Recombinant Mouse ENPP-2/Autotaxin Protein, CF

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

Recombinant Mouse ENPP-2/Autotaxin Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave Bis (p-Nitrophenyl) Phosphate (BPNPP). The specific activity is >5,000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse ENPP-2/Autotaxin protein
  Ser49-Ile862, with an N-terminal 6-His tag
• Accession #: Q9R1E6
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Enpp2
• 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: 94 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: 90-100 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, 20 mM CaCl₂, 0.01% Brij-35 (w/v), pH 8.0
  • Recombinant Mouse ENPP‑2/Autotaxin (Catalog # 6187-EN)
  • Substrate: Bis(p-Nitrophenyl) Phosphate Sodium Salt (BPNPP) (Sigma, Catalog # N3002), 40 mM stock in deionized water (Note: Heating may be necessary to solubilize Substrate.)
  • 0.2 M NaOH
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmENPP-2 to 2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Load into plate 50 µL of 2 ng/µL rmENPP-2, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 2 mM Substrate.
  4. Incubate plate at room temperature for 10 minutes.
  5. Stop the reaction by adding 100 µL of 0.2 M NaOH to all wells.
  6. Read at 410 nm (absorbance) in endpoint mode.
  7. Calculate specific activity:

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

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

  Per Well:
  • rmENPP-2: 0.10 µg
  • Substrate: 0.5 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse ENPP-2/Autotaxin Protein, CF

• ATX
• ATXFLJ26803
• ATX-X
• Autotaxin
• autotaxin-t
• EC 3.1.4.39
• ectonucleotide pyrophosphatase/phosphodiesterase 2
• ectonucleotide pyrophosphatase/phosphodiesterase family member 2
• E-NPP 2
• ENPP2
• ENPP-2
• Extracellular lysophospholipase D
• Lysophosphatidic Acid
• LysoPLD
• NPP2
• PD-IALPHA
• PDNP2
• PDNP2NPP2
• phosphodiesterase I/nucleotide pyrophosphatase 2
• plasma lysophospholipase D

Background

ENPP-2, also known as Autotaxin, belongs to the ectonucleotide pyrophosphatase/phosphodiesterase (NPP) family. Some NPPs hydrolyze phosphates from nucleotides and their derivatives. ENPP-2 shares 40‑50% identity to ENPP-1 & -3, all of which contain an N‑terminal intracellular domain, a single transmembrane domain and a large extracellular domain that includes a catalytic domain, two somatomedin B-like domains, and a C-terminal nuclease like domain (1). Unlike ENPP‑1 and ENPP‑3, ENPP‑2 has weak activity against nucleotides, but exhibits a lysophospholipase D activity which allows the formation of lysophosphatidic acid (LPA) and choline from lysophosphatidylcholine (2). ENPP-2 can also hydrolyze sphingosylphosphorylcholine to produce sphingosine-1-phosphate, a lipid messenger molecule. The hydrolysis of nucleotides and lysophospholipids by ENPP2 is mediated by a single catalytic site (2). LPA and sphingosine-1-phosphate are inhibitors of ENPP-2 (3). ENPP-2 was originally found to stimulate tumor cell motility and has since been found to enhance tumor invasion and metastasis (4) and to be up‑regulated in several types of carcinomas including breast and lung (5). ENPP-2 is most highly expressed in brain and adipose tissue (6). Recombinant mouse ENPP-2 was expressed without its transmembrane and intracellular domains, resulting in the secretion of the recombinant enzyme.

1. Cimpean, A. et al. (2004) Biochem. J. 381:71.
2. Gijsbers, R. et al. (2003) FEBS Letters. 538:60.
3. Van Meeteren, L.A. et al. (2005) J. Biol. Chem. 280:21155.
4. Nam, S.W. et al. (2000) Oncogene 19:241.
5. Jansen, S. et al. (2005) J. Cell Sci. 118:3081.
6. Giganti, A. et al. (2008) J. Biol. Chem. 283:7776.

Publications for ENPP-2/Autotaxin (6187-EN)(3)

Publications using 6187-EN

• Mardjuki, R;Wang, S;Carozza, J;Zirak, B;Subramanyam, V;Abhiraman, G;Lyu, X;Goodarzi, H;Li, L; Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint Cell reports 2024-05-10 [PMID: 38749434] (Bioassay, N/A)
• N Sakai, G Bain, K Furuichi, Y Iwata, M Nakamura, A Hara, S Kitajima, A Sagara, T Miyake, T Toyama, K Sato, S Nakagawa, M Shimizu, S Kaneko, T Wada The involvement of autotaxin in renal interstitial fibrosis through regulation of fibroblast functions and induction of vascular leakage Sci Rep, 2019-05-15;9(1):7414. 2019-05-15 [PMID: 31092842] (Bioassay, Mouse)
• Madan , Damian, Ferguson , Colin G, Lee , Won Yong, Prestwich , Glenn D, Testa , Charles Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate. PLoS ONE, 2013-11-20;8(11):e79065. 2013-11-20 [PMID: 24278115] (Bioassay)

Bioinformatics

• Gene Symbol: Enpp2
• Uniprot:
  • Q9R1E6()