Recombinant Mouse SMPD1 Protein, CF

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

Recombinant Mouse SMPD1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave 2-N-Hexadecanoylamino-4-nitrophenylphosphorylcholine (HNPPC). The specific activity is >1000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse SMPD1 protein
  Tyr58-Leu626, with a C-terminal 10-His tag
• Accession #: Q04519
• N-terminal Sequence: Tyr58
• Structure / Form: Monomer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Smpd1
• 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: 65 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: 70-80 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -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 MES, 0.5 µM ZnCl₂, pH 6.5
  • Developing Buffer: 0.2 M NaOH
  • Recombinant Mouse SMPD-1 (rmSMPD-1) (Catalog # 5680-PD)
  • Substrate: 2-N-Hexadecanoylamino-4-nitrophenylphosphorylcholine (HNPPC) (Gojira Fine Chemicals, Catalog # HN1004), 50 mM stock solution in methanol. Note: Incubate at 37 °C for 5 minutes and vortex to dissolve.
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmSMPD-1 to 2 µg/mL in Assay Buffer and incubate at 37 °C for 10 minutes.
  2. Dilute Substrate to 1 mM in Assay Buffer and incubate at 37 °C for 10 minutes.
  3. Combine 50 µL of rmSMPD-1 and 50 µL of Substrate in a plate. Create Substrate Blanks by using Assay Buffer in place of rmSMPD-1.
  4. Cover plate and incubate at room temperature for 20 minutes in the dark.
  5. Stop the reaction by adding 100 µL of Developing Buffer to each well.
  6. Read (top read) absorbance in endpoint mode at 410 nm.
  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:
  • rmSMPD-1: 0.10 µg
  • Substrate: 250 µM

Notes

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

Alternate Names for Recombinant Mouse SMPD1 Protein, CF

• Acid sphingomyelinase
• aSMase
• ASMsphingomyelin phosphodiesterase
• EC 3.1.4.12
• NPD
• SMPD1
• sphingomyelin phosphodiesterase 1, acid lysosomal

Background

SMPD1 is a lysosomal phosphodiesterase which belongs to the acid sphingomyelinase family (1). SMPD1 catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Ceramide, a bioactive lipid, has emerged into an important signaling molecule involved in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (2). Activation of SMPD1 occurs by the removal, chemical modification or dimerization of its C-terminal cysteine residue (Cys 627) (3). Deficiencies of SMPD1 result in a lysosomal storage disorder referred to as Niemann-Pick disease (4). rmSMPD1 was expressed without its C-terminal cysteine residue, and is therefore constitutively active.

1. Schuchman, E.H. et al. (1991) J. Biol. Chem. 266:8531.
2. Melendez, A.J. et al. (2008) Biochim. Biophys. Acta. 1784:66.
3. Qui, H. et al. (2003) J. Biol. Chem. 278:32744.
4. Smith, E.L. and E.H. Schuchman (2008) FASEB J. 22:3419.

Bioinformatics

• Gene Symbol: Smpd1
• Uniprot:
  • Q04519()