Ectonucleotide pyrophosphatase/phosphodiesterase 4 (ENPP-4 or NPP4) belongs to a group of ecto-enzymes which regulate the availability of extracellular nucleotides (1). This enzyme family forms a subgroup of a larger family that also includes arylsulfatases, phosphopentomutases, 2,3-bisphosphoglycerate-independent phosphoglycerate mutases (iPGM), and alkaline phosphatases (2). Mature mouse ENPP-4 consists of a 392 amino acid (aa) ectodomain that contains the catalytic domain with a zinc-coordinated substrate binding pocket, a 21 aa transmembrane segment, and a 25 aa cytoplasmic tail (3). It shares 86% and 90% aa sequence identity with human and rat ENPP-4, respectively. Alternative splicing generates a short isoform with a 32 aa deletion in the phosphodiesterase domain. ENPP-4 hydrolyzes phosphodiester bonds in nucleotides with a preference for adenine nucleotides (3). It cleaves the diadenosine compounds Ap3A and Ap4a which are released from the dense granules of thrombin-activated platelets (3, 4). These reactions generate AMP and ADP from Ap3A cleavage, and AMP and ATP from Ap4A cleavage (4). ENPP-4 is expressed on the surface of vascular endothelial cells where its activity prolongs platelet aggretation and contributes to thrombus formation (4).
Recombinant Mouse ENPP-4 Protein, CF
R&D Systems | Catalog # 8996-EN
Key Product Details
- R&D Systems HEK293-derived Recombinant Mouse ENPP-4 Protein (8996-EN)
- 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
Accession Number
Applications
Product Specifications
Source
Tyr19-Ala410, with a C-terminal 6-His tag
Purity
Endotoxin Level
N-terminal Sequence Analysis
Predicted Molecular Mass
SDS-PAGE
Activity
The specific activity is >22,000 pmol/min/μg, as measured under the described conditions.
Formulation, Preparation, and Storage
8996-EN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Background: ENPP-4
References
- Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
- Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.
- Albright, R.A. et al. (2014) J. Biol. Chem. 289:3294.
- Albright, R.A. et al. (2012) Blood 120:4432.
Long Name
Alternate Names
Gene Symbol
UniProt
Additional ENPP-4 Products
Product Documents for Recombinant Mouse ENPP-4 Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse ENPP-4 Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Mouse ENPP-4 Protein, CF
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Protocols
View specific protocols for Recombinant Mouse ENPP-4 Protein, CF (8996-EN):
- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Mouse ENPP-4 (rmENPP-4) (Catalog # 8996-EN)
- Substrate: Thymidine 5’-monophosphate p-nitrophenyl ester (Sigma, Catalog # T4510), 100 mM stock in deionized water
- NaOH, 0.2 M in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmENPP-4 to 0.1 ng/µL in Assay Buffer.
- Dilute Substrate to 10 mM in Assay Buffer.
- Load 50 µL of 0.1 ng/µL rmENPP-4 in a clear strip well plate, and start the reaction by adding 50 µL of 10 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 10 mM Substrate.
- Incubate sealed plate at room temperature for 30 minutes.
- Stop reactions by adding 100 µL of 0.2 M NaOH to all wells, including Substrate Blank wells.
- Read at 410 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326). Per Reaction:
- rmENPP-4: 0.005 µg
- Substrate: 5 mM