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Recombinant Human Glucosylceramidase/GBA Protein, CF

R&D Systems | Catalog # 7410-GHB

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human Glucosylceramidase/GBA Protein (7410-GHB)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

P04062.3

Applications

Enzyme Activity
View all Glucosylceramidase/GBA Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human Glucosylceramidase/GBA protein
Ala40-Gln536, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ala40

Predicted Molecular Mass

56 kDa

SDS-PAGE

60-75 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze 4-methylumbelliferyl-beta -D-glucopyranoside.
The specific activity is >200 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human Glucosylceramidase/GBA Protein, CF

Recombinant Human Glucosylceramidase/GBA Protein Enzyme Activity Diagram.

Recombinant Human Glucosylceramidase/GBA Protein, CF (Catalog # 7410-GHB) cleaves the beta-glucosidic linkage of glucosylceramide, an intermediate in glycolipid metabolism.

Recombinant Human Glucosylceramidase/GBA Protein Enzyme Activity.

Recombinant Human Glucosylceramidase/GBA Protein, CF (Catalog # 7410-GHB) is measured by its ability to hydrolyze 4-methylumbelliferyl-beta -D-glucopyranoside.

Recombinant Human Glucosylceramidase/GBA Protein SDS-PAGE.

2 μg/lane of Recombinant Human Glucosylceramidase/GBA Protein (Catalog # 7410-GHB) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 60-75 kDa.

Formulation, Preparation, and Storage

7410-GHB
Formulation Supplied as a 0.2 μm filtered solution in Sodium Citrate.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: Glucosylceramidase/GBA

Glucosylceramidase is a lysosomal enzyme that cleaves the beta-glucosidic linkage of glucosylceramide (1, 2), an intermediate in glycolipid metabolism. The mature enzyme has 497 amino acids with a molecular weight of 62 kDa (3). Glycosylation occurs at four of five N-glycosylation sites and is essential for the trafficking and activity of the enzyme (4). The enzyme is activated in lysosomes by saposin C, although the mechanism of activation is not well understood (5). Defects in Glucosylceramidase are the cause of Gaucher disease, also known as glucocerebrosidase deficiency (6). Gaucher disease is the most prevalent lysosomal storage disease, characterized by accumulation of glucosylceramide in the reticulo-endothelial system. Symptoms of Gaucher disease may include enlarged spleen and liver, liver malfunction, skeletal disorders and bone lesions, severe neurologic complications, swelling of lymph nodes, anemia, low blood platelets and yellow fatty deposits on the white of the eye (7). Currently, enzyme replacement therapy is used to treat patients with the disease (8, 9).

References

  1. Sorge, J. et al. (1985) Proc. Natl. Acad. Sci. USA 82:7289.
  2. Ginns, E. I. et al. (1984) Biochem. Biophyl. Res. Commun. 123:574.
  3. Horowitz, M. et al. (1989) Genomics 4:87.
  4. Grace, M.E. et al. (1994) J. Biol. Chem. 269:2283.
  5. Bruhn, h. (2005) Biochem. J. 389:249.
  6. Liou, B. et al. (2006) J. Biol. Chem. 281:4242.
  7. Grabowski, G.A. (2008). Lancet 372:1263.
  8. Zheng, W. et al. (2007) Proc. Natl. Acad. Sci. USA 104:13192.
  9. Beutler, E. and Gelbart, T. (1996) Hum. Mutat. 8:207.

Alternate Names

Alglucerase, GBA, GBA1, Imiglucerase

Entrez Gene IDs

2629 (Human); 14466 (Mouse)

Gene Symbol

GBA

UniProt

P04062.3

Additional Glucosylceramidase/GBA Products

Product Documents for Recombinant Human Glucosylceramidase/GBA Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Glucosylceramidase/GBA Protein, CF

For research use only

Citations for Recombinant Human Glucosylceramidase/GBA Protein, CF

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Protocols

View specific protocols for Recombinant Human Glucosylceramidase/GBA Protein, CF (7410-GHB):

Materials
  • Assay Buffer: 50 mM Sodium Citrate, 25 mM Sodium Cholate, 5 mM DTT, pH 6.0
  • Stop solution: 0.5 M Glycine, 0.3 M NaOH (~pH 10)
  • Recombinant Human Glucosylceramidase/GBA (rhGBA) (Catalog # 7410-GHB)
  • Substrate: 4-Methylumbelliferyl-beta -D-glucopyranoside (Sigma, Catalog # M3633), 10 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 rhGBA to 0.2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. Load 25 µL of 0 2 ng/µL rhGBA into wells of a plate, and start the reactions by adding 25 μL of 6 mM Substrate. Include a Substrate Blank containing 25 µL of Assay Buffer and 25 µL of 6 mM Substrate.
  4. Seal plate and incubate at 37 °C for 20 minutes.
  5. After incubation, stop the reactions by adding 50 µL of Stop Solution to each well.
  6. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
  7. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
Incubation time (min) x amount of enzyme (µg)

     *Adjusted for Substrate Blank
** Derived using calibration standard 4methylumbelliferone (Sigma, Catalog # 69580).

Per Well:
  • rhGBA: 0.005 µg
  • Substrate: 1.5 mM

FAQs for Recombinant Human Glucosylceramidase/GBA Protein, CF

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  • Q: Why is DTT (dithiothreitol) added to the assay buffer in the assay described for Recombinant Human Glucosylceramidase/GBA Protein, CF, Catalog # 7410-GHB?

    A: The 5 mM DTT is added to the assay buffer in the assay protocol for catalog #  7410-GHB to prevent interchain disulfide bond formation that can cause aggregation of the protein and loss of enzyme activity over time. 

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