ADP-Sugar Pyrophosphatase (NUDT5) is a member of the nudix superfamily of enzymes. Members of this family are pyrophosphohydrolases that act upon substrates with the general structure of a nucleoside (Nu) diphosphate (di) linked to another moiety, X (NDP-X) to yield NMP plus P-X (1). Human NUDT5 is a homodimeric enzyme present in the cytosol of most cell types (2). Glu166 and three magnesium ions are important for stabilizing the transition state during the hydrolysis of ADPR (3). NUDT5 has been suggested to play a role in regulating the intracellular levels of ADPR by NO activation through ADP-ribosylation at cysteine residues of the enzyme in macrophages (4). It also may play defensive role against the mutagenesis induced by oxidized deoxyribonucleosides (5, 6).
Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF
R&D Systems | Catalog # 6414-NH
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Key Product Details
- R&D Systems E. coli-derived Recombinant Human ADP-sugar Pyrophosphatase/NUDT5 (6414-NH)
- 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
E. coli
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
E. coli-derived human ADP-Sugar Pyrophosphatase/NUDT5 protein
Glu2-Phe219, with an N-terminal Met and 6-His tag
Glu2-Phe219, with an N-terminal Met and 6-His tag
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Met
Predicted Molecular Mass
25 kDa
SDS-PAGE
32-37 kDa, reducing conditions
Activity
Measured by its ability to hydrolyze ADP-ribose to AMP and ribose-5-phosphate.
The specific activity is >1,000 pmol/min/μg, as measured under the described conditions.
The specific activity is >1,000 pmol/min/μg, as measured under the described conditions.
Formulation, Preparation, and Storage
6414-NH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, DTT and Glycerol. |
| 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.
|
Background: ADP-Sugar Pyrophosphatase/NUDT5
References
- McLennan, A.G. (2006) Cell Mol. Life Sci. 63:123.
- Zha, M. et al. (2006) J. Mol. Biol. 364:1021.
- Zha, M. et al. (2008) J. Mol. Biol. 379:568.
- Yu, H.N. et al. (2007) Biochem. Biophys. Res. Comm. 354:764.
- Hori, M. et al. (2010) Free Radic. Biol. Med. 48:1197.
- Kamiya, H. et al. (2009) DNA Repair (Amst) 8:1250.
Alternate Names
Nudix motif 5, YSA1, YSA1H
Entrez Gene IDs
11164 (Human)
Gene Symbol
NUDT5
UniProt
Additional ADP-Sugar Pyrophosphatase/NUDT5 Products
Product Documents for Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF
For research use only
Related Research Areas
Citations for Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF
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Protocols
View specific protocols for Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF (6414-NH):
Materials
- Assay Buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5
- Recombinant Human ADP‑Sugar Pyrophosphatase/NUDT5 (rhNUDT5) (Catalog # 6414-NH)
- Coupling Enzyme: 5'-Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
- Substrate: Adenosine 5’-diphosphate ribose (ADPR) (Sigma, Catalog # A0752), 20 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute Substrate to 2.5 mM in Assay Buffer.
- Dilute rhCD73 to 3.33 µg/mL in Assay Buffer.
- Dilute rhNUDT5 to 0.667 µg/mL in Assay Buffer.
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 15 µL of 0.667 µg/mL rhNUDT5 into the plate. Include a Control containing 15 µL of Assay Buffer.
- Add 15 µL of 3.33 µg/mL rhCD73 to the wells, excluding the standard curve and the curve blank.
- Start the reaction by adding 20 µL of 2.5 mM Substrate to the wells, exluding the standard curve and the curve blank.
- Cover the plate with parafilm or a plate sealer and incubate at room temperature for 30 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells.
- Add 100 µL of deionized water to all wells.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Phosphate released* (nmol) x (1000 pmol/nmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
Per Well:
- rhNUDT5: 0.01 µg
- rhCD73: 0.05 µg
- Substrate: 0.238 mM
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