96 Assays, Storage: -80°C
A Fluor de LysTM Assay System
Yeast Sir2 (Silent information regulator 2)1 is the founding exemplar of the 'sirtuins', an apparently ancient group of enzymes that occurs in eukaryotes, the archaea and eubacteria2. In yeast3 and C. elegans4, added copies of sirtuin genes extend lifespan and Sir2 is required for the lifespan extension conferred by caloric restriction in yeast5. There are seven human sirtuins, which have been designated SIRT1-SIRT76. SIRT1, which is located in the nucleus, is the human sirtuin with the greatest homology to Sir2 and has been shown to exert a regulatory effect on p53 by deacetylation of lysine-3827-9. Dr. Konrad Howitz at BIOMOL carried out a screen for modulators of SIRT1 activity which yielded a number of small molecule activators, all of which were plant polyphenols. Several of these Sirtuin Activating Compounds (STACs) extended yeast lifespan in a way that mimicked caloric restriction. Resveratrol, the most potent of these STACs (see Fig. 1) activated SIRT1 in human cells and enhanced the survival rate of cells stressed by irradiation10.
The SIRT1 Fluorescent Activity Assay is based on the unique Fluor de Lys-SIRT1 Substrate/Developer II combination. The Fluor de Lys-SIRT1 Substrate is a unique peptide comprising amino acids 379-382 of human p53 (Arg-His-Lys-Lys(Ac)). The assay's fluorescence signal is generated in proportion to the amount of deacetylation of the lysine corresponding to Lys-382, a known in vivo target7-9 of SIRT1 activity. Fluor de Lys-SIRT1 was the substrate deacetylated most efficiently by SIRT1 from among a panel of substrates patterned on p53, histone H3 and histone H4 acetylation sites (see Fig. 2)10. Includes: Recombinant human SIRT1, Fluor de Lys-SIRT1 Substrate and Developer II, Assay Buffer, NAD+ solution, nicotinamide and suramin (SIRT1 inhibitors), resveratrol (SIRT1 activator), white and clear 1/2 vol. 96-well plates, and detailed instructions.
Fig. 1: Relative SIRT1 activity (25 µM peptide, 25 µM NAD+) in the absence (Control) or presence of 100 µM of various plant polyphenol sirtuin activating compounds (STACs).
Fig. 2: Deacetylation site preferences of recombinant SIRT1. Initial rates of deacetylation were determined for a series of fluorogenic acetylated peptide substrates based on short stretches of human histone H3, H4 and p53 sequence Recombinant human SIRT1 (1 µg), was incubated for 10 min at 37°C with 25 µM of the indicated fluorogenic acetylated peptide substrate and 500 µM NAD+. Reactions were stopped by the addition of 1 mM nicotinamide and the deacetylation-dependent fluorescent signal was determined.