Journal: The Journal of Biological Chemistry

Article Title: Mechanism of Sirt1 NAD + -dependent Protein Deacetylase Inhibition by Cysteine S -Nitrosation *

doi: 10.1074/jbc.M116.754655

Figure Lengend Snippet: Sirt1 S-nitrosation of the Zn2+-tetrathiolate results in Zn2+ release. a, relative Sirt1 S-nitrosation levels were assessed for Sirt1 WT and Sirt1 tetrathiolate mutants (C371S, C374S, C395S, C398S, and C395S/C398S) treated with 100 μm GSNO for 1 h at 37 °C. Full blot and loading control gel are shown in supplemental Fig. S2. b, Sirt1 tetrathiolate mutants do not display deacetylase activity significantly above background. Deacetylase activity of Sirt1 WT and tetrathiolate mutants (1 μm) was assessed via continuous enzyme-coupled assay (n = 3 ± S.E.). c, Sirt1 S-nitrosation results in Zn2+ release. Sirt1 (7.5 μm) was treated with varying concentrations of GSNO in the presence of Zincon (40 μm). Rates of Zn2+ release were measured by following changes in absorbance at 529 and 620 nm for 30 min at 37 °C. Data were plotted as rate of Zn2+ release versus log10 of the GSNO concentration (n = 3 ± S.E.).

Article Snippet: Fmoc amino acids, reduced glutathione, oxidized glutathione, α-ketoglutaric acid, NADH, and Zincon monosodium were purchased from Chem-Impex (Wood Dale, IL).

Techniques: Histone Deacetylase Assay, Activity Assay, Concentration Assay