• 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.  

• Activity

  Measured 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.

• Source

  E. coli-derived Met1-Leu396, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met1

• Predicted Molecular Mass

  43 kDa

• SDS-PAGE

  38-46 kDa, reducing conditions

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

1. Prepare 1X Assay Buffer by diluting 10X stocks 10 fold with deionized water.

2. 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.

3. 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.

4. Prepare reaction mixture containing 0.4 mM ATP (supplied in kit) and 2 mM Mevalonic Acid in 1X Assay Buffer.

5. Dilute rhMVK to 3.33 ng/μL in 1X Assay Buffer.

6. Dilute Coupling Phosphatase 4 (supplied in kit) to10 μg/mL in 1X Assay Buffer.

7. Load 50 μL of each dil6ution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.

8. 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.

9. Add 10 μL of 10 μg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.

10. Add 25 μL of reaction mixture to the wells, excluding the standard curve.

11. Incubate sealed plate at room temperature for 10 minutes.

12. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.

13. Add 100 μL of deionized water to all wells. Mix briefly.

14. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

15. Read plate at 620 nm (absorbance) in endpoint mode.

16. Calculate specific activity:

   *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.

Per Reaction:  
 

• 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:

1. Green, T.R. and Baisted, D.J. (1970) Anal. Biochem. 38:130.

2. Redding-Johanson A.M., et al. (2011) Metab. Eng. 13:194.

3. Hager, E.J. and Gibson, K.M. (2007) N. Engl. J. Med. 357:1871.

4. Favier, L.A. and Schulert, G.S. (2016) Appl. Clin. Genet. 9:101.

5. Stoffels, M. et al. (2014) Rheumatol. Int. 34:295.

6. 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