Recombinant Human Glucosamine (N-acetyl)-6-Sulfatase, CF

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

Recombinant Human Glucosamine (N-acetyl)-6-Sulfatase, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the substrate 4-Nitrocatechol Sulfate (PNCS). The specific activity is >250 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Glucosamine (N-acetyl)-6-Sulfatase/GNS protein
  Val37-Leu552 & Thr44-Leu552, both with a C-terminal 10-His tag
• Accession #: P15586
• N-terminal Sequence: Val37 & Thr44
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GNS
• 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: 59 & 60 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: 94 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, 250 mM NaCl, pH 5.0
  • Recombinant Human Glucosamine (N-acetyl)‑6-Sulfatase/GNS (rhGNS) (Catalog # 2484-SUC)
  • Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in deionized water
  • 0.2 M NaOH
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhGNS to 2 µg/mL in Assay Buffer.
  2. Dilute PNCS substrate to 4 mM in Assay Buffer.
  3. Combine 75 µL of 2 µg/mL rhGNS and 75 µL 4 mM PNCS. Include a Substrate Blank containing Assay Buffer in place of rhGNS.
  4. Incubate at 37 °C for 2 hours.
  5. Stop reactions and develop color by adding 150 µL of 0.2 M NaOH to each reaction.
  6. Load into the wells of a clear 96-well plate 200 µL from each reaction.
  7. Read at 510 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x Conversion Factor** (pmole/OD)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard p-Nitrocatechol (Sigma, Catalog # N15553).

  Per Well:
  • rhGNS: 0.1 µg
  • Substrate: 1 mM

Notes

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

Alternate Names for Recombinant Human Glucosamine (N-acetyl)-6-Sulfatase, CF

• EC 3.1.6
• EC 3.1.6.14
• G6Sglucosamine-6-sulfatase
• glucosamine (N-acetyl)-6-sulfatase
• Glucosamine6Sulfatase
• Glucosamine-6-Sulfatase
• GNS
• MGC21274
• N-acetylglucosamine-6-sulfatase

Background

A member of the sulfatase family, GNS is required for the lysosomal degradation of the glycosaminoglycans (GAG) heparan sulfate and keratan sulfate (1, 2). It hydrolyzes the 6-sulfate group of the N-acetyl-D-glucosamine 6-sulfate units of the GAG. GNS deficiency results in mucopolysaccharidosis type IIID (MPS IIID or Sanfilippo D Syndrome), an inborn error leading to lysosomal accumulation of heparan sulfate. MPS IIID has profound mental deterioration, hyperactivity, and relatively mild somatic manifestations. The deduced amino acid sequence of human GNS consists of a signal peptide (residues 1-36) and a mature chain (residues 37-552) that may be further processed into N-terminal and C-terminal fragments (3). rhGNS corresponds to the single chain and has sulfatase activity described in Activity Assay Protocol.

1. Parenti, G. et al. (1997) Curr. Opin. Genet. & Dev. 7:386.
2. Neufeld, E.F. and J. Muenzer (2001) in The Metabolic and Molecular Basis of Inherited Disease, Scriver, C.R. et al. (eds.) pp. 3421, New York, McGraw-Hill.
3. Robertson, D.A. et al. (1992) Biochem. J. 288:539.

Publications for Glucosamine (N-acetyl)-6-Sulfatase/GNS (2484-SUC)(1)

Publication using 2484-SUC

• T Zhang, S Xie, Z Wang, R Zhang, Q Sun, X Liu, L Chi, JP Li, H Li, T Tan Oligosaccharides mapping of nitrous acid degraded heparin through UHPLC-HILIC/WAX-MS Carbohydr Polym, 2019-12-02;231(0):115695. 2019-12-02 [PMID: 31888803] (Cell Culture, Human)

Bioinformatics

• Gene Symbol: GNS
• Uniprot:
  • P15586()