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A Microtiter-Plate Assay of Human NOS Isoforms

  • John Dawson
  • Richard G. Knowles
Part of the Methods in Molecular Biology™ book series (MIMB, volume 100)

Abstract

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP+ (reviewed in ref. 1). The neuronal (n) and endothelial (e) NOS isozymes are highly regulated by Ca2+ and calmodulin (CaM), whereas the iNOS has CaM tightly bound. NOS are heme proteins that also contain tightly bound flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and require tetrahydrobiopterin (BH4) for activity, whereas NADPH is used as a substrate.

Keywords

Extraction Buffer Flavin Adenine Dinucleotide Nicotinamide Adenine Dinucleotide Phosphate Flavin Adenine Dinucleotide Flavin Mononucleotide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249–258.PubMedGoogle Scholar
  2. 2.
    Feelisch, M., Kubitzek, D., and Werringloer, J. (1996) The oxyhaemoglobin assay, in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, pp. 472–473.Google Scholar
  3. 3.
    Gross, S. S., Jaffe, E. A., Levi, R., and Kilbourn, R. G. (1991) Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-dependent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages. Biochem. Biophys. Res. Comm. 178(3), 823–829PubMedCrossRefGoogle Scholar
  4. 4.
    Stuehr, D. J. and Griffith, O. W. (1996) Purification, assay and properties of mammalian nitric oxide synthases in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, p. 185.Google Scholar
  5. 5.
    Stuehr, D. J. and Griffith, O. W. (1996) Purification, assay and properties of mammalian nitric oxide synthases, in Methods in Nitric Oxide Research (Feelisch, M. and Stamler, J. S., eds.), Wiley, New York, pp. 183–184.Google Scholar
  6. 6.
    Charles, I. C., Scorer, C. A., Angeles Moro, M., Fernandez, C. Chubb, A., Dawson, J., Foxwell, N., Knowles, R. G., and Baylis, S. A. (1996) Expression of the three human NO synthase isozymes. Methods Enzymol. 268, 449–460PubMedCrossRefGoogle Scholar
  7. 7.
    Komori, Y., Hyun, J., Chiang, K., and Fukuto, J. M. (1995). The role of thiols in the apparent activation of rat brain nitric oxide synthase (NOS). J. Biochem 117, 923–927PubMedGoogle Scholar
  8. 8.
    Hofmann, H. and Schmidt, H. H. H. W. (1995). Thiol dependence of nitric oxide synthase. Biochemistry 34(41), 13,443–13,452.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • John Dawson
    • 1
  • Richard G. Knowles
    • 2
  1. 1.Glaxo-Wellcome Research and DevelopmentStevenageUK
  2. 2.Enzyme Pharmacology GroupGlaxo-Wellcome ResearchStevenageUK

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