• Purity

  >75%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

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

• Activity

  Measured by its ability to transfer phosphate from ATP to glycerol. The specific activity is >1,500 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Met1-Glu502, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Thr2

• Predicted Molecular Mass

  57 kDa

• SDS-PAGE

  52-55 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer (1X): 25 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.0

• Recombinant E. coli Glycerol Kinase (rE.coli glpK) (Catalog # 7849-GK)

• Donor Substrate: Adenosine triphosphate (provided in kit)

• Acceptor Substrate: Glycerol (J.T. Baker, Catalog # 4043), 100 mM in deionized water

• Universal Kinase Activity Kit (Catalog # )

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard by adding 40 μL of the 1 mM Phosphate Standard to 360 μL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.

2. Continue 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. Create a reaction mixture containing 0.5 mM ATP and 12.5 mM Glycerol in Assay Buffer.

4. Dilute rE.coli glpK to 2.5 μg/mL in Assay Buffer.

5. Dilute Coupling Enzyme to 10 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 2.5 μg/mL rE.coli glpK into the plate. Include a Control containing 20 μL of Assay Buffer.

8. Load 10 μL of the 10 μg/mL Coupling Phosphatase into the wells containing enzyme and blanks.

9. Load 20 μL of the reaction mixture into the wells containing enzyme and blanks to start the reactions.

10. Incubate sealed plate 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:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
     **Experimentally derived (6). Under the assay conditions, the coupling rate is 0.475.

Per Reaction:

• rE.coli glpK: 0.05 μg

• ATP: 10 nmol

• Glycerol: 250 nmol

• Coupling Phosphatase 4: 0.1 μg

Background: Glycerol Kinase

Glycerol kinase from   E. coli (glpK) catalyzes the ATP-dependent phosphorylation of glycerol to produce   sn-glycerol-3-phosphate (G3P), the first and rate-limiting step in the utilization of glycerol (1). In the presence of glycerol, glpK is stimulated by interaction with the membrane-bound glycerol facilitator (2). In the presence of glucose, glpK activity is allosterically inhibited by fructose-1,6-bisphosphate (FBP) of the glycolytic pathway (3). Under physiological conditions, the enzyme is in an equilibrium between the active dimer and the inactive tetramer. FBP binds to and stabilizes the inactive form, therefore shifting the usage of glycerol metabolic pathway to glycolytic pathway (4). glpK is also inhibited by phosphocarrier protein IIA  ^Glc by a regulatory mechanism distinct from that of FBP (5). GlpK is a member of a superfamily of ATPases that includes actin, hexokinase and the heat shock protein hsc70. Although these proteins are dissimilar in amino acid sequence and function, they share similar tertiary folds and likely the same catalytic mechanism. The enzyme activity was measured using a phosphatase-coupled kinase assay (6).

• References:

1. Pettigrew, D.W. et al. (1988) J. Biol. Chem. 263: 135.

2. Voegele, R.T. et al. (1993) J. Bacteriol. 175:1087.

3. Applebee, M.K. et al. (2011) J. Biol. Chem. 286: 23150.

4. Hurley, J.H. et al. (1993) Science 259:673.

5. Feese, M.D. et al. (1998) Structure 6:1407.

6. Wu, Z.L. (2011) PLoS ONE 6(8): e23172.

• Entrez Gene IDs:

  2710 (Human); 14933 (Mouse); 948423 (E. coli)

• Alternate Names:

  ATP:glycerol 3-phosphotransferase; EC 2.7.1.30; GK; GK1; GKD; glpK; Glycerokinase; Glycerol Kinase