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

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

Recombinant Human 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 >14,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human ENPP-7/Alk-SMase protein
  Ala22-Ser439, with a C-terminal 10-His tag
• Accession #: NP_848638
• 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: 49 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: 60-70 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
  • rhALPL Buffer: 50 mM Tris, 2 mM MgCl₂, pH 9.0
  • Recombinant Human ENPP‑7/Alk‑SMase (rhENPP-7) (Catalog # 4924-EN)
  • Substrate: Sphingomyelin (egg, chicken) (SPM) (Avanti Polar Lipids, Inc., Catalog # 860061C)
  • Phosphocholine Chloride (Sigma, Catalog # P0378), 100 mM stock in deionized water
  • Recombinant Human Alkaline Phosphatase/ALPL (rhALPL) (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 for proper solubilization. Vortex well.
  2. Dilute rhENPP-7 to 0.2 µg/mL in Assay Buffer.
  3. Mix 50 µL of SPM and 50 µL of rhENPP-7 to start the reaction. For blank, substitute SPM with Assay Buffer and keep on ice during incubation.
  4. Incubate at 37 °C for 30 minutes.
  5. Stop the reaction by heating all vials at 95‑100 °C for 6 minutes. Cool on ice for 1 minute followed by brief centrifugation.
  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). Complete the standard curve by preparing the following dilutions in Assay Buffer: 80, 60, 40, 20, 10, and 5 μM.
  7. Dilute rhALPL to 4 µg/mL in rhALPL Buffer.
  8. Combine 100 µL of 4 µg/mL rhALPL with each of the reaction mixture, Blank, and each dilution of the standard curve (100 μL). The total volume of all vials should be 200 µL.
  9. Incubate at room temperature for 20 minutes.
  10. Mix equal volumes of the Malachite Green Reagent A and B together for enough volume to add 80 µL of the Malachite green A and B mixture to all vials. Add the mixture to vials and vortex.
  11. Incubate at room temperature for 10 minutes. Note: Do not let mixtures incubate longer that the recommended time, precipitation may occur.
  12. Load 70 µL from each vial into a plate.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. Calculate specific activity:

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

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

  Per Well:
  • rhENPP-7: 0.0025 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 Human 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 human 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 also 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.

Bioinformatics

• Gene Symbol: ENPP7
• Uniprot:
  • NP_848638()