I. Monitoring enzymatic activity of purified CD73 and CD39

Determination of enzyme activity of purified CD73. In a 25 μl reaction buffer (10mM HEPES, pH 7.4, 2mM MgCl₂, 1mM CaCl₂, and 0.1mg/ml BSA) containing enzyme and AMP substrate at 1μM to 10μM (96-well format), enzyme activity was performed at 23°C for specified time period and terminated by the addition of equal volume (25μl) of AMP-Glo^™ Reagent I containing 50 μM (final concentration) of known CD73 inhibitor, such as AMPCP. Reactions were mixed well and incubated for additional 30 min at 23°C. This was followed by the addition of 50 μl of AMP Detection Solution (10μl of AMP-Glo^™ Reagent II per ml of Kinase-Glo^® One Solution) and incubated for additional 60 min at 23°C before reading luminescence.

The assay format is flexible, i.e., volumes of the standards (AMP) or any AMP utilizing reactions as well as both reagents volumes can be maintained at the ratio of 1:1:2 regardless of the absolute volumes used (CD73 reaction: AMP-Glo^™ Reagent I: Detection Solution). Thus, for 96-well format, we recommend using 25μl:25μl:50μl or multiples of these volumes.

Determination of enzyme activity of purified cytosolic cN-II. Activity of purified cN-II (Human cytosolic 5'-Nucleotidase) was determined using 25μl reaction buffer (50mM HEPES, pH 7.0, 100mM KCl, 20mM MgCl₂, 5mM DTT, 2.5mM dATP, and 0.1mg/ml BSA) containing enzyme and AMP substrate at 10μM (96-well format). Reaction continued at 37°C for 60 minutes or for a certain time period and terminated by the addition of equal volume (25μl) of AMP-Glo^™ Reagent I. Reactions were mixed well and incubated for additional 30 min at 23°C. This was followed by the addition of 50 μl of AMP Detection Solution (10μl of AMP-Glo^™ Reagent II per ml of Kinase-Glo^® One Solution) and incubated for additional 60 min at 23°C before reading luminescence.

Determination of enzyme activity of purified CD39. Activity of CD39 was determined using reaction buffer that contains 25mM Tris, pH 7.5, 5mM CaCl₂, and 0.1mg/ml BSA), with 1μM to 5μM ATP or ADP as substrate, respectively. When ATP is used as a substrate, an equal volume of Kinase-Glo^® One Solution was added to CD39 reactions and incubated for 15 min before reading luminescence using a luminometer. When ADP is used as a substrate for CD39, after enzyme reaction completion, an equal volume of AMP Detection Solution with a known CD39 known inhibitor, such as POM 1 at 50μM final concentration was added. Reactions were mixed well and incubated for 60min before reading luminescence using luminometer. For enzyme dilution, reaction buffer can be used for dilution of enzymes.