PURIFICATION AND CHARACTERIZATION OF PARTICULATE ENDOTHELIUM-DERIVED RELAXING FACTOR SYNTHASE FROM CULTURED AND NATIVE BOVINE AORTIC ENDOTHELIAL-CELLS
PURIFICATION AND CHARACTERIZATION OF PARTICULATE ENDOTHELIUM-DERIVED RELAXING FACTOR SYNTHASE FROM CULTURED AND NATIVE BOVINE AORTIC ENDOTHELIAL-CELLS
复制标题
DOI：
10.1073/pnas.88.23.10480
复制
发表时间：
1991-12-01
影响因子：
11.1
通讯作者：
MURAD, F
中科院分区：
文献类型：
--
作者：
POLLOCK, JS;FORSTERMANN, U;MURAD, F
负责合成内皮来源的放松因子的颗粒酶已从培养和天然（非培养）牛主动脉内皮细胞中纯化。对溶解颗粒酶制备物采用2′，5′-二磷酸腺苷偶联Sepharose亲和层析纯化，然后采用Superose 6凝胶过滤层析纯化，变性聚丙烯酰胺凝胶电泳后得到一条蛋白质条带，几乎等于135 kDa。通过对大鼠胎肺成纤维细胞（RFL-6细胞）可溶性瓜氨酸环化酶的刺激作用、l -精氨酸生成l -瓜氨酸、测定亚硝酸盐/硝酸盐的生成以及对内皮剥离血管条的生物测定来测定各组分的酶活性。从培养的牛主动脉内皮细胞粗颗粒中纯化了3419倍的内皮来源的松弛因子合成酶，回收率为12% （RFL-6法）。纯化的内皮衍生的放松因子合成酶需要l -精氨酸、NADPH、Ca2+、钙调素和5,6,7,8-四氢生物蝶呤才能充分发挥活性。
The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to almost-equal-to 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation Of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.