Recombinant Mouse ENPP-7/Alk-SMase Protein, CF

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

Recombinant Mouse ENPP-7/Alk-SMase Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze sphingomyelin to ceramide and phosphorylcholine. The phosphorylcholine is cleaved by Recombinant Human Alkaline Phosphatase/ALPL (Catalog # 2909-AP) and the phosphate is detected/measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >13,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse ENPP-7/Alk-SMase protein
  Ala22-Gln421, with a C-terminal 10-His tag
• Accession #: NP_001025462
• N-terminal Sequence: Ala22
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Enpp7
• 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 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: 60 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 silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM Sodium Taurocholate (Sigma, Catalog # T4009), 150 mM NaCl, pH 9.0
  • rhTNAP Buffer: 50 mM Tris, 2 mM MgCl₂, pH 9.0
  • Recombinant Mouse ENPP‑7/Alk‑SMase (rmENPP-7) (Catalog # 5160-EN)
  • Substrate: Sphingomyelin (egg, chicken) (SPM) (Avanti Polar Lipids, Inc., Catalog # 860061C), 35 mM in chloroform
  • Phosphocholine Chloride (Sigma, Catalog # P0378), 100 mM stock in deionized water
  • Recombinant Human Alkaline Phosphatase/ALPL/TNAP (rhTNAP) (Catalog # 2909-AP)
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute SPM to 0.4 mM in Assay Buffer. Note: Heat buffer to 100 °C and then add SPM to help keep SPM solubilized. Vortex well.
  2. Dilute rmENPP-7 to 0.1 µg/mL in Assay Buffer.
  3. Mix equal volumes of diluted SPM and diluted rmENPP-7.
  4. Incubate at 37 °C for 30 minutes. As a Substrate Control substitute SPM with Assay Buffer and keep on ice during incubation.
  5. Stop the reaction by heating all vials at 95-100 °C for 6 min. Cool on ice for 1 min followed by brief centrifugation in microcentrifuge.
  6. Prepare a standard curve from the 100 mM Phosphocholine Chloride stock by adding 10 µL of the 100 mM Phosphocholine Chloride Standard to 990 µL of Assay Buffer for a 1 mM stock. Continue by adding 100 µL of the 1 mM Phosphocholine Chloride stock to 900 µL of Assay Buffer for a 100 µM stock (This is the first dilution to use as a standard).
  7. Make the following serial dilutions from the 100 µM Phosphocholine Chloride stock in Assay Buffer to complete standard curve: 80, 60, 40, 20, 10 and 5 μM.
  8. Dilute rhTNAP to 4 µg/mL in rhTNAP Buffer.
  9. Combine 100 µL of the 4 µg/mL rhTNAP with 100 µL each of the diluted rmENPP-7/SPM mixture, the dilutions of the standard curve, and the Substrate Control.
  10. Incubate at room temperature for 20 min.
  11. Mix equal volumes of the Malachite Green Reagents A and B together for enough volume to add 80 µL of the mixture to all vials.
  12. Add the Malachite Green Reagent A and B mixture to vials and vortex.
  13. Incubate at room temperature for 10 min. Note: Do not let mixtures incubate longer, precipitation may occur.
  14. Load 70 µL from each vial into a plate.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/1 nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for the Substrate Control.

  Per Well:
  • rmENPP-7: 0.00125 µg
  • Sphingomyelin: 0.071 mM
  • Phosphocholine Chloride Curve: 0, 125, 250, 500, 1000, 1500, 2000, and 2500 pmol

Notes

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

Alternate Names for Recombinant Mouse ENPP-7/Alk-SMase Protein, CF

• Alkaline sphingomyelin phosphodiesterase
• alkaline sphingomyelinase
• Alk-SMase
• EC 3.1.4.12
• ectonucleotide pyrophosphatase/phosphodiesterase 7
• ectonucleotide pyrophosphatase/phosphodiesterase family member 7
• E-NPP 7
• ENPP7
• ENPP-7
• Intestinal alkaline sphingomyelinase
• MGC50179
• NPP7
• NPP-7

Background

ENPP-7 (ectonucleotide pyrophosphatase/phosphodiesterase 7), also known as alkaline sphingomyelinase (Alk-SMase) is expressed in the intestines and in bile (1). It shares 30 - 36% homology with the members of the nucleotide pyrophosphatase/phosphodiesterese (NPP) family while sharing no similarities with neutral or acid SMase (2). Its main function is the digestion of dietary sphingomyelin by hydrolyzing sphingomyelin into ceramide and phosphorylcholine. ENPP-7 is reported to hydrolyse and inactivate platelet-activating factor (PAF) by a phospholipase C-type activity (3). Studies show a decrease in ENPP-7 activity in human colorectal adenocarcinomas and human colorectal carcinomas, which indicate a potential role of ENPP-7 in human colon cancer (4, 5).

1. Duan, R-D. et al. (2006) Biochim. Biophys. 1761:281.
2. Duan, R-D. et al. (2003) J. Biol. Chem. 278:38528.
3. Wu, J. et al. (2006) Biochem J. 394:299.
4. Hertervig, E. et al. (1996) Cancer. 79:448.
5. Hertervig, E. et al. (1999) Br. J. Cancer. 81:232.

Publications for ENPP-7/Alk-SMase (5160-EN)(1)

Publication using 5160-EN

• 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: Enpp7
• Uniprot:
  • NP_001025462()