Quantification of CD73 Nucleotidase Activity

Nucleotidase activity assays were performed with purified recombinant enzyme by quantifying the inorganic phosphate produced from CD73-catalyzed AMP hydrolysis. Briefly, recombinant CD73 (soluble form, residues 27-549 including a His-tag at the C-terminus) was produced in Sf9 insect cells using the pFastBac baculovirus system (ThermoFisher Scientific) and purified by two consecutive steps (Ni-MTA affinity and size exclusion chromatography) as previously described^{78 (link)}. To determine the enzymatic activity, recombinant CD73 (2 nM, final concentration) was incubated in a reaction buffer (Tris 50 mM, pH7.5, NaCl 100 mM, MgCl₂ 1 mM, CaCl₂ 1 mM and ZnCl₂ 5 µM) and reaction was started by addition of either the natural substrate (AMP) or the test substrates NAD⁺, NADH or NMN (nicotinamide mononucleotide), with concentrations ranging from 0 to 250 µM, and incubated for 2.5 minutes at 37 °C under gentle shaking. Then, the reaction was stopped by addition of the Green Malachite reagent (Phosphate assay kit, Sigma) and production of inorganic phosphate was quantified by reading the optical density at 630 nm on a plate reader (Tecan Sunrise). A standard phosphate concentration range (0–50 µM) for normalizing raw data was included and the results correspond to the average of three independent experiments (GraphPad Prism 8 was used for analyzing and plotting the data).