使用与底物无关的荧光烟酰胺测定法测量去乙酰化酶活性。
Measurement of sirtuin enzyme activity using a substrate-agnostic fluorometric nicotinamide assay.
作者信息
Hubbard Basil P, Sinclair David A
机构信息
Department of Genetics, Harvard Medical School, Boston, MA, USA.
出版信息
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).
摘要
沉默调节蛋白是依赖烟酰胺腺嘌呤二核苷酸（NAD⁺）的多功能赖氨酸脱酰基酶，在细胞内稳态中发挥关键作用。人们越来越多地发现它们作用于多种底物，包括乙酰赖氨酸、丁酰赖氨酸、丙二酰赖氨酸和琥珀酰赖氨酸。早期用于体外测量沉默调节蛋白活性的检测方法受到批评，因为其在所用肽底物上使用了荧光团，这可能会改变所得结果，且不能代表体内情况。我们描述了一种通过检测烟酰胺（NAM）的产生来测量沉默调节蛋白活性的新方法。该检测方法适用于任何底物以及沉默调节蛋白去除的任何修饰。该检测方法还可用于测量糖水解酶（如CD38）和ADP - 核糖基转移酶活性（如mARTs和PARP）。
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本文引用的文献
1
Evidence for a common mechanism of SIRT1 regulation by allosteric activators.
Science. 2013 Mar 8;339(6124):1216-9. doi: 10.1126/science.1231097.
2
Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.
Science. 2011 Nov 11;334(6057):806-9. doi: 10.1126/science.1207861.
3
Negative regulation of STAT3 protein-mediated cellular respiration by SIRT1 protein.
J Biol Chem. 2011 Jun 3;286(22):19270-9. doi: 10.1074/jbc.M110.200311. Epub 2011 Apr 5.
4
Catalysis and mechanistic insights into sirtuin activation.
Chembiochem. 2011 Jan 24;12(2):281-9. doi: 10.1002/cbic.201000434. Epub 2010 Nov 9.
5
Capillary electrophoresis-based sirtuin assay using non-peptide substrates.
J Pharm Biomed Anal. 2011 Mar 25;54(4):772-8. doi: 10.1016/j.jpba.2010.10.010. Epub 2010 Oct 21.
6
Structure-based mechanism of ADP-ribosylation by sirtuins.
J Biol Chem. 2009 Nov 27;284(48):33654-61. doi: 10.1074/jbc.M109.024521. Epub 2009 Sep 30.
7
A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide.
Anal Biochem. 2009 Dec 15;395(2):205-10. doi: 10.1016/j.ab.2009.08.011. Epub 2009 Aug 13.
8
A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.
Anal Biochem. 2009 Nov 1;394(1):101-9. doi: 10.1016/j.ab.2009.07.019. Epub 2009 Jul 16.
9
CBP/p300-mediated acetylation of histone H3 on lysine 56.
Nature. 2009 May 7;459(7243):113-7. doi: 10.1038/nature07861. Epub 2009 Mar 8.
10
Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2.
J Biol Chem. 2008 Feb 29;283(9):5317-26. doi: 10.1074/jbc.M707613200. Epub 2007 Dec 27.
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