Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.

摘要: Abstract 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 solubilized preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed Superose 6 gel filtration resulted in a single protein band after denaturing polyacrylamide electrophoresis that corresponded approximately 135 kDa. The activity various fractions was assayed its stimulatory effect soluble guanylyl cyclase rat fetal lung fibroblasts (RFL-6 cells), formation L-citrulline L-arginine, measuring nitrite/nitrate formation, bioassay endothelium-denuded vascular strips. Endothelium-derived synthase 3419-fold crude fraction cells with 12% recovery assay). Purified required NADPH, Ca2+, calmodulin, 5,6,7,8-tetrahydrobiopterin full activity.