Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF
Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF Summary
Met1-Leu264, with a C-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 20 mM Tris, pH 8.5
- Hydrolysis Buffer: 100 mM HEPES pH 7.0
- Recombinant Human Nicotinamide N‑Methyltransferase/NNMT (rhNNMT) (Catalog # 7736-MT)
- Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water
- Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
- Recombinant Human Adenosine Deaminase/ADA, (rhADA) (Catalog # 7048-AD)
- S-adenosylmethionine (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO in deionized water
- Nicotinamide (Sigma, Catalog # 72340), 100 mM stock in 50 mM Tris, 100 mM NaCl, 30% Isopropanol, pH 8.0
- ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute the stock of reduced glutathione to 40 µM (40 pmol/µL) in Assay Buffer. This is the first point of the standard curve.
- Continue standard curve by performing six ½ serial dilutions of the 40 µM glutathione in Assay Buffer. The standard curve has a range of 31.25 to 2000 pmol per well.
- Dilute rhNNMT to 2 µg/mL in Assay Buffer.
- Create Substrate Mixture containing 200 µM S-adenosylmethionine and 4 mM Nicotinamide in Assay Buffer.
- Combine equal volumes of 2 µg/mL rhNNMT and Substrate Mixture. As a Control, combine equal volumes of Substrate Mixture with Assay Buffer.
- Incubate reactions at 37 °C for 30 minutes.
- To stop the reaction, boil samples at 100 °C for 5 minutes. Then cool reactions on ice for 1 minute.
- Create a Hydrolysis Mixture containing 25 µg/mL rhAHCY and 2.5 µg/mL rhADA in Hydrolysis Buffer.
- Combine equal volumes of cooled reactions from step 7 and Hydrolysis Mixture.
- Incubate mixtures at 37 °C for one hour.
- Load 50 µL of each point of the standard curve into a plate. Include a curve blank containing 50 µL of Assay Buffer.
- Load 50 µL of each reaction mixture into a plate.
- Dilute ThioGlo to 100 µM in DMSO.
- Add 50 µL of 100 µM ThioGlo to each well.
- Incubate at room temperature for 5 minutes in the dark.
- Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted thiol produced* (pmol)|
|Incubation time (min) x amount of enzyme (µg)|
*Derived from the reduced glutathione standard curve using linear fitting and adjusted for Control.Per Well:
- rhNNMT: 0.025 µg
- rhAHCY: 0.625 µg
- rhADA: 0.0625 µg
- S-adenosylmethionine: 0.025 mM
- Nicotinamide: 0.5 mM
- ThioGlo: 50 µM
Background: Nicotinamide N-Methyltransferase/NNMT
NNMT (Nicotinamide N-Methyltransferase) is a cytosolic enzyme that catalyzes the N-methylation of nicotinamide and other pyridines using S-adenosyl-L-methionine (AdoMet) as the methyl group donor (1, 2). The enzyme is highly expressed in liver (1) but is also detected in other organs. NNMT plays a significant role in nicotinamide metabolism (3) and in the detoxification of xenobiotics. The association with thyroid cancer and renal carcinoma makes NNMT useful as a tumor biomarker.
- Cantoni, G.L. et al. (1951) J. Biol. Chem. 189:203.
- Aksoy S. et al. (1994) J. Biol. Chem. 269:14835.
- D’Souza J. et al. (1980) Xenobiotica 10:151.
Citation for Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
N-methylation of BI 187004 by thiol S-methyltransferase
Authors: HH Maw, X Zeng, S Campbell, ME Taub, AM Teitelbaum
Drug Metab. Dispos., 2018;0(0):.
Sample Types: Recombinant Protein
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