Recombinant Mouse ENPP-6 Protein, CF

R&D Systems | Catalog # 8990-EN

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Key Product Details

  • R&D Systems HEK293-derived Recombinant Mouse ENPP-6 Protein (8990-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

AAH96376

Applications

Enzyme Activity
View all ENPP-6 Proteins and Enzymes »

Product Specifications

Source

Human embryonic kidney cell, HEK293-derived mouse ENPP-6 protein
His23-Ser419, 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

His23

Predicted Molecular Mass

47 kDa

SDS-PAGE

50-64 kDa, reducing conditions

Activity

Measured by its ability to cleave O-(4-Nitrophenylphosphoryl) choline.
The specific activity is >3,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8990-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-6

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). Mouse 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 90% and 96% aa sequence identity with the human and rat orthologs, respectively (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 upregulated during oligodendrocyte differentiation (8, 9).

References

  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.

Long Name

Ectonucleotide Pyrophosphatase/Phosphodiesterase 6

Alternate Names

ENPP6, GPC-Cpde, NPP6

Entrez Gene IDs

133121 (Human); 320981 (Mouse); 306460 (Rat)

Gene Symbol

ENPP6

UniProt

AAH96376

Additional ENPP-6 Products

Product Documents for Recombinant Mouse ENPP-6 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Mouse ENPP-6 Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Mouse ENPP-6 Protein, CF (8990-EN):

Materials
  • Assay Buffer: 50 mM Tris, 0.5 M NaCl, pH 9.0
  • Recombinant Mouse ENPP-6 (rmENPP-6) (Catalog # 8990-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 rmENPP-6 to 0.2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Load 50 µL of 0.2 µg/mL rmENPP-6 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. Read at 405 nm (absorbance) in kinetic mode for 5 minutes.
  5. 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:

  • rmENPP-6: 0.01 µg
  • Substrate: 1 mM

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