Recombinant Human a-N-acetylgalactosaminidase Protein, CF

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human a-N-acetylgalactosaminidase Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave alpha -N-acetylgalactosaminyl from 4-Nitrophenyl N-acetyl-alpha -D-galactosaminide. The specific activity is >1800 pmol/min/μg, as measured under the described condition.
• Source: Chinese Hamster Ovary cell line, CHO-derived human alpha-N-acetylgalactosaminidase/NAGA protein
  Leu18-Gln411, with a C-terminal 6‑His tag
• Accession #: P17050
• N-terminal Sequence: Leu18
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NAGA
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 46 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: 50-60 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, NaCl, Brij and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.1 M Sodium Citrate, 0.2 M NaCl, pH 4.0
  • Recombinant Human alpha ‑N‑acetylgalactosaminidase/NAGA (rhNAGA) (Catalog # 6717-GH)
  • Substrate: 4-Nitrophenyl-N-acetyl-alpha -D-galactosaminide (Sigma, Catalog # N4264), 15 mM stock in DMSO
  • 0.2 M Sodium Hydroxide
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhNAGA to 2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 2 mM in Assay Buffer.
  3. Load 50 µL of 2 ng/µL rhNAGA and 50 µL of 2 mM Substrate into a clear 96-well plate. Also create a Substrate Blank by loading 50 µL of Assay Buffer and 50 µL of 2 mM Substrate.
  4. Incubate plate at room temperature for 10 minutes.
  5. Add 100 μL of 0.2 M NaOH to each well used in order to stop the reaction and develop the color.
  6. Read absorbance in endpoint mode at 402 nm.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x volume^† (L) x 10^12 pmol/mol) / (Inc. time (min) x epsilon ** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Using the extinction coefficient 17700 M^-1cm^-1
  ***Using the path corr. 0.6 cm
  ^†Based upon a 0.0002 L Volume

  Per Reaction:
  • rhNAGA: 0.1 μg
  • Substrate: 1 mM

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 a-N-acetylgalactosaminidase Protein, CF

• alpha-N- (alpha-galactosidase B)
• alphaNacetylgalactosaminidase
• alpha-N-acetylgalactosaminidase
• EC 3.2.1
• EC 3.2.1.49
• GALB
• N-acetylgalactosaminidase, alpha-
• NAGA

Background

NAGA is a lysosomal alpha -N-acetylgalactosaminidase that cleaves non-reducing alpha -N-acetylgalactosaminyl moieties from glycoconjugates (1). Mature NAGA has 394 amino acids and is trafficked to the lysosome via the mannose-6-phosphate receptor‑mediated pathway (2). The enzyme is a retaining exoglycosidase, where both the substrate and product of the enzymatic reaction have the same anomeric configuration (3). Deficiency in NAGA results in increased urinary excretion and tissue accumulation of glycopeptides and oligosaccharides containing terminal alpha ‑N‑acetylgalactosaminyl moieties (4), manifesting as Schindler’s disease, an autosomal recessive disease with neuroaxonal dystrophy and other neurological symptoms (5). The enzyme can be used to remove alpha ‑N‑acetylgalactosaminyl residues present on red blood cells thus converting blood type A to blood type O (6, 7, 8).

1. Wang, A.M. et al. (1990) J. Biol. Chem. 265:21859.
2. Sweeley, C.C. et al. (1983) Arch. Biochem. Biophys. 223:158.
3. Garman, S.C. et al. (2002) Structure. 10:425.
4. Eng, C.M. et al. (2001) N. Engl. J. Med. 345:9.
5. Wang, A.M. et al. (1990) J. Clin. Invest. 86:1752.
6. Liu, Q.P. et al. (2007) Nature Biotechnol. 25:454.
7. Calcutt, M. J. et al. (2002) FEMS Microbiol. Lett. 214:77.
8. Zhu, A. et al. (1996) Arch. Biochem. Biophys. 327:324.

Publications for alpha-N-acetylgalactosaminidase/NAGA (6717-GH)(1)

Publication using 6717-GH

• S Cecioni, RA Ashmus, PA Gilormini, S Zhu, X Chen, X Shan, C Gros, MC Deen, Y Wang, R Britton, DJ Vocadlo Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates Nature Chemical Biology, 2022-02-24;18(3):332-341. 2022-02-24 [PMID: 35210619] (Bioassay, Human)

Bioinformatics

• Gene Symbol: NAGA
• Uniprot:
  • P17050()