Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Prepare 1X Assay Buffer by diluting 10X Assay Buffer in 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.
Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
Prepare Substrate Mixture composed of 0.4 mM ATP and 20 mM Choline Chloride in 1X Assay Buffer. Dilute rhCHK beta to 20 ng/µL in 1X Assay Buffer.
Load 15 µL of the 20 ng/µL rhCHK beta into the plate in triplicate. Include a Control containing 15 µL of 1X Assay Buffer.
Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 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 Substrate 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) =
Adjusted 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 fitting and adjusted for Control. **The coupling rate is 0.475 under these conditions.
rhCHK beta : 0.3 µg
Coupling Phosphatase 4: 0.1 µg
ATP: 0.2 mM
Choline Chloride: 10 mM
Background: Choline Kinase beta
Choline kinase catalyzes the first reaction in the choline pathway for the biosynthesis of phosphatidylcholine and phosphatidylethanolamine, the major phospholipids in eukaryotic membranes. Phosphatidylcholine is important for a variety of function in eukaryotes such as facilitating the transport of cholesterol through the organism, acting as a precursor for second messengers and as a cofactor for several membrane-related enzymes (1, 2). Choline kinase also plays a vital role in the production of sphingomyelin, another important membrane phospholipid (3). While choline kinase alpha (CHKA) is more active on choline, Choline Kinase beta (CHKB) is more active on ethanolamine (4). These two enzymes can also form active heterodimer and oligomer (5, 6). CHKA is associated with breast, lung and prostate carcinomas (7, 8) and is a promising target for cancer diagnosis and treatment (9). Mice lacks CHKA die early in embryogenesis. In contrast, mice that lack CHKB survive to adulthood, but develop hindlimb muscular dystrophy and forelimb bone deformity (10, 11), suggesting that CHKB is a rate-limiting enzyme in skeletal muscles. The enzymatic activity of recombinant human CHKB is measured using a phosphatase-coupled method (12).
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