| Assay Method Information | |
| | Enzyme Inhibition Assay |
| Description: | A general phosphate detection assay method using Malachite Green reagent was adapted to measure the activity of NaMN adenylyltransferaseA14. Briefly, the byproduct of NadD catalyzed reaction, inorganic pyrophosphate (PPi), was hydrolysed by inorganic pyrophosphatase and the resulting orthophosphate was detected by the Malachite Green dye. The reaction mixture contained 2.3 nM ecNadD (or 1.2 nM baNadD) in 100 mM Hepes, pH 7.5 buffer, 0.2 mM ATP, 0.07 or 0.2 mM NaMN, 10 mM MgCl2, 0.1 mg/ml Bovine Serum Albumin, 0.2 U inorganic pyrophosphatase. Appropriate amount of inhibitors were added to the reaction mixture to assess their effect on enzyme activity. After preincubation of the enzyme with the compounds for 5 min at room temperature, the reaction was started by adding NaMN substrate. The reaction was quenched with two volumes of Malachite Green Reagent in 1.2 M sulfuric acid prepared as described by Cogan et al.A41. |
| Affinity data for this assay | |
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