• 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 4-Nitrocatechol Sulfate (PNCS). The specific activity is >100 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ser18-Ser506, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser18

• Predicted Molecular Mass

  54 kDa

• SDS-PAGE

  60 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Sodium Acetate, pH 5.0

• Recombinant Mouse Arylsulfatase A/ARSA (rmARSA) (Catalog # 2858-SU)

• Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in diH2O

• 0.2 M Sodium Hydroxide

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rmARSA to 20 μg/mL in Assay Buffer.

2. Dilute Substrate to 2 mM in Assay Buffer.

3. Combine in triplicate 75 μL of 20 μg/mL rmARSA and 75 μL of 2 mM Substrate. Include a Substrate Blank containing Assay Buffer in place of rmARSA.

4. Incubate reactions for 30 minutes at 37 °C.

5. Stop reactions by adding 150 μL 0.2 M sodium hydroxide to each for a final concentration of 0.1 M sodium hydroxide per reaction.

6. Load into a clear 96-well plate 200 μL from each reaction vial.

7. Read at 510 nm (absorbance) in endpoint mode.

8. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Nitrocatechol (PNC) (Sigma, Catalog # N15553).

Per Well:

• rmARSA: 1 μg

• Substrate: 0.5 mM

Background: Arylsulfatase A/ARSA

As a member of the sulfatase family, arylsulfatase A is encoded by the ARSA gene and required for the lysosomal degradation of cerebroside-3-sulfate, a sphingolipid sulfate ester and a major constituent of the myelin sheet (1). ARSA deficiency results in metachromatic leukodystrophy (MLD), a lysosomal storage disease in the central and peripheral nervous systems with severe and progressive neurological symptoms (2). The deduced amino acid sequence of mouse ARSA consists of a signal peptide (residues 1-17) and a mature chain (residues 18-506) (3). Recombinant mouse ARSA corresponds to the mature chain and has sulfatase activity as described in Activity Assay Protocol.

• References:

1. Lukatela, G. et al. (1998) Biochemistry 37:3654.

2. Parenti, G. et al. J. (1997) Curr. Opin. Genet. & Dev. 7:386.

3. Kreysing, J. et al. (1994) Genomics 19:249.

• Entrez Gene IDs:

  410 (Human); 11883 (Mouse)

• Alternate Names:

  ARSA; Arylsulfatase A; ASA; Cerebroside 3-sulfatase; Cerebroside-sulfatase; EC 3.1.6; EC 3.1.6.8; MLD