NITRIC-OXIDE SYNTHASE ISOZYMES - CHARACTERIZATION, PURIFICATION, MOLECULAR-CLONING, AND FUNCTIONS

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozy mes I (in neuronal and epithelial cells), II (in cytokine-induced cell s), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity . Across species, amino acid sequences for each isoform are well conse rved (> 90% for isoforms I and III, > 80% for isoform II). All isoform s use L-arginine and molecular oxygen as substrates and require the co factors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucl eotide, and flavin mononucleotide. They air bind calmodulin and contai n heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulati on of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO syntha se can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not r egulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzyme s and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is c onstitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endo thelial cells keeps blood vessels dilated, prevents the adhesion of pl atelets and white cells, and probably inhibits vascular smooth muscle proliferation.