Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF Summary
Details of Functionality
Measured by its ability to hydrolyze 4-methylumbelliferyl-N-acetyl-beta -D-glucosaminide (4-MU-GlcNAc) The specific activity is >3500 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived b. thetaiotaomicron O-GlcNAcase/OGA protein Gln22-Lys737, with an N-terminal Met and 6-His tag
>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
83 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
66-76 kDa, reducing conditions
Publications
Read Publications using 6779-GH in the following applications:
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 under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
Assay Buffer: 50 mM MES, 100 mM NaCl, pH 5.5
Recombinant B. thetaiotaomicron O-GlcNAcase/OGA (rBtOGA) (Catalog # 6779-GH)
Substrate: 4-Methylumbelliferyl-N-acetyl-beta -D-glucosaminide (Sigma, Catalog # M2133), 50 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rBtOGA to 2 ng/μL in Assay Buffer.
Dilute Substrate to 2 mM in Assay Buffer.
Load into a plate 50 μL of 2 ng/μL rBtOGA, and start the reaction by adding 50 μL of 2 mM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 2 mM Substrate.
Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.
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:
rBtOGA: 0.1 μg
Substrate: 1 mM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant B. thetaiotaomicron O-GlcNAcase/OGA Protein, CF
bifunctional protein NCOAT
FLJ11229
GH84
HEXC
HEXC3
Hexosaminidase B
hyaluronidase in meningioma
KIAA0679
MEA5FLJ23355
meningioma expressed antigen 5 (hyaluronidase)
Meningioma-expressed antigen 5
MGEA5
NCOAT
Nuclear cytoplasmic O-GlcNAcase and acetyltransferase
OGA
OGlcNAcase
O-GlcNAcase
Background
The addition of the monosaccharide beta -N-acetyl-D-glucosamine to serine and threonine residues in proteins (O‑GlcNAc glycosylation) is a dynamic, intracellular, post‑translational modification that shares features with phosphorylation (1). Almost all major classes of intracellular proteins are modified with O‑GlcNAc glycosylation. O‑GlcNAc is known to regulate gene transcription, act as an energy sensor to desensitize insulin response, and coordinate phosphorylation to control protein activity (2, 3, 4, 5). In humans, O‑GlcNAc is introduced by a single O‑linked N‑acetylglucosamine transferase, OGT, and removed by a single glycosidase, OGA. Both OGT and OGA are cytosolic. Enzymes with high sequence homology to human OGA have been found in human pathogens and symbionts (6, 7, 8), where these enzymes are proposed to metabolize O‑GlcNAc in human proteins. OGA from the human gut symbiont Bacteroides thetaiotaomicron and its human counterpart are very similar in structure and function, and both enzymes operate via an unusual 'substrate-assisted' catalytic mechanism (8, 9). Recombinant B. thetaiotaomicron OGA can be used as an enzymatic tool to investigate O‑GlcNAc glycosylation.
Wells, L. et al. (2001) Science 291:2376.
Wells, L. et al. (2003) Cell. Mol. Life Sci. 60:222.
Yang, X. et al. (2008) Nature 451:964-9.
Love, D.C .and Hanover, J.A. (2005). Sci. STKE 312:1.
Hart, G. W. et al. (2011) Annu. Rev. Biochem. in press.
Martinez-Fleites, C. (2008) Nat. Struct. Mol. Biol. 15:764.
Rao, F. V. et al. (2006) The EMBO J. 25:1569.
Dennis, R.J. et al. (2006) Nat. Struct. Mol. Biol. 13:365.
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