Recombinant S. cerevisiae ATP Sulfurylase/MET3 Protein, CF

2 μg/lane of Recombinant Yeast ATP‑Sulfurylase/MET3 (Catalog # 7175-AS) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 57-58 kDa.

• Reactivity: Ye
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant S. cerevisiae ATP Sulfurylase/MET3 Protein, CF Summary

• Details of Functionality: Measured by its ability to sulfurylate ATP. The specific activity is >2,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived yeast ATP-Sulfurylase/MET3 protein
  Pro2-Phe511, with N-terminal Met and 6-His tag
• Accession #: P08536
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: MET3
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 59 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 57-58 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 1 mM MgCl₂, 20 mM Na₂SO₄, pH 8.0
  • Recombinant Yeast ATP Sulfurylase/MET3 (ryMET3) (Catalog # 7175-AS)
  • Recombinant Human Inorganic Pyrophosphatase/PPA1 (rhPPA1) (Catalog # 6557-PP)
  • Recombinant Penicillum chrysogenum APS Kinase/APSK (rAPS Kinase) (Catalog # 7176-SK)
  • Adenosine Triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute ATP to 1.8 mM in Assay Buffer.
  2. Dilute rhPPA1 to 3 µg/mL in Assay Buffer.
  3. Dilute rAPS Kinase to 120 µg/mL in Assay Buffer.
  4. Prepare Reaction Mixture by combining equal volumes of 1.8 mM ATP, 3 µg/mL rhPPA1, and 120 µg/mL rAPS Kinase.
  5. Dilute ryMET3 to 0.2 µg/mL in Assay Buffer.
  6. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  7. 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.
  8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  9. Load 25 µL of the 0.2 µg/mL ryMET3 into the plate. Include a Control containing 25 µL of Assay Buffer.
  10. Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
  11. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells.
  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:

  Specific Activity (pmol/min/µg) =	Phosphate released* (nmol) x (1000 pmol/nmol)	÷ 2**
  	Incubation time (min) x amount of enzyme (µg)

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
  **Two inorganic phosphates are generated from each MET3 produced pyrophosphate

  Per Reaction:
  • ryMET3: 0.005 µg
  • ATP: 0.3 mM
  • rhPPA1: 0.025 µg
  • rAPS Kinase: 1 µg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant S. cerevisiae ATP Sulfurylase/MET3 Protein, CF

• ATPSulfurylase
• ATP-Sulfurylase
• MET3

Background

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) and generates pyrophosphate (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 ATP Sulfurylase reaction is highly unfavorable energetically with the standard free-energy change of approximately +46 kJ/mol (4). To drive the reaction forward, APSK and pyrophosphatase are generally added into the reaction simultaneously to remove the products. The recombinant S. cerevisiae ATP Sulfurylase can be used for in vitro PAPS synthesis (5, 6).

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. Hawes, C.S. and Nicholas, D.J.D. (1973) Biochem. J. 133: 541.
5. Wu, Z.L. et al. (2002) The FASEB J. 16:539.
6. Lin, C.H. et al. (1995) J. Am.Chem. Soc. 117:8031.

Publications for ATP-Sulfurylase/MET3 (7175-AS)(1)

Publication using 7175-AS

• Rubera, I;Clotaire, L;Laurain, A;Destere, A;Martin, L;Duranton, C;Leftheriotis, G; A Plasma Pyrophosphate Cutoff Value for Diagnosing Pseudoxanthoma Elasticum International journal of molecular sciences 2024-06-13 [PMID: 38928212] (Enzyme Assay)

Bioinformatics

• Gene Symbol: MET3
• Uniprot:
  • P08536()