详细说明
- Purity>95%, 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.
- ActivityMeasured by its ability to transfer phosphate from ATP to mevalonic acid. The specific activity is >2,400 pmol/min/μg, as measured under the described conditions.
- SourceE. coli-derived Met1-Leu396, with a C-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet1 - Predicted Molecular Mass43 kDa
- SDS-PAGE38-46 kDa, reducing conditions
| 9349-VK | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl 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
Materials
- Universal Kinase Activity Kit (Catalog # )
- 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl2, 100 mM CaCl2, pH 7.0
- Recombinant Human MVK (rhMVK) (Catalog # 9349-VK)
- RS-Mevalonic Acid (Sigma, Catalog # 90469), 50 mM stock in deionized water
- 96-well Clear Plate (Catalog # )
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer by diluting 10X stocks 10 fold with deionized water.
- 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. This is the first point of the standard curve.
- Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 0.4 mM ATP (supplied in kit) and 2 mM Mevalonic Acid in 1X Assay Buffer.
- Dilute rhMVK to 3.33 ng/µL in 1X Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to10 µg/mL in 1X Assay Buffer.
- Load 50 µL of each dil6ution of the standard curve into a plate. Include a curve blank containing 50 µL of 1X Assay Buffer.
- Load 15 µL of the 3.33 ng/µL rhMVK into empty wells of the same plate as the curve. Include a control containing 15 µL of 1X Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- 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.
** The coupling rate is 0.475 under these conditions.
- rhMVK: 0.05 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- Mevalonic Acid: 1 mM
Background: MVK
Mevalonate kinase converts (R)-mevalonate to (R)-5-phosphomevalonate using ATP as the donor substrate (1). It is a key early enzyme in isoprenoid and sterol synthesis and may be a regulatory site in cholesterol biosynthetic pathway (2). Deficiency of the enzyme results in mevalonic aciduria, hyperimmunoglobulinaemia D and periodic fever syndrome, a disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal dismay and skin rash (3, 4, 5). The enzymatic activity of the recombinant human MVK is measured using a phosphatase-coupled method (6).
- References:
- Green, T.R. and Baisted, D.J. (1970) Anal. Biochem. 38:130.
- Redding-Johanson A.M., et al. (2011) Metab. Eng. 13:194.
- Hager, E.J. and Gibson, K.M. (2007) N. Engl. J. Med. 357:1871.
- Favier, L.A. and Schulert, G.S. (2016) Appl. Clin. Genet. 9:101.
- Stoffels, M. et al. (2014) Rheumatol. Int. 34:295.
- Wu, Z.L. (2011) PLoS ONE 6:e23172.
- Long Name:Mevalonate Kinase
- Entrez Gene IDs:4598 (Human); 17855 (Mouse); 81727 (Rat)
- Alternate Names:EC 2.7.1.36; LH receptor mRNA-binding protein; LRBP; mevalonate kinase (mevalonic aciduria); mevalonate kinase 1; mevalonate kinase; Mevalonic Aciduria; MKFLJ96772; MVK; MVLK; POROK3
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