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Recombinant Human NMNAT-1 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human NMNAT-1 Protein, CF Summary

Details of Functionality
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.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human NMNAT-1 protein
Met1-Thr279, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Ser4
Structure / Form
Non-covalently linked hexamer
Protein/Peptide Type
Recombinant Enzymes
Gene
NMNAT1
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Enzyme Activity
Theoretical MW
32 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
33 kDa, reducing conditions
Publications
Read Publication using
5865-NT in the following applications:

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 and DTT.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • 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

Notes

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

Alternate Names for Recombinant Human NMNAT-1 Protein, CF

  • EC 2.7.7.1
  • EC 2.7.7.18
  • NaMN adenylyltransferase 1
  • nicotinamide mononucleotide adenylyltransferase 1
  • nicotinamide nucleotide adenylyltransferase 1
  • nicotinamide nucleotide adenylyltransferase
  • Nicotinate-nucleotide adenylyltransferase 1
  • NMN adenylyltransferase 1
  • NMNAT1
  • NMNAT-1
  • NMNATPNAT1
  • PNAT1
  • pyridine nucleotide adenylyltransferase 1

Background

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).
  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.

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Publications for NMNAT-1 (5865-NT)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Enzyme Assay.


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Bioinformatics

Gene Symbol NMNAT1
Uniprot