Recombinant Human Tissue alpha-L-Fucosidase/FUCA1, CF

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

Recombinant Human Tissue alpha-L-Fucosidase/FUCA1, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate 4-methylumbelliferyl-alpha -L-fucopyranoside. The specific activity is >4,800 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Tissue alpha-L-Fucosidase/FUCA1 protein
  Gln32-Lys466 with a C-terminal 6-His tag
• Accession #: P04066
• N-terminal Sequence: Gln32
• Protein/Peptide Type: Recombinant Enzymes
• Gene: FUCA1
• 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: 51 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: 55-60 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 Sodium Acetate, 5 mM MgCl₂, pH 4.5
  • Recombinant Human Tissue alpha ‑L‑Fucosidase/FUCA1 (rhFUCA1) (Catalog # 7039-GH)
  • Substrate: 4-Methylumbelliferyl-alpha -L-fucopyranoside (Research Products International Corp, Catalog # M65200), 50 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhFUCA1 to 2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 1.6 mM in Assay Buffer.
  3. Load into a plate 50 μL of 2 ng/μL rhFUCA1, and start the reaction by adding 50 μL of 1.6 mM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 1.6 mM Substrate.
  4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

  Per Well:
  • rhFUCA1: 0.100 μg
  • Substrate: 0.8 mM

Notes

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

Alternate Names for Recombinant Human Tissue alpha-L-Fucosidase/FUCA1, CF

• Alpha-L-fucosidase 1
• Alpha-L-fucosidase I
• Alpha-L-fucoside fucohydrolase 1
• EC 3.2.1
• EC 3.2.1.51
• FUCA
• FUCA1
• fucosidase, alpha-L- 1, tissue
• Tissue alphaLFucosidase
• Tissue alpha-L-Fucosidase

Background

Fucosylated glycoconjugates play numerous roles in biological processes, such as development, apoptosis and the immune responses, and are involved in the pathology of inflammation, cancer and cystic fibrosis (1, 2, 3, 4). Tissue alpha ‑L‑Fucosidase, also known as FUCA1, is a lysosomal enzyme responsible for hydrolyzing alpha ‑L‑fucose moieties from glycolipids and oligosaccharides (5). The mature enzyme in the liver has a molecular weight of 200 kDa and exists in the native state as a tetramer (6). Defects in FUCA1 are the cause of fucosidosis (7, 8, 9), an autosomal recessive lysosomal storage disease characterized by the accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Serum alpha ‑L‑fucosidase has been identified as a useful marker for oral cancer (10).

1. Hiraishi, K. et al. (1993) Glycobiology 3:381.
2. Solter, D. and Knowles, B.B. (1978) Proc. Natl. Acad. Sci. USA 75:5565.
3. Becker, D.J. and Lowe, J.B. (2003) Glycobiology 13:41R.
4. Liu, T.W. et al. (2009) Proc. Natl. Acad. Sci. USA 106:14581.
5. Fukushima, H. et al. (1985) Proc. Natl. Acad. Sci. USA 82:1262.
6. Alhadeff, J.A. et al. (1975) J. Biol. Chem. 250:7106.
7. O’Brien, J.S. et al. (1987) Enzyme 38:45.
8. Durand, P. et al. (1969) J. Pediatr. 75:665.
9. Johnson, K. and Dawson, G. (1985) Biochem. Biophys. Res. Commun. 133:90.
10. Shah, M. et al. (2008) Cancer 113:336.

Bioinformatics

• Gene Symbol: FUCA1
• Uniprot:
  • P04066()