Department of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People's Republic of China.
Anal Biochem. 2009 Dec 15;395(2):205-10. doi: 10.1016/j.ab.2009.08.011. Epub 2009 Aug 13.
Sirtuins are nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD(+) after the deacetylation was determined by converting NAD(+) to a highly fluorescent cyclized alpha-adduct compound. By this assay, we found that nicotinamide, Cu(2+), and Zn(2+) antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.
去乙酰化酶 Sirtuins 是烟酰胺腺嘌呤二核苷酸 (NAD(+)) 依赖性脱乙酰酶，可催化组蛋白和 p53 等蛋白质的脱乙酰化。本文开发了一种灵敏、便捷的荧光法来评估 SIRT1 的酶活性。具体来说，通过将 NAD(+) 转化为高荧光环化的 α-加合物化合物，来测定脱乙酰化后剩余的 NAD(+)。通过该测定法，我们发现烟酰胺、Cu(2+) 和 Zn(2+) 拮抗 SIRT1 的活性。白藜芦醇可特异性地通过 7-氨基-4-甲基香豆素 (AMC)-标记的乙酰化肽来刺激酶活性。表没食子儿茶素没食子酸酯 (EGCG) 可抑制 SIRT1 活性，无论是 AMC 标记的肽还是非标记的肽。然而，维生素 C 与 EGCG 的组合可逆转 EGCG 对非标记肽的抑制作用，或刺激 SIRT1 对 AMC 标记肽的去乙酰化作用。该测定法不需要任何同位素物质，因此在生物学上是安全的。它可以适应 96 孔微孔板进行高通量筛选。值得注意的是，该测定法可使用带有或不带有荧光标记的乙酰化肽，这有利于体外研究 SIRT1 激活剂或抑制剂的底物特异性。
