Ullrich V, Brugger R, Lottspeich F, Siegle I
Adv Exp Med Biol. 1997;400A:113-9. doi: 10.1007/978-1-4615-5325-0_16.
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.
前列环素合酶（PGIS）是在通过DEAE-琼脂糖凝胶、固定化金属亲和层析和羟基磷灰石层析进行纯化后，经去污剂增溶从牛主动脉微粒体中分离得到的。这种纯蛋白表现出一种血红素硫醇盐蛋白（P450）的光谱特征，就像早期从猪微粒体中纯化得到的酶一样，并且在SDS/PAGE上的表观质量为52 kDa。从一种内肽酶Lys-C消化产物中分离出三个肽段并进行了测序。用兔子制备了抗血清，并通过亲和层析进行纯化。这使得能够对培养的内皮细胞的微粒体进行蛋白质免疫印迹分析。用白细胞介素-1处理后，细胞产生6-酮-PGF1α的活性在27小时内增加了约三倍，同时PGIS的量也增加了。使用单克隆抗体建立了一种酶联免疫吸附测定法（ELISA），用于定量牛组织中的PGIS。
