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
AnalysisHis23
Predicted Molecular Mass
47 kDa
SDS-PAGE
50-64 kDa, reducing conditions
8990-EN |
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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.
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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
Dilute rmENPP-6 to 0.2 µg/mL in Assay Buffer.
Dilute Substrate to 2 mM in Assay Buffer.
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.
Read at 405 nm (absorbance) in kinetic mode for 5 minutes.
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
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:
Stefan, C. et al. (2005) Trends Biochem. Sci. 30:542.
Greiner-Tollersrud, O.K. (2014) Neurochem. Res. 39:2025.
Sakagami, H. et al. (2005) J. Biol. Chem. 280:23084.
Greiner-Tollersrud, L. et al. (2013) Neurochem. Res. 38:300.
Jahn, O. et al. (2009) Mol. Neurobiol. 40:55.
Royland, J.E. et al. (2008) J. Neuroendocrinol. 20:1319.
Bastian, T.W. et al. (2012) Endocrinology 153:5668.
Dugas, J.C. et al. (2006) J. Neurosci. 26:10967.
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
