Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction
Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction
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DOI：
10.1016/j.ab.2004.05.039
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发表时间：
2004-09-01
影响因子：
2.9
通讯作者：
Glaser, KB
中科院分区：
文献类型：
--
作者：
Marcotte, PA;Richardson, PR;Glaser, KB
设计了一种新的荧光底物，用于sirtuin（SIRT）类人蛋白质脱乙酰酶，该酶由含有单个乙酰基赖氨酸残基的肽序列组成。在羧基末端附近具有荧光基团（四甲基罗丹明-6-羧酸，6-塔姆拉），在氨基末端附近具有非荧光猝灭基团（QSY-7）。肽序列模仿p53乙酰化位点，但对胰蛋白酶不反应，因为所有其他赖氨酸和精氨酸残基已被丝氨酸取代。然而，SIRT-脱乙酰化肽容易被胰蛋白酶切割，导致6-塔姆拉荧光的最大30倍增强。在毫摩尔浓度的烟酰胺停止脱乙酰化，但不抑制胰蛋白酶，和微量滴定板测定的SIRT已被设计使用的荧光底物和这些试剂。利用该方法，分析了共底物烟酰胺腺嘌呤二核苷酸和竞争性抑制剂烟酰胺与SIRT 1和SIRT 2反应的动力学。几种烟酰胺类似物也已被测试为抑制剂，并发现对这些酶的亲和力比母体化合物低得多。(C)2004年爱思唯尔公司All rights reserved.
A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue., with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence. Nicotinamide at millimolar concentrations stops the deacetylation but does not inhibit trypsin, and a microtiter plate assay of the SIRTs has been devised using the fluorescent substrate and these reagents. Using this method, the kinetics of the reaction of the cosubstrate nicotinamide adenine dinucleotide and the competitive inhibitor nicotinamide with SIRT1 and SIRT2 has been analyzed. Several nicotinamide analogs have also been tested as inhibitors and found to have much lower affinity for these enzymes than does the parent compound. (C) 2004 Elsevier Inc. All rights reserved.