Particulate and soluble bovine endothelial nitric oxide synthases are structurally similar proteins yet different...

In cultured bovine aortic endothelial cells (BAECs), 95% of the total endothelial nitric oxide (NO) synthase (type III) activity was found in the particulate fraction and only 5% was found in the soluble fraction. The soluble and particulate endothelial NO synthase activities behaved similarly on anion-exchange and gel filtration chromatography, whereas the soluble brain NO synthase (type I) had chromatographic properties different from the type III endothelial NO synthases. We have purified the particulate endothelial NO synthase from cultured and native BAECs using affinity chromatography on 2',5'-ADP Sepharose followed by Superose 6 gel filtration chromatography. Subsequently, monoclonal antibodies were generated against the purified particulate endothelial NO synthase. In protein immunoblotting analyses, crude and partially purified samples of particulate and soluble type III endothelial NO synthase demonstrated a single band at a molecular mass of 135 kDa with monoclonal antibody (MAb) H32. Purified type I soluble brain NO synthase did not cross-react with MAb H32. These data indicate that the soluble and particulate endothelial NO synthase are structurally similar proteins and represent an isozyme that can be distinguished from the brain NO synthase.