Recombinant Human Adenosine Deaminase/ADA Protein, CF

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

Recombinant Human Adenosine Deaminase/ADA Protein, CF Summary

• Details of Functionality: Measured by the ability to catalyze the hydrolytic deamination of adenosine to inosine. The specific activity is >44,000 pmol/min/μg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Adenosine Deaminase/ADA protein
  Met1-Leu363, with a C-terminal 6-His tag
• Accession #: P00813
• N-terminal Sequence: Inconclusive result, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments
• Protein/Peptide Type: Recombinant Enzymes
• Gene: ADA
• 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: 42 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: 40-42 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, pH 7.5
  • Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
  • Substrate: Adenosine (Sigma, Catalog # A9251), 10 mM stock in deionized water
  • UV plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Thaw the Substrate stock and then warm it at 37 °C for 20 minutes. Mix well after incubation.
  2. Dilute rhADA to 0.1 ng/μL in Assay Buffer.
  3. Dilute Substrate to 200 μM in Assay Buffer.
  4. Load into a plate 50 μL of 0.1 ng/μL rhADA and start the reaction by adding 50 μL of 200 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
  5. Read plate at a wavelength of 265 nm (bottom read) in kinetic mode for 5 minutes.
  6. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x -1 x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 8500 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rhADA: 0.005 μg
  • Substrate: 100 μM

Notes

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

Alternate Names for Recombinant Human Adenosine Deaminase/ADA Protein, CF

• ADA
• ADA1
• adenine deaminase
• Adenosine aminohydrolase
• Adenosine Deaminase
• EC 3.5.4.4

Background

Adenosine Deaminase (ADA, adenosine aminohydrolase) is one of the key enzymes of purine nucleotide catabolism. It catalyses the hydrolytic deamination of adenosine and deoxy‑adenosine to inosine and deoxyinosine (1, 2). ADA is expressed in virtually all tissues and is expressed at high levels in T-lymphocytes. Adenosine Deaminase deficiency can cause a form of SCID (severe combined immunodeficiency) and lymphopenia in both B- and T-cell lineages (3, 4). ADA can be used as a sensitive diagnostic marker for tuberculous pleuritis (5). Although it is primarily a cytosolic enzyme, ADA is known to be a positive regulator of T-cell co‑activation due to its binding to CD26 at the cell surface. The interaction of ADA with CD26 regulates lymphocyte-epithelial cell adhesion (6).

1. Wolfenden, R.V. et al. (1969) Biochemistry 6:2412.
2. Lowenstein, J.M. (1972) Physiol. Rev. 52:382.
3. Giblett, E.R. et al. (1972) Lancet 2:1067.
4. Coleman, M.S. et al. (1978) J. Biol. Chem. 253:1619.
5. Baba, K. et al. (2008) PLoS ONE 3:e2788.
6. Gines, S. et al. (2002) Biochem. J. 361:203.

Publications for Adenosine Deaminase/ADA (7048-AD)(1)

Publication using 7048-AD

• N Gourdin, M Bossennec, C Rodriguez, S Vigano, C Machon, C Jandus, D Bauché, J Faget, I Durand, N Chopin, O Tredan, JC Marie, B Dubois, J Guitton, P Romero, C Caux, C Ménétrier- Autocrine Adenosine regulates tumor polyfunctional CD73+CD4+ effector T cells devoid of immune checkpoints Cancer Res., 2018-03-20;0(0):. 2018-03-20 [PMID: 29559470] (Bioassay, Human)

Bioinformatics

• Gene Symbol: ADA
• Uniprot:
  • P00813()