Measurement of sirtuin enzyme activity using a substrate-agnostic fluorometric nicotinamide assay

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...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1077; p. 167
Main Authors Hubbard, Basil P, Sinclair, David A
Format Journal Article
LanguageEnglish
Published United States 01.01.2013
Subjects
Acetylation
Fluorometry - methods
Humans
Kinetics
Lysine - metabolism
Mitochondrial Proteins - metabolism
NAD - metabolism
Nucleotide Transport Proteins - metabolism
Peptide Fragments - metabolism
Sirtuin 1 - metabolism
Substrate Specificity
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Summary: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).
ISSN:1940-6029
DOI:10.1007/978-1-62703-637-5_11