ODC Enzyme Activity Assay

ODC activity was measured using 100 ng of purified ODC protein diluted in a buffer containing 25 mM Tris and 0.1 mM EDTA. Each of the reactions was added to 200 μL of assay mix containing 6.25 mM Tris HCl (pH 7.5), 100 μM l-ornithine, 50 μM pyridoxal-5-phosphate, and 0.1 μCi [1-¹⁴C] l-ornithine (American Radiolabeled Chemicals, Inc., specific activity 55 mCi/mmol) +/– 1.56 mM DTT in a microcentrifuge tube. The microcentrifuge tubes were then placed into scintillation vials containing a piece of filter paper saturated with 200 μL of 0.1 M NaOH to capture the release of radiolabeled carbon dioxide. The samples were incubated in a 37 °C incubator while shaking for 30 min. The enzymatic reaction was stopped by adding 250 μL of 5 M sulfuric acid to each sample and incubating at 37 °C while shaking for 30 min. The microcentrifuge tubes were removed from the scintillation vials, and 5 mL of scintillation fluid was added. Disintegrations per minute (DPM) of each sample were measured using a TriCarb liquid scintillation counter (PerkinElmer). The specific ODC activity was expressed as nmol CO2/min/mg protein.