Figure 1. ATP inhibits Sirt1 activity.

(a) Treatment with 2-deoxyglucose (2-DG) activates Sirt1. 293 HEK cells were co-transfected with the expression vectors for p53 and either HY or WT Sirt1 as indicated. Twenty-four hours later, each plate was divided into two plates and further cultured. Forty-eight hours after transfection, cells were incubated with either 25 mM Glucose (Glu) or 25 mM 2-DG for 2 h. The acetylation status of K382 in p53 was evaluated by immunoblotting with an antibody specific for acetylated K382. The experiment shown in a was repeated using FLAG-tagged p65 protein (a component of NF-κB) (b) and FLAG-tagged acetyl CoA synthestase1 (ACS1) (c). The acetylation of p65 and ACS1 were assessed by immunoprecipitation with FLAG antibody followed by immunoblotting with antibody specific for acetylated K310 for p65 and acetylated lysine antibody for acetylated ACS1. (d) Cells were treated with media containing either 25 mM Glu or 2-DG for 2 h and the intracellular levels of ATP, ADP and AMP were measured by HPLC. (e) The intracellular levels of NAD⁺ after Glu or 2-DG treatment were determined by HPLC (n=4). The HPLC chromatogram is shown in Supplementary Fig. 1a. (f) ATP inhibits Sirt1 activity. Deacetylation of Ac-p53 by recombinant Sirt1 in the presence of 0–8 mM ATP. Deacetylation in the absence of NAD⁺ is shown as a negative control. (g) Sirt1 activity against Ac-H4 as a function of ATP (0–10 mM) (n=4). Results are presented as % of control (0 mM ATP). (h) Sirt1 activity against Ac-H4 in the presence of either 1 or 5 mM of ATP, AMP, adenosine, CTP and UTP as a percentage of the Sirt1 activity in the absence of the nucleotides (n=4). (i) Lineweaver–Burk plot of recombinant Sirt1 activity in the presence of 0 (⧫), 4 (✓), 6 (▴) and 8 (× ) mM ATP for the range of NAD⁺ (n=3). (j) The activities of yeast Sir2 and Sirt2 are relatively resistant to ATP. The relative activities of Sirt1, yeast Sir2 and Sirt2 against Ac-H4 in the presence of 0 or 5 mM ATP is shown (n=4).