| Assay Method Information | |
| | NNMT Enzymatic Activity Assay |
| Description: | Enzyme activity assays were performed as previously described with NNMT (16.25 ug/mL, 550 nM) in 50 mM Tris buffer (pH 8.6) containing 1 mM DTT (all final concentrations). The enzyme was incubated with the substrate pyridine compound for 5 min at 37 °C using a heat block before the reaction was initiated by addition of AdoMet (for substrates that were not soluble in water, stocksolutions were prepared in DMSO and diluted to a final DMSO concentration of <5% in the assay mixture). As a control, the methylation of nicotinamide was also measured in the presence of 5% DMSO, revealing no difference in enzymatic activity. For these screens, the final reagent concentrations were 100 uM AdoMet and 2.0 mM substrate and a series of time points were initially investigated (5, 15, 30, and 60 min). At each time point, 15 uL aliquots of the reaction mixture were taken and added to 70 uL of acetonitrile, which served to quench the reaction by causing precipitation of the enzyme. |
| Affinity data for this assay | |
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