Recombinant Human B3GAT3 Protein, CF

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

Recombinant Human B3GAT3 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze UDP-GlcA. The specific activity is >1 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human beta-1,3-Glucuronyltransferase 3/B3GAT3 protein
  Glu72-Val335, with an N-terminal Met and 6-His tag
• Accession #: O94766
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: B3GAT3
• Purity: >80%, 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: 30 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: 30-35 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Buffer A: 25 mM MES, 1 mM MnCl₂ (supplied in kit), pH 7.0
  • Buffer B: 100 mM Tris, 5 mM CaCl₂, pH 7.5
  • Recombinant Human beta -1,3-Glucuronyltransferase 3/B3GAT3 (rhB3GAT3) (Catalog # 6808-GT)
  • Substrate: UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in DMSO
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute the 1 mM Phosphate standard stock by adding 40 µL to 360 µL of Buffer A for a 100 µM stock. This is the first point of the standard curve.
  2. Prepare standard curve by performing six additional one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Dilute Substrate to 1.25 mM in Buffer A.
  4. Dilute rhB3GAT3 to 80 µg/mL in Buffer A.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
  6. Load 25 µL of the 80 µg/mL rhB3GAT3 into the plate. Include 25 µL of Buffer A for a blank control.
  7. Start the reaction by adding 25 µL of Substrate to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 4 hours.
  9. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
  10. Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to the reaction wells and blanks, excluding the standard curve. Add 50 µL of Buffer B to the standard curve.
  11. Tap to mix and incubate for 10 minutes at room temperature.
  12. Add 30 µL of the Malachite Green Reagent A to all wells.
  13. Add 50 µL of deionized water to all wells.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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 Substrate Blank.

  Per Reaction:
  • rhB3GAT3: 2 µg
  • Coupling Phosphatase 1: 100 ng
  • Substrate: 0.625 mM

Notes

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

Alternate Names for Recombinant Human B3GAT3 Protein, CF

• B3GAT3
• beta-1,3-glucuronyltransferase 3 (glucuronosyltransferase I)
• Beta-1,3-glucuronyltransferase 3
• EC 2.4.1.135
• galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 3
• GLCATI
• GlcAT-I
• GlcUAT-I
• Glucuronosyltransferase I
• Sqv-8-like protein
• UDP-GlcUA:Gal beta-1,3-Gal-R glucuronyltransferase

Background

B3GAT3 is an essential enzyme involved in the synthesis of the linkage region of heparan sulfate and chondroitin sulfate proteoglycans (1). It transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcA) to the trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. B3GAT3 knockout mice are embryonic lethal before the 8-cell stage due to failed cytokinesis, suggesting critical roles for chondroitin sulfate and heparan sulfate in embryonic cell division (2). Unlike the broad substrate specificity exhibited by B3GAT1, B3GAT3 shows strict specificity for Gal beta 1-3Gal beta 1 (3). The crystal structure shows the enzyme is an alpha / beta protein with two subdomains that constitute the donor and acceptor binding sites with the active site lying in the cleft between the two subdomains (4). Like most known glycosyltransferases, B3GAT3 is a type II Golgi-resident transmembrane protein with a short N‑terminal cytoplasmic domain and a single-pass transmembrane domain followed by an enzymatic domain in the lumen of Golgi apparatus. In the current assay, hydrolase activity against UDP-GlcA was measured using a phosphatase-coupled method (5).

1. Kitagawa, H. et al. (1998) J. Biol. Chem. 273:6615.
2. Isumikawa, T. et al. (1998) J. Biol. Chem. 285:12190.
3. Fondeur-Gelinotte, M. et al. (2007) Glycobiology 17:857.
4. Pedersen, L.C. et al. (2000) J. Biol. Chem. 275:34580.
5. Wu, Z.L, et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: B3GAT3
• Uniprot:
  • O94766()