Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF

R&D Systems | Catalog # 8020-GK

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Key Product Details

  • R&D Systems E. coli-derived Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK (8020-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

P75959

Applications

Enzyme Activity
View all N-Acetyl-D-Glucosamine Kinase/NAGK Proteins and Enzymes »

Product Specifications

Source

E. coli-derived e. coli N-Acetyl-D-Glucosamine Kinase/NAGK protein
Met1-Asp303 with C-terminal 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<0.01 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met1

Predicted Molecular Mass

34 kDa

SDS-PAGE

37-38 kDa, reducing conditions

Activity

Measured by its ability to phosphorylate N-acetyl-D-glucosamine.
The specific activity is >10,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8020-GK
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol, Brij-35 and DTT.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: N-Acetyl-D-Glucosamine Kinase/NAGK

N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) are repeating sugar units of peptidoglycan, the major component of bacterial cell wall structure and a drug target of various antibiotics including penicillin (1). Recently, interest has been generated regarding cell wall peptidoglycan catabolism, because as much as 50% of the peptidoglycan is turned over in one generation of bacterial growth (2). N-acetylglycosamine kinase (nagK) is a key enzyme for the recycling of GlcNAc in E. coli (3). Due to its high activity, it can be used for efficient conversion of GlcNAc to GlcNAc-6-phosphate. The enzyme is assayed using a phosphatase-coupled kinase assay (4).

References

  1. Plumbridge, J. (2009). J. Bacteriol. 191:5641.
  2. Reith, J. et al (2011). J. Bacteriol. 193:5386.
  3. Uehara, T. and Park, J.T.(2011) J. Bacteriol. 186:7273.
  4. Wu, Z.L. (2011) PLoS One 6:e23172.

Long Name

N-Acetyl-D-Glucosamine Kinase

Alternate Names

GlcNAc Kinase, GNK, N-Acetylglucosamine Kinase

Entrez Gene IDs

55577 (Human); 56174 (Mouse); 297393 (Rat); 945664 (E. coli)

Gene Symbol

NAGK

UniProt

P75959

Additional N-Acetyl-D-Glucosamine Kinase/NAGK Products

Product Documents for Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF

Certificate of Analysis

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Product Specific Notices for Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF

For research use only

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Protocols

View specific protocols for Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF (8020-GK):

Materials
  • Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0
  • Recombinant E.coli N‑Acetyl‑D‑Glucosamine Kinase/NAGK (rE.coli NAGK) (Catalog # 8020-GK)
  • Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog # 1079), 1 M stock in deionized water
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. 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 Assay Buffer for a 100 µM stock.
  2. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare a reaction mixture composed of 0.5 mM ATP and 12.5 mM GlcNAc in Assay Buffer.
  4. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
  5. Dilute rE.coli NAGK to 1 µg/mL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 20 µL of the 1 µg/mL rE.coli NAGK into the plate. Include a control containing 20 µL of Assay Buffer.
  8. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.
  9. Add 20 µL of reaction mixture to the wells, excluding the standard curve.
  10. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.
  11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  12. Add 100 µL of deionized water to all wells. Mix briefly.
  13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
  14. Read plate at 620 nm (absorbance) in endpoint mode.
  15. 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 or 4-parameter fitting and adjusted for control.
     ** Under these conditions, the coupling rate is 0.475.

Per Reaction:

  • rE.coli NAGK: 0.020 µg
  • Coupling Phosphatase 4: 0.1 ug
  • ATP: 0.2 mM
  • GlcNAc: 5 mM

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