Recombinant Human ENPP-6 Protein, CF

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

Recombinant Human ENPP-6 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave O-(4-Nitrophenylphosphoryl) choline. The specific activity is >4,000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human ENPP-6 protein
  Arg23-Ser419, with a C-terminal 10-His tag
• Accession #: Q6UWR7
• N-terminal Sequence: Arg23
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ENPP6
• Purity: >95%, 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: 47 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: 49-60 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 under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.5 M NaCl, pH 9.0
  • Recombinant human ENPP-6 (rhENPP-6) (Catalog # 8489-EN)
  • Substrate: O-(4-Nitrophenylphosphoryl) choline (Sigma, Catalog # N5879), 500 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhENPP-6 to 0.4 µg/mL in Assay buffer.
  2. Dilute room temperature Substrate to 2 mM in Assay buffer.
  3. Load 50 µL of 0.4 ng/µL rhENPP-6 in a clear strip well plate.
  4. Start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
  5. Read at 405 nm (absorbance) in kinetic mode for 5 minutes.
  6. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x Conversion Factor** (pmol/OD)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank.
  **Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326).

  Per Well:
  • rhENPP-6: 0.02 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-6 Protein, CF

• B830047L21Rik
• EC 3.1
• ectonucleotide pyrophosphatase/phosphodiesterase 6
• ectonucleotide pyrophosphatase/phosphodiesterase family member 6
• E-NPP 6
• ENPP6
• ENPP-6
• GPC-Cpde
• MGC33971
• NPP6
• NPP-6

Background

Ectonucleotide Pyrophosphatase/Phosphodiesterase 6 (ENPP-6) is a choline-specific glycerophosphodiester phosphodiesterase. It is a member of a family of membrane-linked glycoproteins that, at an alkaline pH, hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, lysophospholipids, and choline phosphate esters (1). ENPP-6 is a GPI-linked protein that is synthesized as a 440 amino acid (aa) precursor and has a predicted molecular weight of approximately 55 kDa (2). Its extracellular region contains a catalytic domain that is nearly 400 aa in length and shares 88% aa sequence identity with the mouse and rat orthologs (1, 3). A soluble form of ENPP-6 can be proteolytically shed and associate into a disulfide-linked homodimer (2, 4).

The catalytic domain of ENPP-6 specifically recognizes the phosphocholine part of its substrate (2, 3). ENPP-6 has been shown to display preference for choline-containing phospholipids or phosphodiesters such as lysophosphatidylcholine (LPC), glycerophosphorylcholine (GPC), sphingosylphosphorylcholine (SPC), Platelet-Activating Factor (PAF), and lysoPAF (3). Furthermore, ENPP-6 shows preference for LPC with short (12:0 and 14:0) or polyunsaturated (18:2 and 20:4) fatty acids (3). In vitro, ENNP-6 has been shown to efficiently hydrolyze the classical substrate for phospholipase C, p-nitrophenyl phosphorylcholine, but not the classical nucleotide phosphodiesterase substrate, p-nitrophenyl thymidine 5'-monophosphate (3). ENPP-6 is predominantly expressed in brain, where it is localized to myelin and kidney, with lesser expression being found in the heart (3, 5). ENPP-6 expression in the brain has been shown to be regulated by Thyroid Hormone and iron (6, 7). Additionally, in rat brain, ENPP-6 expression has been shown to be up-regulated during oligodendrocyte differentiation (8, 9).

1. Stefan, C. et al. (2005) Trends Biochem. Sci. 30:542.
2. Greiner-Tollersrud, O.K. (2014) Neurochem. Res. 39:2025.
3. Sakagami, H. et al. (2005) J. Biol. Chem. 280:23084.
4. Greiner-Tollersrud, L. et al. (2013) Neurochem. Res. 38:300.
5. Jahn, O. et al. (2009) Mol. Neurobiol. 40:55.
6. Royland, J.E. et al. (2008) J. Neuroendocrinol. 20:1319.
7. Bastian, T.W. et al. (2012) Endocrinology 153:5668.
8. Dugas, J.C. et al. (2006) J. Neurosci. 26:10967.
9. Cahoy, J.D. et al. (2008) J. Neurosci. 28:264.

Bioinformatics

• Gene Symbol: ENPP6
• Uniprot:
  • Q6UWR7()