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. 2019 Oct 25;14(10):e0220094. doi: 10.1371/journal.pone.0220094

Fig 4. Determination of enzyme activity of purified cytosolic cN-II and inhibitor selectivity.

Fig 4

A) Enzyme reaction for cN-II (Human cytosolic 5'-Nucleotidase) was carried out using increasing concentrations of enzyme and 10μM AMP for 60min at 37°C and activity was determined following AMP-Glo assay as described in Materials and Methods section. The results are shown as net RLU after subtraction of no enzyme control. The experiment was done in triplicates; results shown are mean ± SD. B) Selectivity of the inhibitor AMP-CP against CD73 and cytosolic cN-II was determined using purified CD73 (0.1 ng/Rx) and cN-II (0.6 mU/Rx) by AMP-PC using 10μM AMP substrate at 23°C for 5 min (CD73) and at 37°C for 30 min (cN-II). Activities were determined following AMP-Glo protocol. The inhibitor inhibited CD73 activity with an IC50 value of 3x10-7M with minimal or no inhibition of cN-II. Experiments were done in triplicates; results shown are mean ± SD. C) Monitoring enzymatic activity of membrane-associated CD73 in cell-based assay.