• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• 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 >0.5 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Ala27-His522, with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His & Thr56

• Predicted Molecular Mass

  56 kDa

• SDS-PAGE

  53-59 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Sodium Actetate, 250 mM NaCl,  pH 5.0

• Recombinant Human N?Acetylgalactosamine-6-Sulfatase/GALNS (rhGALNS) (Catalog # 8269-SU)

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

• Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water

• 96-well Clear Plate (Catalog # )

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

1. Dilute rhGALNS to 40 μg/mL in Assay Buffer.

2. Dilute PNCS to 4 mM in Assay Buffer.

3. Combine 200 μL of 40 μg/mL of rhGALNS and 200 μL of 4 mM of PNCS in microtubes.  Include a Substrate Blank with 200 μL of Assay Buffer and 200 μL of 4 mM of PNCS.

4. Incubate at 37 °C overnight (16-20 hours).

5. Load 100 μL from each reaction and Substrate Blank vial into a plate in triplicate, minimally.

6. Stop reaction by adding 100 μL of 0.2 M NaOH to each well containing reactions and Substrate Blank.

7. Read plate 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:

• rhGALNS: 2 μg

• Substrate: 1 mM

Background: N-Acetylgalactosamine-6-Sulfatase/GALNS

GALNS is a lysosomal sulfatase that hydrolyzes the 6-sulfate groups of the N-acetyl-D-galactosamine 6-sulfate units of chondroitin sulfate and of the D-galactose 6-sulfate units of keratan sulfate (1). The conversion to 3-oxoalanine (also known as C-formylglycine, FGly) of a cysteine residue is critical for catalytic activity of the enzyme (2). Deficiencies of this enzyme lead to Morquio A syndrome or mucopolysaccharidosis 4A (MPS4A), a lysosomal storage disorder characterized by intracellular accumulation of keratan sulfate and chondroitin-6-sulfate (3). Key clinical features of this disease include short stature, skeletal dysplasia, dental anomalies, and corneal clouding (4). Current therapeutic method for lysosomal storage diseases is enzyme replacement therapy (5).

• References:

1. Tomatsu, S. et al. (1991) Biochem. Biophys. Res. Commun. 181:677.

2. Dierks, T. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94:11963.

3. Rivera-Colon, Y. et al. (2013) J. Mol. Biol. 423:736.

4. Fukuda, S. et al. (1992) J. Clin. Invest. 90:1049.

5. Ohashi, T. (2012) Pediatr. Endocrinol. Rev. 10 Suppl 1:26.

• Entrez Gene IDs:

  2588 (Human); 50917 (Mouse); 292073 (Rat)

• Alternate Names:

  Chondroitinase; Chondroitinsulfatase; EC 3.1.6; EC 3.1.6.4; FLJ17434; FLJ42844; FLJ98217; GA6S; galactosamine (N-acetyl)-6-sulfate sulfatase; Galactose-6-Sulfate Sulfatase; GalNAc6S Sulfatase; GALNAC6S; GALNS; GAS; MPS4A; NAcetylgalactosamine6Sulfatase; N-Acetylgalactosamine-6-Sulfatase; N-acetylgalactosamine-6-sulfate sulfatase