Recombinant Human NMNAT-1 Protein, CF

Catalog # Availability Size / Price Qty
5865-NT-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human NMNAT-1 Protein, CF Summary

Product Specifications

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.
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.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human NMNAT-1 protein
Met1-Thr279, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Ser4
Structure / Form
Non-covalently linked hexamer
Predicted Molecular Mass
32 kDa
SDS-PAGE
33 kDa, reducing conditions

Product Datasheets

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5865-NT

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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.

Assay Procedure

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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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
Entrez Gene IDs
64802 (Human)
Alternate Names
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

Citation for Recombinant Human NMNAT-1 Protein, 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

  1. Expression and regulation of nampt in human islets.
    Authors: Kover K, Tong P, Watkins D, Clements M, Stehno-Bittel L, Novikova L, Bittel D, Kibiryeva N, Stuhlsatz J, Yan Y, Ye S, Moore W
    PLoS ONE, 0;8(3):e58767.
    Species: Human
    Sample Types: Cell Culture Supernates
    Applications: Enzyme Assay

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