Recombinant Human ENPP-2/Autotaxin Protein, CF

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

Recombinant Human ENPP-2/Autotaxin Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave Bis (p-Nitrophenyl) Phosphate (BPNPP). The specific activity is >8,000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ENPP-2/Autotaxin protein
  Asp49-Ile863, with an N-terminal 6-His tag
• Accession #: Q13822
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ENPP2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• 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: 99 kDa and 103 kDa, migrates as a doublet under 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 silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 5 mM MgCl₂, 0.02% Brij-35 (v/v), pH 8.5
  • Recombinant Human ENPP‑2/Autotaxin (rhENPP-2) (Catalog # 5255-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.)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhENPP-2 to 0.1 ng/µL in Assay Buffer.
  2. Dilute Substrate BPNPP to 2 mM in Assay Buffer.
  3. Load into the wells of a microplate 50 µL of 0.1 ng/µL rhENPP-2 and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
  4. Read at 400 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-Aldrich, Catalog # 241326).

  Per Well:
  • rhENPP-2: 0.005 µg
  • Substrate: 1 mM

Notes

This product may be covered by one or more of the following US issued patents 5,449,753; 5,731,167; 6,084,069; 6,417,338 and US pending applications.

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

Alternate Names for Recombinant Human 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 ENPP1 & 3, all of which contain a 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). The hydrolysis of nucleotides and lysophospholipids by ENPP-2 is mediated by a single catalytic site (2). Evidence shows LPA and sphingosine 1-phosphate to be specific 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).

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.

Publications for ENPP-2/Autotaxin (5255-EN)(4)

Publications using 5255-EN

• T Pleli, A Mondorf, N Ferreiros, D Thomas, K Dvorak, RM Biondi, DMZ Heringdorf, S Zeuzem, G Geisslinge, H Zimmermann, O Waidmann, A Piiper Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors Cell. Physiol. Biochem., 2018;45(6):2516-2528. 2018 [PMID: 29587249] (Bioassay, N/A)
• S She, M Yang, H Hu, P Hu, Y Yang, H Ren Proteomics Based Identification of Autotaxin As An Anti-Hepatitis B Virus Factor and a Promoter of Hepatoma Cell Invasion and Migration Cell. Physiol. Biochem., 2018;45(2):744-760. 2018 [PMID: 29414802] (Bioassay, Human)
• G Zhang, Y Cheng, Q Zhang, X Li, J Zhou, J Wang, L Wei ATX?LPA axis facilitates estrogen?induced endometrial cancer cell proliferation via MAPK/ERK signaling pathway Mol Med Rep, 2018;0(0):. 2018 [PMID: 29328374] (Bioassay, Human)
• Ryu J, Han H Autotaxin-LPA axis regulates hMSC migration by adherent junction disruption and cytoskeletal rearrangement via LPAR1/3-dependent PKC/GSK3beta/beta-catenin and PKC/Rho GTPase pathways. Stem Cells, 2015;33(3):819-32. 2015 [PMID: 25376707] (Bioassay, Human)

Bioinformatics

• Gene Symbol: ENPP2
• Uniprot:
  • Q13822()