FIGURE 3.

Sirt1 deacetylase inhibition by S-nitrosation is reversible in the presence of reductants and Zn²⁺. a, Sirt1 (2 μm) was treated with buffer or GSNO (100 μm) for 1 h at 37 °C, followed by incubation with DTT (10 mm) and/or ZnCl₂ (1 μm) for 5 min at 37 °C. Sirt1 activity (0.5 μm) was assessed via the enzyme-coupled assay. Data were plotted as the mean deacetylation rate for each condition, and significance was determined using a one-way ANOVA and Tukey post-test (n = 4 ± S.E.). b, Sirt1 (2.5 μm) was treated with buffer or GSNO (60 μm) for 30 min at 37 °C followed by incubation with DTT (5 mm) and ZnCl₂ (1–10 μm) for 5 min at 37 °C. Sirt1 activity (1 μm) was assessed via HPLC as detailed under “Experimental Procedures.” Data were plotted as the mean percent peptide deacetylation for each condition, and significance was determined via Student's t test (n = 3 ± S.E.). c, Sirt1 S-nitrosation is reversed by DTT. Sirt1 was treated with GSNO (100 μm) for 1 h at 37 °C, followed by immediate treatment with DTT (10 mm). The relative level of S-nitrosation was assessed via the biotin switch assay (n = 3, representative blot). d, Sirt1 inhibition by S-nitrosation is recovered by GSH. Sirt1 (2 μm) was treated with buffer or GSNO (100 μm) for 1 h at 37 °C, followed by incubation with varying concentrations of GSH and ZnCl₂ (1 μm) for 5 min at 37 °C. Sirt1 activity (0.5 μm) was assessed via the enzyme-coupled assay, and data were plotted as the mean deacetylation rate versus GSH concentration (n = 4 ± S.E.). e, Sirt1 S-nitrosation is reversed by GSH. Sirt1 was treated with GSNO (100 μm) for 1 h at 37 °C followed by immediate treatment with varying concentrations of GSH for 5 min at 37 °C. The relative level of S-nitrosation was assessed via the biotin switch assay. Full blot and loading control gel are shown in supplemental Fig. S3 (n = 3, representative blot). Values that are significantly different are indicated by bars and asterisks as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001.