• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  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. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Thr34-Thr756, with an N-terminal 6-His tag Accession # NP_061300

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  81 kDa

• SDS-PAGE

  113 kDa, reducing conditions

Assay Procedure

Materials

• 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:

     *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

Background: ASAH2/N-acylsphingosine Amidohydrolase-2

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).

• References:

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.

• Long Name:

  Neutral Ceramidase

• Entrez Gene IDs:

  56624 (Human); 54447 (Mouse); 114104 (Rat)

• Alternate Names:

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