Recombinant Human N-Acetylmannosamine Kinase/GNE Protein, CF

R&D Systems | Catalog # 8268-GK

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human N-Acetylmannosamine Kinase/GNE Protein (8268-GK)
  • 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

E. coli

Accession Number

Q9Y223

Applications

Enzyme Activity
View all N-Acetylmannosamine Kinase/GNE Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human N-Acetylmannosamine Kinase/GNE protein
Thr406-Arg720, with an N-terminal Met and 6-His tag

Purity

>90%, 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

Met & Leu410

Predicted Molecular Mass

34 kDa

SDS-PAGE

31-39 kDa, reducing conditions

Activity

Measured by its ability to phoshorylate N-Acetyl-D-Mannosamine.
The specific activity is >130 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8268-GK
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT.
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: N-Acetylmannosamine Kinase/GNE

Sialic acid modification of cell surface molecules is crucial for many biologic processes, such as cell adhesion (1), formation or masking of recognition determinants (2), and stabilization of glycoprotein structure of glycoproteins (3). Furthermore, sialic acids are over-expressed in many tumor cells (4) and are associated with a higher metastatic potential (5). The rate limiting enzyme of sialic acid biosynthesis is a bifunctional enzyme, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), that converts UDP-GlcNAc to ManNAc and subsequently phosphorylates the ManNAc to ManNAc 6-phosphate (6). The activity of GNE is regulated through feedback inhibition by the downstream product, CMP-sialic acid (7). In humans, GNE mutation causes two genetic diseases, sialuria (8) and hereditary inclusion body myopathy (HIBM). In sialuria, a mutation of either Arg-263 or Arg-266 in the epimerase domain impairs the feedback inhibition of GNE (9). HIBM is an autosomal recessive neuromuscular disorder caused by more than 60 different point mutations (10).The recombinant human N-Acetylmannosamine Kinase only contains the kinase domain (11). The kinase activity was measured using a phosphatase-coupled method (12).

References

  1. Varki, A. (2007) Nature 446:1023.
  2. Karlsson, K.-A. (1995) Curr. Opin. Struct. Biol. 5:622.
  3. Rens-Domiano, S. and Reisine, T. (1991) J. Biol. Chem. 266:20094.
  4. Roth, J. et al. (1988) Proc. Natl. Acad. Sci. U. S. A. 85:2999.
  5. Bresalier, R. S. et al. (1990) Cancer Res. 50:1299.
  6. Stasche, R. et al. (1997) J. Biol. Chem. 273: 24319.
  7. Kornfeld, S. et al. (1964) Proc. Natl.Acad. Sci. U.S.A. 52:371.
  8. Weiss, P. et al. (1989) J. Biol. Chem. 264:17635.
  9. Eisenberg, I. et al. (2001) Nat. Genet. 29:83.
  10. Huizing, M. and Krasnewich, D. M. (2009) Biochim. Biophys. Acta 1792:881.
  11. Martinez, J. et al. (2012) J. Biol. Chem. 287:13656.
  12. Wu, Z. (2011) PLoS ONE 6:e23172.

Long Name

Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine Kinase

Alternate Names

DMRV, GLCNE, GNE, IBM2, NAcetylmannosamine Kinase, Uae1

Entrez Gene IDs

10020 (Human); 50798 (Mouse); 114711 (Rat)

Gene Symbol

GNE

UniProt

Q9Y223

Additional N-Acetylmannosamine Kinase/GNE Products

Product Documents for Recombinant Human N-Acetylmannosamine Kinase/GNE 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 N-Acetylmannosamine Kinase/GNE Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human N-Acetylmannosamine Kinase/GNE Protein, CF (8268-GK):

Materials
  • Assay Buffer (10X): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl2, 100 mM CaCl2 pH 7.0 (supplied in kit)
  • Recombinant Human N‑Acetylmannosamine Kinase/GNE (rhGNE) (Catalog # 8268-GK)
  • N-Acetyl-D-mannosamine (Sigma, Catalog # A8176), 100 mM stock in deionized water
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X Assay Buffer using deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock.
  3. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
  4. Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. Prepare Substrate Mixture composed of 0.4 mM ATP and 2 mM N-Acetyl-D-mannosamine in 1X Assay Buffer.
  6. Dilute rhGNE to 66.67 μg/mL in 1X Assay Buffer.
  7. Load 15 µL of the 66.67 μg/mL rhGNE into the plate in triplicate. Include a control containing 15 µL of 1X Assay Buffer.
  8. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
  9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
  10. Add 25 µL of Substrate Mixture to the wells, excluding the standard curve.
  11. Incubate sealed plate at room temperature for 10 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg) x coupling rate**

     *Derived from the phosphate standard curve using linear fitting and adjusted for Control
     **The coupling rate is 0.475 under these conditions.

Per Reaction:

  • rhGNE: 1 µg
  • Coupling Phosphatase 4: 0.1 µg
  • ATP: 0.2 mM
  • N-Acetyl-D-mannosamine: 1 mM

FAQs

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