• 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 >5,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Chinese Hamster Ovary cell line, CHO-derived Thr15-Ile761, with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  83 kDa

• SDS-PAGE

  132 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM MES, 150 mM NaCl, 1% Sodium Cholate, pH 6.5

• o-PA Buffer: 0.2 M NaOH, 0.1% beta -mercaptoethanol (v/v)

• Recombinant Human ASAH2/N‑acylsphingosine Amidohydrolase-2 (rhASAH2) (Catalog # 3557-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 C12-ceramide stock in 1.99 mL Assay Buffer for a 250 µM Substrate concentration. (Note: Preheat assay buffer to 37 °C and vortex for 30 seconds to dissolve Substrate).

2. Dilute rhASAH2 to 0.05 µg/mL in Assay Buffer.

3. Combine 200 µL of 250 µM Substrate and 50 µL of 0.05 µg/mL rhASAH2. Include one blank containing 50 µL rhASAH2 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.

9. Load 200 µL 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:

• rhASAH2: 0.001 µg

• C12-ceramide: 100 μM

• o-PA: 1 mg/mL

Background: ASAH2/N-acylsphingosine Amidohydrolase-2

The human ASAH2 gene encodes N-acylsphingosine amidohydrolase-2, also known as neutral or non-lysosomal ceramidase. ASAH2 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). An N-terminally truncated form of human ASAH2 has been shown to localize to mitochondria (4). A major physiological function of ASAH2 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.

4. El Bawab, S. et al. (2000) J. Biol. Chem. 275:21508.

• 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