Recombinant P. chrysogenum APS kinase/APSK Protein, CF
Recombinant P. chrysogenum APS kinase/APSK Protein, CF Summary
Met1-Glu211, 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 and NaCl.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Universal Kinase Activity Kit (Catalog # EA004)
- Assay Buffer: 25 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 10 mM MgCl2, pH 7.5
- Recombinant P. chrysogenum APS Kinase/APSK (rAPS Kinase) (Catalog # 7176-SK)
- Adenosine 5'-phosphosulfate (APS) (Sigma, Catalog # A5508), 1 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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 Assay Buffer for a 100 µM stock. This is the first point of the starndard 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 5.0 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Dilute rAPS Kinase to 15 ng/µL in Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.
- Dilute APS and ATP to 0.24 mM in Assay Buffer.
- Prepare substrate mixture by combining equivalent volumes of 0.24 mM APS and 0.24 mM ATP.
- Load 10 µL of the 15 ng/µL rAPS Kinase in triplicate to the plate. Include a control containing 10 µL of Assay Buffer.
- Load 10 µL of 10 µg/mL Coupling Phosphatase 4 into wells containing enzyme and Assay Buffer.
- Add 30 µL of substrate mixture to the wells, excluding the standard curve.
- Seal the plate and incubate at room temperature for 15 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)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.Per Reaction:
- rAPS Kinase: 0.150 µg
- Coupling Phosphatase 4: 0.100 µg
- APS: 3600 pmol
- ATP: 3600 pmol
Background: APS Kinase/APSK
Sulfur metabolism is essential to all organisms. Inorganic sulfate is sequentially activated by ATP sulfurylase and adenosine 5′‑phosphosulfate kinase (APSK). ATP sulfurylase converts ATP to adenosine‑5'‑phosphosulfate (APS) (1). APSK subsequently phosphorylates APS at the 3’ site to generate 3'‑phosphoadenosine‑5'‑phosphosulfate (PAPS) (2). PAPS is the sulfur donor required for all sulfotransferase reactions in humans and is also the sulfur source for synthesis of cysteine, methionine and glutathione (3). The P. chrysogenum APSK is a homodimer that undergoes a temperature‑dependent reversible dissociation into inactive monomers (4). The kinetic mechanism of this enzyme is strictly ordered with ATP bound before APS and PAPS dissociating before MgADP. APS is reported as a potent substrate inhibitor with the formation of a dead‑end E·MgADP·APS ternary complex as the cause of the substrate inhibition (5). The recombinant P. chrysogenum APSK is required for in vitro PAPS synthesis (6, 7) and the enzymatic activity was measured using a phosphatase‑coupled method (8).
- Robbins, P. W. and Lipmann, F. (1958) J. Biol. Chem. 233:686.
- MacRae, I.J. et al. (1998) J. Biol. Chem. 273:28583.
- Strott, C.A. (2002) Endocr. Rev 23:703.
- Renosto, F. et al. (1985) J. Biol. Chem. 260:1535.
- Renosto, F. et al. (1991) Arch. Biochem. Biophys. 284:30.
- Wu, Z.L. et al. (2002) The FASEB J. 16:539.
- Lin, C.H. et al. (1995) J. Am.Chem. Soc. 117:8031.
- Wu, Z.L. (2011) PLoS ONE 6(8): e23172.
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