Recombinant Human NDST2 Protein, CF

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

Recombinant Human NDST2 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >1,250 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived human N-Deacetylase/N-Sulfotransferase 2/NDST2 protein
  Ala81-Gly883, with C-terminal addition of HPGGGSGGGSGGGSHHHHHH
• Accession #: P52849
• N-terminal Sequence: Ala81
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NDST2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 94 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: 90-100 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 and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5 (supplied in kit)
  • Recombinant Human N-Deacetylase/N-Sulfotransferase 2/NDST2 (rhNDST2) (Catalog # 7689-ST)
  • PAPS (3'-Phosphoadenosine-5'-phosphosulfate) (Catalog # ES019)
  • Heparan sulfate (Celsus Labs, Catalog # HO-3105), 50 mg/mL stock in deionized water
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare reaction mixture containing 0.8 mM PAPS, 8 mg/mL heparan sulfate, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rhNDST2 to 1.6 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 1.6 µg/mL rhNDST2 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

  Per Reaction:
  • rhNDST2: 0.04 μg
  • Coupling Phosphatase 3: 0.5 µg
  • PAPS: 0.4 mM
  • Heparan sulfate: 200 µg

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human NDST2 Protein, CF

• bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 2
• EC 2.8.2.8
• Glucosaminyl N-deacetylase/N-sulfotransferase 2
• HSST2MGC129697
• MGC129705
• N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 2
• NDST2
• NDST-2
• N-heparan sulfate sulfotransferase 2
• N-HSST 2
• NST2

Background

Heparan sulfate is a highly sulfated polysaccharide that can be found on the cell surface and within extracellular matrix. It is typically covalently attached to the protein core of proteoglycans, such as syndecans and glypicans (1, 2). Heparin, on the other hand, is a highly sulfated version of heparan sulfate and is predominantly found in mast cells. Both heparin and heparan sulfate contain disaccharide repeats of uronic acid and N‑acetylglucosamine and are modified by the same sulfotransferases (1, 2). The uronic acid residues can be sulfated at the 2-O position by heparan sulfate 2-O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at the N, 3-O, and 6-O positions by N‑deacetylase/N-sulfotransferases (NDSTs), heparan sulfate 3‑O sulfotransferases and heparan sulfate 6‑O sulfotransferases, respectively. All these enzymes are Golgi resident proteins. NDST-mediated modifications are believed to occur before modifications by other sulfotransferases (3). There are four NDSTs in the human genome and all are dual enzymes with both deacetylase and sulfotransferase domains (4, 5). While NDST1 is mainly responsible for heparan sulfate synthesis, NDST2 is mainly responsible for the synthesis of heparin in mast cells (5). Disrupting the NDST2 gene causes production failure of fully sulfated heparin (6, 7).

1. Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
3. Carlsson, P. et al. (2008) J. Biol. Chem. 283:20008.
4. Aikawa, J. and Esko, J. D. (1999) J. Biol. Chem. 274:2690.
5. Dagalv, A. et al. (2011) J. Biol. Chem. 286:44433.
6. Forsberg, E. et al. (1999) Nature 400:773.
7. Humphries, D.E. et al. (1999) Nature 400:769.

Bioinformatics

• Gene Symbol: NDST2
• Uniprot:
  • P52849(Human)