Recombinant P. chrysogenum APS kinase/APSK Protein, CF 20 UG

• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to phosphorylate adenosine 5'-phosphosulfate (APS). The specific activity is >100 pmol/min/μg, as measured under the described conditions, and is subject to known limitations imposed by a factor of coupling rate (8). See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Met1-Glu211, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser2

• Predicted Molecular Mass

  25 kDa

• SDS-PAGE

  29-30 kDa, reducing conditions

Assay Procedure

Materials

• Universal Kinase Activity Kit (Catalog # )

• 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 # )

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. 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.

2. 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.

3. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

4. Dilute rAPS Kinase to 15 ng/µL in Assay Buffer.

5. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.

6. Dilute APS and ATP to 0.24 mM in Assay Buffer.

7. Prepare substrate mixture by combining equivalent volumes of 0.24 mM APS and 0.24 mM ATP.

8. Load 10 µL of the 15 ng/µL rAPS Kinase in triplicate to the plate. Include a control containing 10 µL of Assay Buffer.

9. Load 10 µL of 10 µg/mL Coupling Phosphatase 4 into wells containing enzyme and Assay Buffer.

10. Add 30 µL of substrate mixture to the wells, excluding the standard curve.

11. Seal the plate and incubate at room temperature for 15 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.

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).

• References:

1. Robbins, P. W. and Lipmann, F. (1958) J. Biol. Chem. 233:686.

2. MacRae, I.J. et al. (1998) J. Biol. Chem. 273:28583.

3. Strott, C.A. (2002) Endocr. Rev 23:703.

4. Renosto, F. et al. (1985) J. Biol. Chem. 260:1535.

5. Renosto, F. et al. (1991) Arch. Biochem. Biophys. 284:30.

6. Wu, Z.L. et al. (2002) The FASEB J. 16:539.

7. Lin, C.H. et al. (1995) J. Am.Chem. Soc. 117:8031.

8. Wu, Z.L. (2011) PLoS ONE 6(8): e23172.

• Long Name:

  Adenosine-5'-phosphosulfate 3'-phosphotransferase

• Alternate Names:

  APS Kinase; APSK