Recombinant Mouse CD39L3/ENTPD3 Protein, CF

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

Recombinant Mouse CD39L3/ENTPD3 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the 5’-phosphate groups from the substrate adenosine-5’-triphosphate (ATP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >20,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse CD39L3/ENTPD3 protein
  Gln44-Pro485, with a C-terminal 6-His tag
• Accession #: AAH79871
• N-terminal Sequence: No results obtained, Gln44 predicted.
• Structure / Form: Monomer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Entpd3
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 50 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: 85-100 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, NaCl, CaCl₂ and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 5 mM CaCl₂, pH 7.5
  • Recombinant Mouse CD39L3/ENTPD3 (rmCD39L3) (Catalog # 4464-EN)
  • Substrate: Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 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
  1. Dilute rmCD39L3 to 0.02 µg/mL in Assay Buffer.
  2. Prepare a standard curve from 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer 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. (This is the first dilution to use as a standard.)
  3. Perform six additional 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.
  4. Load 25 µL of 0.02 μg/mL rmCD39L3 and the standard curve into a plate. Include a Substrate Blank containing Assay Buffer.
  5. Dilute the Substrate to 100 µM in Assay Buffer.
  6. Add 25 µL of the 100 µM Substrate to all wells and mix well.
  7. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
  8. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  9. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  10. Read plate at 620 nm (absorbance) in endpoint mode.
  11. 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 Substrate Blank.

  Per Well:
  • rmCD39L3: 0.0005 µg
  • Substrate: 35.7 µM

Notes

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

Alternate Names for Recombinant Mouse CD39L3/ENTPD3 Protein, CF

• ATPDase
• CD39 antigen-like 3
• CD39L3
• CD39L3EC 3.6.1.5
• CD39-like 3
• EC 3.6.1
• Ecto-apyrase 3
• Ecto-ATP diphosphohydrolase 3
• Ecto-ATPase 3
• Ecto-ATPDase 3
• ectonucleoside triphosphate diphosphohydrolase 3
• ENTPD3
• FLJ93839
• HB6
• HB6NTPDase 3
• NTPDase-3

Background

Ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase-3) is an integral membrane protein with an extracellular catalytic domain (1). rhNTPDase-3 was expressed as a protein lacking its N- and C-terminal transmembrane domains, resulting in the secretion of the soluble rhNTPDase-3 ectodomain. NTPDase-3 hydrolyzes the b- and g phosphate groups of nucleotides, preferring ATP, ADP, UTP, and UDP as substrates (1). Through its hydrolysis of extracellular nuceotides, NTPDase-3 is important for the regulation of purinergic signaling (2). The enzyme is expressed at its highest levels in brain, pancreas, spleen, and prostate tissues (3). In the brain, NTPDase-3 may play a role in the regulation of feeding, sleep, and other behaviors (4). 

1. Lavoie, E.G. et al. (2004) Biochem. Pharmacol. 67:1917.
2. Crawford, P.A. et al. (2007) Arch. Biochem. Biophys. 457:7.
3. Chadwick, B.P. and A.M. Frischauf (1998) Genomics 50:357.
4. Belcher, S.M. et al. (2006) Neuroscience 137:1331.

Bioinformatics

• Gene Symbol: Entpd3
• Uniprot:
  • AAH79871()