FIGURE 4.

A, chemical structures of NAD⁺, ADP-ribose, and nicotinamide. B, the levels of covalent [³H]AziPm binding to human SIRT2 in solution are shown, as in Fig. 2B. However, in this experiment, SIRT2 was photolabeled with 4 μm [³H]AziPm ± propofol and also ± substrates (acetylated histones, ADP-ribose, and nicotinamide). Each mean was derived from 3–6 values from separate experiments. Two-way analysis of variance determined a significant effect of propofol and substrates on the means and their interaction (***, p < 0.001 for all comparisons). Bonferroni's post hoc tests comparing the indicated means determined that the substrates significantly increased [³H]AziPm binding (p < 0.001), and that this binding was inhibited by propofol (p < 0.001). ns, not significant. C, the levels of covalent [³H]AziPm binding to human SIRT1 in solution are shown. This experiment was performed identically to that shown in B, but with SIRT1 instead of SIRT2, and the means were from 3–5 values. Two-way analysis of variance did not determine a significant effect of propofol or substrates on the means or their interaction (p > 0.05). D, top, on the apo-SIRT1 structure, residues Tyr³¹⁷, Phe³⁶⁶, and Cys³⁸⁰, which are homologous to the SIRT2 residues that were photolabeled by AziPm, are shown as red sticks outlined by a transparent surface topology; zinc is colored orange. Bottom, enlarged view of the residues, but with the solid surface topology of the protein shown. E, identical views as in D, but with the structure of ADPr-SIRT1. Mean values are shown with S.E.