Recombinant Mouse ENPP-4 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse ENPP-4 Protein, CF Summary

• Details of Functionality: 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.
• Source: Human embryonic kidney cell, HEK293-derived mouse ENPP-4 protein
  Tyr19-Ala410, with a C-terminal 6-His tag
• Accession #: Q8BTJ4
• N-terminal Sequence: Tyr19
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Enpp4
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 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: 56-74 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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

Notes

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

Alternate Names for Recombinant Mouse ENPP-4 Protein, CF

• AP3Aase
• EC 3.1
• ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative)
• E-NPP 4
• ENPP4
• ENPP-4
• KIAA0879ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function)
• NPP-4
• NPP4ectonucleotide pyrophosphatase/phosphodiesterase family member 4

Background

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).

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.

Publications for ENPP-4 (8996-EN)(1)

Publication using 8996-EN

• Mardjuki, R;Wang, S;Carozza, J;Zirak, B;Subramanyam, V;Abhiraman, G;Lyu, X;Goodarzi, H;Li, L; Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint Cell reports 2024-05-10 [PMID: 38749434] (Bioassay, N/A)

Bioinformatics

• Gene Symbol: Enpp4
• Uniprot:
  • Q8BTJ4()