Measurement of Sirtuin Enzyme Activity Using a Substrate-Agnostic Fluorometric Nicotinamide Assay
Measurement of Sirtuin Enzyme Activity Using a Substrate-Agnostic Fluorometric Nicotinamide Assay
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DOI：
10.1007/978-1-62703-637-5_11
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发表时间：
2013-01-01
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影响因子：
--
通讯作者：
Sinclair, David A.
中科院分区：
文献类型：
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作者：
Hubbard, Basil P.;Sinclair, David A.
Sirtuins是NAD(+)依赖的多功能赖氨酸脱酰酶，在细胞内稳态中发挥关键作用。它们越来越多地被发现针对各种底物，包括乙酰基、丁酰基、丙二酰基和琥珀酰赖氨酸。早期在体外测量sirtuin活性的方法受到批评，因为它们在所使用的多肽底物上使用荧光素，这可能会改变所获得的结果，并且不能代表体内的情况。我们描述了一种通过检测烟酰胺(NAM)的产生来测量sirtuin活性的新方法。该分析方法适用于任何底物和被sirtuins去除的任何修饰。该方法还可用于测定糖水解酶(如CD38)和ADP-核糖基转移酶活性(如MARTS和PAPS)。
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).