Recombinant Mouse ENPP-4 Protein, CF

R&D Systems | Catalog # 8996-EN

R&D Systems
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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

HEK293

Accession Number

Applications

Enzyme Activity
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Product Specifications

Source

Human embryonic kidney cell, HEK293-derived mouse ENPP-4 protein
Tyr19-Ala410, with a C-terminal 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

Tyr19

Predicted Molecular Mass

45 kDa

SDS-PAGE

56-74 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze thymidine 5'-monophosphate p-nitrophenyl ester.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: ENPP-4

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).

References

  1.  Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
  2. Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.
  3. Albright, R.A. et al. (2014) J. Biol. Chem. 289:3294.
  4. Albright, R.A. et al. (2012) Blood 120:4432.

Long Name

Ectonucleotide Pyrophosphatase/Phosphodiesterase 4

Alternate Names

AP3Aase, ENPP4, NPP-4

Entrez Gene IDs

22875 (Human); 224794 (Mouse); 301261 (Rat)

Gene Symbol

ENPP4

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):

Materials
  • 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
  1. Dilute rmENPP-4 to 0.1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 10 mM in Assay Buffer.
  3. 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.
  4. Incubate sealed plate at room temperature for 30 minutes.
  5. Stop reactions by adding 100 µL of 0.2 M NaOH to all wells, including Substrate Blank wells.
  6. Read at 410 nm (absorbance) in endpoint mode.
  7. 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

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