FIGURE 1.
Nitric oxide inhibits KDM3A activity. In vitro demethylase reactions were conducted at 25 °C in 10 μl of Tris, pH 8.0, containing 80 μm FeSO4, 1 mm α-ketoglutarate, 220 μm O2, 2 mm ascorbate, 40 ng of recombinant KDM3A protein, and 125 nm histone 3 peptide fragment containing a dimethyl lysine 9 (H3K9me2peptide). Representative MS-MALDI spectra are shown of H3K9me2peptide fragment alone (A), H3K9me2peptide + KDM3A at 60 min (B), H3K9me2peptide + KDM3A + •NO (100 μm Sper/NO; [•NO]ss = 1.4 ± 0.8 μm) at 60 min (C), and H3K9me2peptide + KDM3A + •NO (10 μm Proli/NO; [•NO] = 20 μm bolus) at 60 min (D). E, time and •NO concentration effects on KDM3A activity. Sper/NO was added at 25, 50, 100, and 200 μm to the reaction. H3K9me0 and H3K9me1 product formation was then measured and quantified at the indicated time points. These concentrations of Sper/NO gave a range of initial steady-state •NO concentrations of ≈0.2–2.0 μm. n = 3.