Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase.
Although nitric oxide (NO) appears to be one of the oxidation products of L-arginine catalyzed by NO synthase (NOS; EC 1.14.13.39), past studies on the measurement of NO in cell-free enzymatic assays have not been based on the direct detection of the free NO molecule. Instead, assays have relied on indirect measurements of the stable NO oxidation products nitrite and nitrate and on indirect actions of NO such as guanylate cyclase activation and oxyhemoglobin oxidation. Utilizing a specific chemiluminescence assay, we report here that the gaseous product of L-arginine oxidation, catalyzed by both inducible macrophage and constitutive neuronal NOS, is indistinguishable from authentic NO on the basis of their physicochemical properties. NO gas formation by NOS was dependent on L-arginine, NADPH, and oxygen and inhibited by NG-methyl-L-arginine and cyanide anion. Superoxide dismutase (SOD) caused a marked, concentration-dependent increase in the production of free NO by mechanisms that were unrelated to the dismutation of superoxide anion or activation of NOS. These observations indicate that free NO is formed as a result of NOS-catalyzed L-arginine oxidation and that SOD enhances the generation of NO without directly affecting NO itself. SOD appears to elicit a novel biological action, perhaps accelerating the conversion of an intermediate in the L-arginine-NO pathway such as nitroxyl (HNO) to NO.
Hobbs
A J
AJ
Department of Pharmacology, University of California, School of Medicine, Los Angeles, 90024.
Fukuto
J M
JM
Ignarro
L J
LJ
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D002474:Cell-Free System; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D000594:Amino Acid Oxidoreductases