Recombinant Human Glucokinase/GCK Protein, CF Summary
Product Specifications
| Met | 6-His tag | Human
Cystatin A (Met1-Phe98) Accession # P01040 | GS | Human Glucokinase/GCK (Val16-Gln465) Accession # P35557 |
| N-terminus | C-terminus | |||
Analysis
Product Datasheets
Carrier Free
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.
7840-GK
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, 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 Procedure
- Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, pH 7.0
- Recombinant Human Glucokinase/GCK (rhGCK) (Catalog # 7840-GK)
- Acceptor Substrate: Glucose (Sigma, Catalog # G5767), 2 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
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit to 100 µM by adding 40 µL of standard to 360 µL of Assay Buffer. This is the first point of the standard curve.
- Perform six additional one‑half serial dilutions in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Dilute Coupling Phosphatase 4 to 10 µg/mL in Assay Buffer.
- Dilute rhGCK to 10 µg/mL in Assay Buffer.
- Prepare a substrate mixture containing 0.5 mM ATP and 250 mM glucose in Assay Buffer.
- Load 50 µL of each point of the standard curve to empty wells.
- Load 20 µL dilute rhGCK to wells. Include Control containing 20 µL Assay Buffer.
- Load 10 µL dilute Coupling Enzyme to wells used, excluding the standard curve.
- Start the reaction by adding 20 µL substrate mixture to wells used, excluding the standard curve.
- Cover the plate with a plate sealer and incubate 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.
**Under these conditions, the coupling rate is 0.475 (4).
- rhGCK: 0.2 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM (10 nmol)
- Glucose: 100 mM (5000 nmol)
Reconstitution Calculator
Background: Glucokinase/GCK
Hexokinases phosphorylate hexose to form hexose 6‑phosphate, the first step for hexose metabolism. There are four mammalian hexokinases (I, II, III and IV) and hexokinase IV is commonly known as glucose kinase (GCK). Unlike hexokinase I, II and III, which have high affinity for glucose and are strongly inhibited by the product, glucose‑6‑phosphate (1), GCK has much lower affinity for glucose and is not inhibited by the product (2, 3). Consequently, GCK has a Km for glucose of approximately 7 mM (4), which is 100 times higher than that of hexokinase I, II, and III. This unique enzymatic property of GCK allows it to respond to blood glucose levels and contribute to the maintenance of blood glucose levels within the normal physiological range of 4 mM to 6 mM. In the pancreatic islets, GCK serves as a glucose sensor to control insulin release in the beta cells, and to control glucagon release in the alpha cells (5). In hepatocytes, GCK responds to changes of ambient glucose levels by increasing or reducing glycogen synthesis. Mutations in GCK have been associated with non‑insulin‑dependent diabetes mellitus (6, 7), maturity‑onset diabetes of the young type 2 (8), and hyperinsulinemia of infancy (9). The enzyme activity was measured using a phosphatase coupled kinase assay (4).
- Aleshin, A.E. et al.(1998) Structure 6:39.
- Takeda, J. et al. (1993) J. Biol. Chem. 268:15200.
- Lange, A.J. et al. (1991) Biochem. J. 277:159.
- Wu, Z.L. (2011) PLoS ONE 6(8):e23172.
- Xu, L.Z. et al. (1995) J. Biol. Chem. 270:9939.
- Gidh-Jain, M. et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90:1932.
- Stoffel, M. et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7698.
- Hattersley, A.T. et al. (1992) Lancet 339:1307.
- Gloyn, A.L. et al. (2003) Diabetes 52:2433.
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