Abstract
We describe a fluorescence assay for nitric oxide synthase activity based on a new indicator, 4,5-diaminofluorescein (DAF-2). The method offers the advantage of being safer and more convenient than the citrulline radioassay in common use. The rapid and irreversible binding of DAF-2 to oxidized nitric oxide (NO) enables NO production to be measured in real time. The protocol is applied to the measurement of nitric oxide synthase in crude extracts of skeletal muscle.
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References
Hevel, J. M. and Marletta, M. A. (1994) Nitric-oxide synthase assays. Methods Enzymol. 233, 250–258.
Reiser, P. J., Kline, W. O., and Vaghy, P. L. (1997) Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82, 1250–1255.
Sutherland, H., Khundkar, R., Zolle, O., et al. (2001) A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle. Nitric Oxide 5, 475–481.
Kojima, H., Nakatsubo, N., Kikuchi, K., et al. (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem. 70, 2446–2453.
Nakatsubo, N., Kojima, H., Kikuchi, K., et al. (1998) Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett. 427, 263–266.
Nagano, T. and Yoshimura, T. (2002) Bioimaging of nitric oxide. Chem. Rev. 102, 1235–1270.
Kobzik, L., Stringer, B., Balligand, J.-L., et al. (1995) Endothelial type nitric oxide synthase in skeletal muscle fibers: mitochondrial relationships. Biochem. Biophys. Res. Commun. 211, 375–381.
Frandsen, U., Lopez-Figueroa, M., and Hellsten, Y. (1996) Localization of nitric oxide synthase in human skeletal muscle. Biochem. Biophys. Res. Commun. 227, 88–93.
Thompson, M., Becker, L., Bryant, D., et al. (1996) Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle. J. Appl. Physiol. 81, 2415–2420.
Southam, E. and Garthwaite, J. (1991) Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices. Neurosci. Lett. 130, 107–111.
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© 2004 Humana Press Inc.
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Sutherland, H., Zolle, O., Khundkar, R., Simpson, A.W.M., Jarvis, J.C., Salmons, S. (2004). A Nonradioactive Assay for Nitric Oxide Synthase Activity in Tissue Extracts. In: Hassid, A. (eds) Nitric Oxide Protocols. Methods in Molecular Biology™, vol 279. Humana Press. https://doi.org/10.1385/1-59259-807-2:105
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DOI: https://doi.org/10.1385/1-59259-807-2:105
Publisher Name: Humana Press
Print ISBN: 978-1-58829-237-7
Online ISBN: 978-1-59259-807-6
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