Recombinant Human Glucosylceramidase/GBA Protein, CF

Product Discontinued

Recombinant Human Glucosylceramidase/GBA Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze 4-methylumbelliferyl-beta -D-glucopyranoside. The specific activity is >180 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Glucosylceramidase/GBA protein
  Met1-Gln536, with C-terminal 6-His tag
• Accession #: P04062
• N-terminal Sequence: Ala40
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GBA1
• 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:
  • Enzyme Activity
• Theoretical MW: 56 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: 58-75 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, NaCl, Glycerol and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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 10.0)
  • Recombinant Human Glucosylceramidase/GBA (rhGBA) (Catalog # 7410-GH)
  • 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 0.4 ng/μL in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. Load in a plate 25 μL of 0.4 ng/μL rhGBA, and start the reaction 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 4-methylumbelliferone (Sigma, Catalog # 69580).

  • rhGBA: 0.010 μg
  • Substrate: 1.5 mM

Notes

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

Alternate Names for Recombinant Human Glucosylceramidase/GBA Protein, CF

• Acid beta-glucosidase
• Alglucerase
• beta-glucocerebrosidase
• D-glucosyl-N-acylsphingosine glucohydrolase
• EC 3.2.1.45
• GBA
• GBA1
• GC
• GCB
• GLUC
• glucosidase, beta, acid
• glucosidase, beta; acid (includes glucosylceramidase)
• Glucosylceramidase
• Imiglucerase
• lysosomal glucocerebrosidase

Background

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).

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: 1263–1271.
8. Zheng, W. et al. (2007) Proc. Natl. Acad. Sci. USA 104:13192.
9. Beutler, E. and Gelbart, T. (1996) Hum. Mutat. 8:207.

Publications for Glucosylceramidase/GBA (7410-GH)(9)

Publications using 7410-GH

• A Brown, J Zhang, B Lawler, B Lu Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1 Genes, 2021-07-15;12(7):. 2021-07-15 [PMID: 34356092] (Cell Culture, Human)
• MC Babcock, CR Mikulka, B Wang, S Chandriani, S Chandra, Y Xu, K Webster, Y Feng, HR Nelvagal, A Giaramita, BK Yip, M Lo, X Jiang, Q Chao, JC Woloszynek, Y Shen, S Bhagwat, MS Sands, BE Crawford Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor Scientific Reports, 2021-07-14;11(1):14486. 2021-07-14 [PMID: 34262084] (Bioassay, Human)
• L Oftedal, J Maple-Grød, MGG Førland, G Alves, J Lange Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid Scientific Reports, 2020-12-16;10(1):22098. 2020-12-16 [PMID: 33328543] (Cell Culture, Human)
• MA Do, D Levy, A Brown, G Marriott, B Lu Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles Sci Rep, 2019-11-21;9(1):17274. 2019-11-21 [PMID: 31754156] (Enzyme Assay, N/A)
• H Li, A Ham, TC Ma, SH Kuo, E Kanter, D Kim, HS Ko, Y Quan, SP Sardi, A Li, O Arancio, UJ Kang, D Sulzer, G Tang Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations Autophagy, 2018-10-12;15(1):113-130. 2018-10-12 [PMID: 30160596] (Mouse)
• SP Yun, D Kim, S Kim, S Kim, SS Karuppagou, SH Kwon, S Lee, TI Kam, S Lee, S Ham, JH Park, VL Dawson, TM Dawson, Y Lee, HS Ko ?-Synuclein accumulation and GBA deficiency due to L444P GBA mutation contributes to MPTP-induced parkinsonism Mol Neurodegener, 2018-01-08;13(1):1. 2018-01-08 [PMID: 29310663] (Bioassay, Mouse)
• RA Ashmus, DL Shen, DJ Vocadlo Fluorescence-Quenched Substrates for Quantitative Live Cell Imaging of Glucocerebrosidase Activity Meth. Enzymol., 2017-10-31;598(0):199-215. 2017-10-31 [PMID: 29306435] (Bioassay, Human)
• Choi S, Kim D, Kam T, Yun S, Kim S, Park H, Hwang H, Pletnikova O, Troncoso J, Dawson V, Dawson T, Ko H Lysosomal Enzyme Glucocerebrosidase Protects against Abeta1-42 Oligomer-Induced Neurotoxicity. PLoS ONE, 2015-12-02;10(12):e0143854. 2015-12-02 [PMID: 26629917] (Bioassay, Human)
• Berger , Zdenek, Perkins , Sarah, Ambroise , Claude, Oborski , Christin, Calabrese , Matthew, Noell , Stephen, Riddell , David, Hirst , Warren D Tool compounds robustly increase turnover of an artificial substrate by glucocerebrosidase in human brain lysates. PLoS ONE, 2015-03-12;10(3):e0119141. 2015-03-12 [PMID: 25763858] (Enzyme Assay, Human, Mouse)

Bioinformatics

• Gene Symbol: GBA1
• Uniprot:
  • P04062()