• 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.  

• 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. See Activity Assay Protocol on .

• Source

  Human embryonic kidney cell, HEK293-derived His23-Ser419, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His23

• Predicted Molecular Mass

  47 kDa

• SDS-PAGE

  50-64 kDa, reducing conditions

Assay Procedure

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 # )

• 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:
   

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

• rmENPP-6: 0.01 μg

• Substrate: 1 mM

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

• Entrez Gene IDs:

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

• Alternate Names:

  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