In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 94, No. 23 ( 1997-11-11), p. 12705-12710
Abstract:
Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO − ) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe 2+ - N -methyl- d -glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and a N = 12.7 G, whereas NO − is undetectable. In the presence of l -arginine ( l -Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N -nitro- l -arginine methyl ester. Isotope-labeling experiments with l -[ 15 N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of l -Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of l -citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from l -Arg.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.94.23.12705
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
1997
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
