FIG. 2.

Stimulation of Sir2 HDAC activity by Pnc1 in vitro. (A) HDAC assays were carried out with a Biomol fluorescent HDAC kit (see Materials and Methods). Each reaction mixture contained 1 μg (11.2 pmol) of GST-Sir2 and 200 μM NAD⁺ and was incubated for 30 min at 30°C. Where indicated, 50 μM NAM and 0.5 μg (17.8 pmol) of recombinant His₆-Pnc1 or C167A mutant Pnc1 was added to the reaction mixture. Activity for the Sir2-only reaction was set at 100%. (B) Sir2 activity measured over time in the absence of exogenously added NAM. The amount of activity at the 0.5-h time point without Pnc1 added was assigned an arbitrary value of 1.0. For later time points, the average fold increase in HDAC activity compared to the 0.5-h time point is plotted. (C) Sir2 activity measured over time in the presence of 7.5 μM NAM. Data are plotted in the same way as in panel B. Error bars represent the average deviation from three independent experiments.