A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide |
| |
Authors: | Yu Feng Jiahui Wu Lei Chen Chen Luo Xu Shen Kaixian Chen Hualiang Jiang Dongxiang Liu |
| |
Affiliation: | aDepartment of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China;bCenter for Drug Design and Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China |
| |
Abstract: | 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 α-adduct compound. By this assay, we found that nicotinamide, Cu2+, and Zn2+ 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. |
| |
Keywords: | Sirtuins NAD+ Deacetylation Fluorescence Cyclized α -adduct |
本文献已被 ScienceDirect 等数据库收录! |
|