Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme (endothelium-derived relaxing factor/arginine/cGMP)

Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neu- trophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citruline requires NADPH and Ca2 . We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affmity col- umn eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer. Endothelium-derived relaxing factor, a labile substance formed by endothelial cells, which mediates vasodilation, has been shown to be identical to nitric oxide (NO) (1-3). In addition to relaxing blood vessels, NO has multiple messen- ger functions as it has been demonstrated in macrophages (4) and in brain tissue (5-7). NO appears responsible for the cytotoxic effects of macrophages and neutrophils (8). We have obtained direct evidence for NO as a messenger for the influences of the excitatory amino acid glutamate on cGMP in the cerebellum (7). We showed a striking enhancement by glutamate and other excitatory amino acids of the conversion of arginine to NO and the associated formation of citrulline. Moreover we observed that Nw-monomethyl-L-arginine (MeArg), an inhibitor of the enzymatic conversion of arginine to NO, inhibits glutamate-elicited cGMP formation, an influ- ence selectively reversed by excess arginine. Evidence that NO mediates functions of tissues as diverse as the brain, endothelium, and blood cells suggests a wide- spread role for NO as a messenger molecule. Localizing NO formation at a cellular level throughout the body would be greatly facilitated by immunohistochemical identification of NO synthetase, the NO-forming enzyme. The mechanism of conversion of arginine to NO, presently unclear (9), would be greatly clarified by purification of NO synthetase. Charac- terization of this enzyme has been hampered by the complex assays required to assay the enzyme by monitoring NO formation directly. We have shown that NO formation is accompanied by the stoichiometric conversion of arginine to citrulline, permitting a simple, sensitive, and specific enzyme assay measuring the transformation of (3H)arginine to (3H)citrulline (7). Utilizing this assay in the present study, we have purified NO synthetase to homogeneity. We demon- strate that NO synthetase is a calmodulin-requiring enzyme, explaining numerous reports of a crucial role for calcium in endothelium-dependent smooth muscle relaxation.