• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

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

• Activity

  Measured by its ability to hydrolyze the 5’-phosphate group from the substrate adenosine-5’-monophosphate (AMP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # ). The specific activity is >10,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Chinese Hamster Ovary cell line, CHO-derived Trp29-Lys549, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Trp29

• Predicted Molecular Mass

  59 kDa

• SDS-PAGE

  60 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM Tris, 0.5 mM MgCl₂, pH 7.5

• Recombinant Mouse 5'‑Nucleotidase/CD73 (rmCD73) (Catalog # 4488-EN)

• Substrate: AMP (Sigma, Catalog # A1752), 10 mM stock in deionized water

• Malachite Green Phosphate Detection Kit (Catalog # )

• 96-well Clear Plate (Costar, Catalog # 92592)

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

1. Dilute rmCD73 to 0.04 µg/mL in Assay Buffer.

2. Prepare a standard curve from the 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer 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 dilution to use as a standard).

3. Perform six additional one-half serial dilutions of the 100 µM phosphate stock. The standard curve has a range of 0.039 to 2.5 nmol per well.

4. Load 25 µL of 0.04 µg/mL rmCD73, standard curve, and blanks (Assay Buffer) into a plate.
   Dilute Substrate to 200 µM in Assay Buffer.

5. Add 25 µL of the Substrate to all wells. Mix well.

6. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.

7. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.

8. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

9. Read plate at 620 nm (absorbance) in endpoint mode.

10. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.

Per Reaction:

• rmCD73: 0.001 µg

• Substrate: 100 µM

Background: 5'-Nucleotidase/CD73

CD73, an ecto-5’-Nucleotidase, is an ectoenzyme that is attached to the cell membrane by a glycosyl phophatidylinositol anchor (1, 2). The enzyme is expressed by most cell types. The 5’-Nucleotidase activity of CD73 converts extracellular nucleoside-5’-monophosphates to nucleosides. CD73 is one of several enzymes responsible for the production of extracellular adenosine, a signaling molecule that is involved in responses to inflammation and tissue injury (3).

• References:

1. Resta, R. et al. (1993) Gene 133:171.

2. Resta, R. et al. (1998) Immunol. Rev. 161:95.

3. Pilcher, M. et al. (2003) J. Biol. Chem. 278:13468.

• Entrez Gene IDs:

  4907 (Human); 23959 (Mouse); 58813 (Rat)

• Alternate Names:

  5' nucleotidase (CD73); 5'-NT; 5-NT; 5'Nucleotidase; 5'-Nucleotidase; 5'-nucleotidase, ecto (CD73); CD73 antigen; CD73; E5NT; EC 3.1.3.5; ecto-5'-nucleotidase; eN; eNT; NT; NT55'-nucleotidase; NT5E; NTE; Purine 5-Prime-Nucleotidase