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

Methods Mol Biol. 2013;1077:167-77. doi: 10.1007/978-1-62703-637-5_11.

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).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Fluorometry / methods*
  • Humans
  • Kinetics
  • Lysine / metabolism*
  • Mitochondrial Proteins / metabolism*
  • NAD / metabolism*
  • Nucleotide Transport Proteins / metabolism*
  • Peptide Fragments / metabolism*
  • Sirtuin 1 / metabolism*
  • Substrate Specificity

Substances

  • Mitochondrial Proteins
  • Nucleotide Transport Proteins
  • Peptide Fragments
  • SLC25A33 protein, human
  • NAD
  • SIRT1 protein, human
  • Sirtuin 1
  • Lysine