Recombinant Human NMNAT-1 Protein, CF

R&D Systems | Catalog # 5865-NT

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant Human NMNAT-1 Protein (5865-NT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 21 (baculovirus)

Accession Number

Q9HAN9

Structure / Form

Non-covalently linked hexamer

Applications

Enzyme Activity
View all NMNAT-1 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human NMNAT-1 protein
Met1-Thr279, with a C-terminal 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ser4

Predicted Molecular Mass

32 kDa

SDS-PAGE

33 kDa, reducing conditions

Activity

Measured by the production of NAD+, which is converted to NADH by alcohol dehydrogenase.
The specific activity is >2,500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

5865-NT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: NMNAT-1

NMNAT-1 is expressed in the nuclei of all human tissues, with highest expression in skeletal muscle, heart, kidney, pancreas, and brain (1). The enzyme transfers adenylate from ATP to nicotinamide ribonucleotide or nicotinate ribonucleotide to generate NAD+ or deamido-NAD+, and is an essential enzyme for the production of nuclear NAD+ (2). Nuclear NAD+ is required by poly(ADP-ribose) polymerase 1 (PARP-1), which poly-ADP-ribosylates chromatin in response to DNA strand breaks. NMNAT-1 is known to interact with PARP-1, resulting in its activation, but this interaction with PARP-1 is prevented when NMNAT-1 is phosphorylated at Ser136 (3). Nuclear NAD+ levels are also important for the regulation of SIR2 histone deacetylases (4). A naturally occurring Ube4b/NMNAT-1 chimeric protein is directly involved in slowing the degeneration of injured neurons in mice (5). NMNAT activity is required for the activation of tiazofurin, a drug used to treat leukemia (6). Two other NMNAT enzymes are present in humans. NMNAT-2 is localized in the Golgi complex and cytoplasm, and NMNAT-3 is a mitochondrial enzyme (7).

References

  1. Emanuelli, M. et al. (2001) J. Biol.Chem. 276:406.
  2. Schweiger, M. et al. (2001) FEBS Lett. 492:95.
  3. Berger, F. et al. (2007) Proc. Natl. Acad. Sci. USA 104:3765.
  4. Revollo, J.R. et al. (2004) J. Biol. Chem. 279:50754.
  5. Mack, T.G. et al. (2001) Nature Neurosci. 4:1199.
  6. Boulton, S. et al. (1997) Br. J. Cancer 76:845.
  7. Berger, F. et al. (2005) J. Biol. Chem. 280:36334.

Long Name

Nicotinamide mononucleotide adenylyltransferase-1

Alternate Names

NMN adenylyltransferase 1, NMNAT1, PNAT1

Entrez Gene IDs

64802 (Human)

Gene Symbol

NMNAT1

UniProt

Q9HAN9

Additional NMNAT-1 Products

Product Documents for Recombinant Human NMNAT-1 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human NMNAT-1 Protein, CF

For research use only

Citations for Recombinant Human NMNAT-1 Protein, CF

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Protocols

View specific protocols for Recombinant Human NMNAT-1 Protein, CF (5865-NT):

Materials
  • Assay Buffer: 50 mM HEPES, pH 7.5
  • Recombinant Human NMNAT-1 (rhNMNAT-1) (Catalog # 5865-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-1 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 Assay Buffer.
  3. In a plate, load 50 µL of 0.5 ng/µL rhNMNAT-1, 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 1012 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-1: 0.025 µg
  • Substrate Mixture: 0.5 mM beta -NMN, 2 mM ATP, 15 mM MgCl2, 0.05 mg/mL ADH, and 1% Ethanol

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