Recombinant Human ENPP-5 Protein, CF

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

Recombinant Human ENPP-5 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave O-(4-Nitrophenylphosphoryl) choline. The specific activity is >300 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human ENPP-5 protein
  Pro25-Ser430, with a C-terminal 10-His tag
• Accession #: Q9UJA9
• N-terminal Sequence: Pro25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ENPP5
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 48 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: 70-80 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: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, pH 5.5
  • Recombinant Human ENPP-5 (rhENPP-5) (Catalog # 8655-EN)
  • Substrate: O-(4-Nitrophenylphosphoryl) choline (Sigma, Catalog # N5879), 500 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 rhENPP-5 to 5 µg/mL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Load 50 µL of 5 µg/mL rhENPP-5 in a clear strip well plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 2 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 each well used.
  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 Well:
  • rhENPP-5: 0.25 µg
  • Substrate: 1 mM

Notes

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

Alternate Names for Recombinant Human ENPP-5 Protein, CF

• EC 3.1
• ectonucleotide pyrophosphatase/phosphodiesterase 5 (putative function)
• ectonucleotide pyrophosphatase/phosphodiesterase 5 (putative)
• ectonucleotide pyrophosphatase/phosphodiesterase family member 5
• E-NPP 5
• ENPP5
• ENPP-5
• KIAA0879
• NPP5
• NPP-5

Background

Ectonucleotide pyrophosphatase/phosphodiesterase 5 (ENPP-5 or NPP5) 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 human ENPP-5 is an approximately 50 kDa protein that consists of a 407 amino acid (aa) extracellular region with one phosphodiesterase/nucleotide pyrophosphatase domain, a 21 aa transmembrane segment, and a 25 aa cytoplasmic tail (3, 4). Within the ectodomain, human ENPP-5 shares 85% aa sequence identity with mouse and rat ENPP-5. R&D Systems in-house testing indicates that recombinant human ENPP-5 exhibits phosphodiesterase activity with the substrate O-(4-Nitrophenylphosphoryl) choline.

1. Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
2. Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.
3. Ohe, Y. et al. (2003) Biochem. Biophys. Res. Commun. 308:719.

Publications for ENPP-5 (8655-EN)(1)

Publication using 8655-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: ENPP5
• Uniprot:
  • Q9UJA9()