Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF Summary

• Details of Functionality: Measured by its ability to methylate nicotinamide. The specific activity is >65 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Nicotinamide N-Methyltransferase/NNMT protein
  Met1-Leu264, with a C-terminal 6-His tag
• Accession #: P40261
• N-terminal Sequence: Met1
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NNMT
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 30 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 25-29 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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
  1. Dilute the stock of reduced glutathione to 40 µM (40 pmol/µL) in Assay Buffer. This is the first point of the standard curve.
  2. 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.
  3. Dilute rhNNMT to 2 µg/mL in Assay Buffer.
  4. Create Substrate Mixture containing 200 µM S-adenosylmethionine and 4 mM Nicotinamide in Assay Buffer.
  5. Combine equal volumes of 2 µg/mL rhNNMT and Substrate Mixture. As a Control, combine equal volumes of Substrate Mixture with Assay Buffer.
  6. Incubate reactions at 37 °C for 30 minutes.
  7. To stop the reaction, boil samples at 100 °C for 5 minutes. Then cool reactions on ice for 1 minute.
  8. Create a Hydrolysis Mixture containing 25 µg/mL rhAHCY and 2.5 µg/mL rhADA in Hydrolysis Buffer.
  9. Combine equal volumes of cooled reactions from step 7 and Hydrolysis Mixture.
  10. Incubate mixtures at 37 °C for one hour.
  11. Load 50 µL of each point of the standard curve into a plate. Include a curve blank containing 50 µL of Assay Buffer.
  12. Load 50 µL of each reaction mixture into a plate.
  13. Dilute ThioGlo to 100 µM in DMSO.
  14. Add 50 µL of 100 µM ThioGlo to each well.
  15. Incubate at room temperature for 5 minutes in the dark.
  16. Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
  17. 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Nicotinamide N-Methyltransferase/NNMT, CF

• EC 2.1.1.1
• Nicotinamide NMethyltransferase
• Nicotinamide N-Methyltransferase
• NNMT

Background

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.

1. Cantoni, G.L. et al. (1951) J. Biol. Chem. 189:203.
2. Aksoy S. et al. (1994) J. Biol. Chem. 269:14835.
3. D’Souza J. et al. (1980) Xenobiotica 10:151.

Publications for Nicotinamide N-Methyltransferase/NNMT (7736-MT)(1)

Publication using 7736-MT

• HH Maw, X Zeng, S Campbell, ME Taub, AM Teitelbaum N-methylation of BI 187004 by thiol S-methyltransferase Drug Metab. Dispos., 2018-03-07;0(0):. 2018-03-07 [PMID: 29514826] (Bioassay, Human)

Bioinformatics

• Gene Symbol: NNMT
• Uniprot:
  • P40261(Human)