Aj. Hobbs et al., FORMATION OF FREE NITRIC-OXIDE FROM L-ARGININE BY NITRIC-OXIDE SYNTHASE - DIRECT ENHANCEMENT OF GENERATION BY SUPEROXIDE-DISMUTASE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(23), 1994, pp. 10992-10996
Although nitric oxide (NO) appears to be on of the oxidation products
of L-arginine catalyzed by NO synthase (NOS; EC 1.14.13.39), past stud
ies on the measurement of NO in cell-free, enzymatic assays have not b
een based on the direct detection of the free NO molecule. Instead, as
says have relied on indirect measurements of the stable NO oxidation p
roducts nitrite and nitrate and on indirect actions of NO such as guan
ylate cyclase activation and oxyhemoglobin oxidation. Utilizing a spec
ific chemiluminescence assay, we report here that the gaseous product
of L-arginine oxidation, catalyzed by both inducible macrophage and co
nstitutive neuronal NOS, is indistinguishable from authentic NO on the
basis of their physicochemical properties. NO gas formation by NOS wa
s dependent on L-arginine, NADPH, and oxygen and inhibited by N-G-meth
yl-L-arginine and cyanide anion. Superoxide dismutase (SOD) caused a m
arked, concentration-dependent increase in the production of free NO b
y mechanisms that were unrelated to the dismutation of superoxide anio
n or activation of NOS. These observations indicate that free NO is fo
rmed as a result of NOS-catalyzed L-arginine oxidation and that SOD en
hances 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 nit
roxyl (HNO) to NO.