Recombinant Human NMNAT-2 Protein, CF

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

Recombinant Human NMNAT-2 Protein, CF Summary

• Details of Functionality: Measured by the production of NAD⁺, which is converted to NADH by alcohol dehydrogenase. The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human NMNAT-2 protein
  Met1-Glu307, with a C-terminal 6-His tag
• Accession #: Q9BZQ4
• N-terminal Sequence: Thr2, Thr133, Thr145
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NMNAT2
• Purity: >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 35 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: 34-38 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, Glycerol, Brij-35 and DTT.
• Purity: >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 5 mM DTT, pH 7.5
  • Recombinant Human NMNAT‑2 (rhNMNAT-2) (Catalog # 6279-NT)
  • beta -Nicotinamide mononucleotide ( beta -NMN) (Sigma, Catalog # N3501), 50 mM stock in deionized water
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • Yeast Alcohol Dehydrogenase (ADH) (Sigma, Catalog # A3263), 5 mg/mL stock in 25 mM MES, 20% Glycerol, pH 6.5
  • 1 M Magnesium Chloride
  • 95-100% Ethanol
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhNMNAT-2 to 0.5 ng/µL in Assay Buffer.
  2. Prepare Substrate Mixture: 30 mM MgCl₂, 1 mM beta -NMN, 4 mM ATP, 0.1 mg/mL ADH, and 2% Ethanol in 50 mM HEPES, pH 7.5.
  3. In a plate, load 50 µL of 0.5 ng/µL rhNMNAT-2, and start the reaction by adding 50 µL of Substrate Mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Read absorbance at a wavelength of 339 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 6220 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhNMNAT-2: 0.025 µg
  • Substrate Mixture: 0.5 mM beta -NMN, 2 mM ATP, 15 mM MgCl₂, 0.05 mg/mL ADH, and 1% Ethanol

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human NMNAT-2 Protein, CF

• C1orf15
• chromosome 1 open reading frame 15
• EC 2.7.7.1
• EC 2.7.7.18
• KIAA0479MGC2756
• NaMN adenylyltransferase 2
• nicotinamide mononucleotide adenylyltransferase 2
• nicotinamide nucleotide adenylyltransferase 2
• Nicotinate-nucleotide adenylyltransferase 2
• NMN adenylyltransferase 2
• NMNAT2
• NMNAT-2
• PNAT2
• pyridine nucleotide adenylyltransferase 2

Background

Humans produce three nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymes. All three enzymes transfer adenylate from ATP to nicotinamide ribonucleotide or nicotinate ribonucleotide to generate NAD⁺ or deamido-NAD⁺, and are important enzymes in the NAD biosynthetic pathway (1). The three enzymes differ in tissue expression patterns and subcellular location, indicating that they each play a unique role in NAD homeostasis (2). NMNAT-2 is expressed primarily in the central nervous system and in muscle tissue in contrast to NMNAT-1, which has a broad tissue distribution (3). NMNAT-2 is found in the cytosol and Golgi complex, while NMNAT-1 is a nuclear enzyme and NMNAT-3 is mitochondrial (1). A number of studies have implicated the NMNAT proteins in cancer and neurodegenerative diseases (4). NMNAT‑2 has been shown to be essential for the maintenance of healthy axons. When the level of this labile protein falls below a critical threshold in axons, the process of axonal degeneration begins (5). The axon-protective function of NMNAT-2 is dependent on its NAD⁺ synthesis activity (6).

 

1. Berger, F. et al. (2005) J. Biol. Chem. 280:36334.
2. Raffaelli, N. et al. (2002) Biochem. Biophys. Res. Commun. 297:835.
3. Yalowitz, J.A. et al. (2004) Biochem. J. 377:317.
4. Lau, C. et al. (2009) Front. Biosci. 14:410.
5. Gilley, J. and M.P. Coleman (2010) PLoS Biol. 8:1.
6. Yan, T. et al. (2010) Neurochem. Int. 56:101.  

Publications for NMNAT-2 (6279-NT)(1)

Publication using 6279-NT

• YO Ali, G Bradley, HC Lu Screening with an NMNAT2-MSD platform identifies small molecules that modulate NMNAT2 levels in cortical neurons Sci Rep, 2017-03-07;7(0):43846. 2017-03-07 [PMID: 28266613] (ELISA (Standard), Mouse)

Bioinformatics

• Gene Symbol: NMNAT2
• Uniprot:
  • Q9BZQ4()