Recombinant Mouse ASAH2 Protein, CF

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

Recombinant Mouse ASAH2 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate C12:0 ceramide into sphingosine and dodecanoic acid. The specific activity is > 3,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse ASAH2/N-acylsphingosine Amidohydrolase-2 protein
  Thr34-Thr756, with an N-terminal 6-His tag
  Accession # NP_061300
• Accession #: NP_061300
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Asah2
• 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: 81 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: 113 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 MES, NaCl and Sodium Cholate.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 25 mM MES, 150 mM NaCl, 1% (w/v) Sodium Cholate, pH 6.5
  • o-PA Buffer: 0.2 M NaOH, 0.1% beta -mercaptoethanol (v/v)
  • Recombinant Mouse ASAH2/N‑acylsphingosine Amidohydrolase-2 (rmASAH2) (Catalog # 3558-AH)
  • Substrate: C12-ceramide (Avanti Polar Lipids, Catalog # 860512P), 50 mM stock in Chloroform
  • o-Phthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dissolve 10 µL of 50 mM stock of Substrate in 1.99 mL Assay Buffer for a 250 µM concentration. (Note: Preheat assay buffer to
     37 °C and vortex for 30 seconds to dissolve Substrate).
  2. Dilute rmASAH2 to 0.05 µg/mL in Assay Buffer.
  3. Combine 200 µL of 250 µM Substrate and 50 µL of 0.05 µg/mL rmASAH2. Include one blank containing 50 µL rmASAH2 and 200 µL Assay Buffer and another blank containing 200 µL Substrate and 50 µL Assay Buffer.
  4. Incubate at 37 °C for 1 hour.
  5. Stop reactions by heating them at 95-100 °C for 5 minutes.
  6. Dilute o-PA to 2 mg/mL in o-PA Buffer.
  7. Add 250 µL of the o-PA mixture to all reaction vials, including controls. Mix well.
  8. Incubate at room temperature for 10 minutes. Note: It is important not to deviate from this incubation time.
  9. Load 200 µL (in duplicate) of reaction mixtures and controls in a plate.
  10. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  11. Calculate specific activity using the following equation:

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

  *Average duplicates, use the control with the higher RFU value to adjust fluorescence.
  **Derived using calibration standard Sphingosine (Avanti Polar Lipids, Catalog # 860490P).

  Per Well:
  • rmASAH2: 0.001 µg
  • C12-ceramide: 100 μM
  • o-PA: 1 mg/mL

Notes

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

Alternate Names for Recombinant Mouse ASAH2 Protein, CF

• ASAH2 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2
• ASAH2
• BCDase
• HNAC1
• LCDase
• Nacylsphingosine Amidohydrolase2
• NCDase
• N-CDase

Background

The mouse ASAH2 gene encodes acylsphingosine amidohydrolase-2, also known as neutral ceramidase. Neutral ceramidase is a type II integral membrane protein that can be cleaved to produce a soluble secreted protein (1). The enzyme is abundant in the brush border membranes of the intestine, but is also expressed in tissues such as kidney, brain and liver (2, 3). A major physiological function of neutral ceramidase is the metabolism of dietary sphingolipids, but the enzyme may also be involved in the generation of messenger molecules such as sphingosine and sphingosine 1-phosphate (3).

1. Tani, M. et al. (2003) J. Biol. Chem. 278:10523.
2. Kono, M. et al. (2006) J. Biol. Chem. 281:7324.
3. Mitsutake, S. et al. (2001) J. Biol. Chem. 276:26249.

Publications for ASAH2/N-acylsphingosine Amidohydrolase-2 (3558-AH)(3)

Publications using 3558-AH

• R Sun, X Gu, C Lei, L Chen, S Chu, G Xu, MA Doll, Y Tan, W Feng, L Siskind, CJ McClain, Z Deng Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection Cell Reports, 2022-03-29;38(13):110560. 2022-03-29 [PMID: 35354041] (Bioassay, Mouse)
• Sphingosine rescues burn-injured mice from pulmonary Pseudomonas aeruginosa infection J Leukoc Biol, 2016-07-14;0(0):. 2016-07-14 [PMID: 27418352] (In Vivo, Mouse)
• Chen BC, Chang HM, Hsu MJ, Shih CM, Chiu YH, Chiu WT, Lin CH Peptidoglycan induces cyclooxygenase-2 expression in macrophages by activating the neutral sphingomyelinase-ceramide pathway. J. Biol. Chem., 2009-06-15;284(31):20562-73. 2009-06-15 [PMID: 19531467] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: Asah2
• Uniprot:
  • NP_061300()