FIGURE 1.

SIRT6 deacetylation assays. A, charcoal-binding assay measuring the production of OAADPr. All assays were carried out in the presence of 4 μm SIRT6 WT (●) or H131Y (■), 300 μm [³H]H3K9Ac peptide, and 600 μm NAD⁺. Shown is the average of three experiments for WT and H131Y. The rate of OAADPr production was 3.2 ± 0.2 μm/h based on linear regression analysis with an R² of 0.98612. B, HPLC separation of ³H-labeled products monitoring deacetylation of a [³H]H3K9Ac peptide. Reactions were carried out in the presence of 300 μm [³H]H3K9Ac peptide, 2 mm NAD⁺, and either 4 μm SIRT6, 4 μm Hst2 or no enzyme. Shown are all counts for the Hst2 deacetylation reaction (green), SIRT6 (brown), and a no enzyme control (blue) as well as a zoomed in view displaying counts for [³H]acetate, [³H]OAADPr, and H3K9 ([³H]acetylated N terminus). C, continuous assay monitoring the release of nicotinamide over time. Shown is the average of three trials with assays carried out in the presence of 8 μm (gray) or 0 μm (white) SIRT6, 300 μm H3K9Ac, and 0.6 mm NAD⁺. The rate of nicotinamide release in the presence of SIRT6 was 10.5 ± 0.4 μm/h and the rate in its absence was 5.9 ± 0.8 μm/h. Error bars, S.D.