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Measurement of Sirtuin Enzyme Activity Using a Substrate-Agnostic Fluorometric Nicotinamide Assay

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Sirtuins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1077))

Abstract

The sirtuins are NAD+-dependent, multifunctional lysine deacylases that play key roles in cellular homeostasis. They are increasingly being found to target a variety of substrates including acetyl-, butyryl-, malonyl-, and succinyl-lysines. Early assays for measuring sirtuin activity in vitro were criticized for their use of fluorophores on the peptide substrates used, which may alter the results obtained and not be representative of the in vivo situation. We describe a new protocol for the measurement of sirtuin activity by detecting the production of nicotinamide (NAM). The assay is amenable to any substrate and any modification removed by sirtuins. The assay may also be used to measure glycohydrolase (e.g., CD38) and ADP-ribosyltransferase activity (e.g., mARTs and PARPs).

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Acknowledgements

This work was supported by an NSERC PGS-D Fellowship to B.P.H., the Paul F. Glenn Foundation for Medical Research, the United Mitochondrial Disease Foundation, The Juvenile Diabetes Research Foundation, and NIA/NIH grants to D.A.S. B.P.H. and D.A.S. are inventors on a patent licensed to Millipore. D.A.S. is a consultant to GlaxoSmithKline.

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Basil P. Hubbard & David A. Sinclair

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  1. Basil P. Hubbard
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  2. David A. Sinclair
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  1. Duke University Medical Center Institute of Molecular Physiology, Durham, North Carolina, USA

    Matthew D. Hirschey

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Hubbard, B.P., Sinclair, D.A. (2013). Measurement of Sirtuin Enzyme Activity Using a Substrate-Agnostic Fluorometric Nicotinamide Assay. In: Hirschey, M. (eds) Sirtuins. Methods in Molecular Biology, vol 1077. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-637-5_11

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  • DOI: https://doi.org/10.1007/978-1-62703-637-5_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-636-8

  • Online ISBN: 978-1-62703-637-5

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