Recombinant E. coli Glycerol Kinase Protein, CF Summary
Met1-Glu502, with a C-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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.
- 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 # EA004)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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.
- 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.
- Create a reaction mixture containing 0.5 mM ATP and 12.5 mM Glycerol in Assay Buffer.
- Dilute rE.coli glpK to 2.5 µg/mL in Assay Buffer.
- Dilute Coupling Enzyme to 10 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 20 µL of the 2.5 µg/mL rE.coli glpK into the plate. Include a Control containing 20 µL of Assay Buffer.
- Load 10 µL of the 10 µg/mL Coupling Phosphatase into the wells containing enzyme and blanks.
- Load 20 µL of the reaction mixture into the wells containing enzyme and blanks to start the reactions.
- Incubate sealed plate at room temperature for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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.
**Experimentally derived (6). Under the assay conditions, the coupling rate is 0.475.
- 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 IIAGlc 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).
- Pettigrew, D.W. et al. (1988) J. Biol. Chem. 263: 135.
- Voegele, R.T. et al. (1993) J. Bacteriol. 175:1087.
- Applebee, M.K. et al. (2011) J. Biol. Chem. 286: 23150.
- Hurley, J.H. et al. (1993) Science 259:673.
- Feese, M.D. et al. (1998) Structure 6:1407.
- Wu, Z.L. (2011) PLoS ONE 6(8): e23172.
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Kinase Activity Assays
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