Recombinant Human ENPP-6 Protein, CF Summary
Arg23-Ser419, with a C-terminal 10-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- 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
- Dilute rhENPP-6 to 0.4 µg/mL in Assay buffer.
- Dilute room temperature Substrate to 2 mM in Assay buffer.
- Load 50 µL of 0.4 ng/µL rhENPP-6 in a clear strip well plate.
- Start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 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).
- rhENPP-6: 0.02 µg
- Substrate: 1 mM
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).
- 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.
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