NNMT His Tag Protein, Rhesus macaque

NNMT (Nicotinamide N-methyltransferase) is a cytosolic methyltransferase whose crystal structure has been resolved in rhesus macaques and mice at a resolution of 1.88 Å, belonging to the methyltransferase superfamily. This enzyme utilizes S-adenosylmethionine as a methyl donor to catalyze the methylation of nicotinamide into 1-methylnicotinamide, thereby regulating NAD⁺ metabolic homeostasis; it is highly expressed in the liver, adipose tissue, and brain, and participates in energy expenditure, lipid metabolism, and insulin sensitivity modulation through the NAD⁺/Sirt1 pathway. Clinical studies have demonstrated that upregulated NNMT expression is closely associated with obesity, type 2 diabetes, and metabolic syndrome, while its levels are also significantly elevated in specific brain regions of patients with Parkinson's disease and Alzheimer's disease, suggesting that NNMT serves not only as a biomarker for metabolic disorders but also as a promising therapeutic target for drug intervention.

Assay protocol

Principle: The MTase-Glo™ Methyltransferase Assay can measure the activity of NNMT based on the reaction product S-adenosyl homocysteine (SAH). After the methyltransferase reaction is complete, just add the MTase-Glo™ Reagent and MTase-Glo™ Detection Solution to convert SAH to ATP and measure the light from the luciferase reaction.

Materials

1.Assay Buffer: 20mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 0.1 mg/ml BSA. Add 1mM DTT prior to use.

2.NNMT His tag Protein, Rhesus macaque

3.MTase-Glo^TM Methyltransferase Assay (Promega, Catalog #: V7601)

4.Substrate: Nicotinamide (Sigma, Catalog #: N481907-100GM)

5.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

6.Plate Reader (PerkinElmer)

Produce

1. Thaw the active NNMT and all Methyltransferase-Glo^TM Assays kit reagents on ice.

2. Prepare the following working solutions with Methyltransferase Reaction Buffer on ice:

2X final concentration of Active NNMT: 0.4 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL, 0 μg/mL

2X Substrate Cocktail: 40 μM of SAM and 80 μM of Nicotinamide in Reaction Buffer

3. In a 384-well plate, add the following components to bring the initial reaction volume to 20 μL:

Component 1. 10 μL of 2X Substrate Cocktail

Component 2. 10 μL of 2X Active NNMT

4. Mix the reaction on an orbital shaker for 2 minutes. Seal the plate with a plate seal and incubate at 37°C for 60 minutes.

5. Dilute 10X Methyltransferase-Glo^TM Reagent with equal volume of nanopure water, and add 5 μL of the 5X Methyltransferase-Glo^TM Reagent to all reaction wells.

6. Mix on an orbital shaker for 2 minutes and then incubate at room temperature for 30 minutes.

7. Add 25 μL of MTase-Glo^TM Detection Solution to all reaction wells. Mix for 2 minutes and then incubate at room temperature for 30 minutes

8. Read at luminescence, respectively in endpoint mode.

9. Using the SAH standard curve, determine the concentration of SAH produced (pmol) and calculate the methyltransferase specific activity as outlined below.