isolated and sequenced. An antiserum was prepared from rabbits and purified by affinity chromatography. This allowed Western blots of microsomes from cultured endothelial cells. After treatment with IL-1 the activity of the cells in producing 6-keto-PGF1 alpha increased about threefold over 27 h which was accompanied by an increase in PGIS mass. A monoclonal antibody was used to set up an ELISA which served for the quantitation of PGIS in bovine tissues.
Ullrich
V
V
Brugger
R
R
Lottspeich
F
F
Siegle
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001011:Aorta / Q000201:enzymology*; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D016415:Sequence Alignment; D013552:Swine
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase