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Related Experiment Videos

No .NO from NO synthase

H H Schmidt1, H Hofmann, U Schindler

  • 1Clinical Biochemistry and Pathobiochemistry, Julius-Maximilians-Universität, Würzburg, Germany. schmidt@toxi.uni-wuerzburg.de

Proceedings of the National Academy of Sciences of the United States of America
|December 10, 1996
PubMed
Summary
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Nitric-oxide synthase (NOS) does not primarily produce nitric oxide (.NO). Superoxide dismutase (SOD) addition suggests NOS forms a precursor, like nitroxyl (NO-), which SOD then converts to .NO.

Area of Science:

  • Biochemistry
  • Enzymology
  • Nitric Oxide Biology

Background:

  • Nitric-oxide synthase (NOS) catalyzes L-arginine oxidation to L-citrulline and nitric oxide (.NO).
  • Current NOS activity analysis relies on indirect measurements like nitrite/nitrate or L-citrulline quantification.
  • Direct quantification of .NO formation from purified NOS is challenging.

Purpose of the Study:

  • To directly quantify nitric oxide (.NO) formation from purified nitric-oxide synthase (NOS).
  • To investigate the role of superoxide dismutase (SOD) in NOS activity assays.
  • To elucidate the primary reaction products and stoichiometry of NOS-catalyzed L-arginine turnover.

Main Methods:

  • Direct quantification of .NO using two independent methods.
  • Experiments involving purified NOS, tetrahydrobiopterin (H4Bip), L-arginine (Arg), NADPH, and superoxide dismutase (SOD).

Related Experiment Videos

  • Analysis of NOS-derived products including nitrous oxide (N2O) and hydroxylamine (NH2OH), and S-nitrosoglutathione in the presence of glutathione.

    • Main Results:

    • Purified NOS did not produce detectable .NO without SOD.
    • SOD presence affected internal .NO standard recovery and NADPH consumption, suggesting scavenging or alternative pathways.
    • Detection of NOS-derived N2O and NH2OH, and S-nitrosoglutathione indicated formation of an .NO precursor, likely nitroxyl (NO-).

    Conclusions:

    • Nitric oxide (.NO) is not the primary product of NOS-catalyzed L-arginine turnover.
    • Superoxide dismutase (SOD) likely converts a NOS-derived precursor, such as nitroxyl (NO-), into .NO.
    • Alternative reaction mechanisms and stoichiometry for NOS activity need consideration.