Figure 4.
Sir2 transfers the acetyl group in the substrate to a breakdown product of NAD. Shown are ion-exchange chromatograms depicting fractionation of reactions that contained Sir2, 500 μM Ac-K16 peptide, and ≈30,000 cpm of 3H-labeled, acetylated histone H3. Incubation was for 6 h at 25°C. The labeled substrates remained bound to the column under the elution conditions used. Red traces represent cpm, blue traces represent A254. The green trace in A represents the salt gradient from 0 to 500 mM used in these experiments; it was omitted in the two lower panels for simplicity. The standards indicated at the top elute as follows: NAD, fraction 10; acetate, fraction 11; product 2, fraction 14; ADP-ribose, fraction 15 (also indicated by tick marks at the bottom of each panel). (A) No NAD was included in the reaction. (B) NAD (100 μM) was included in the reaction. (C) The reaction was carried out at pH 10 and included 100 μM NAD.