Purified UCKL-1 (500 ng) and UCK2 (1 ng) were dispensed into 384-well plates and mixed with a substrate/ATP buffer. Final concentration of ATP was 100 µM. ADP formation was measured at various substrate concentrations. (A) UCKL-1 with uridine as substrate; (B) UCK2 with uridine as substrate; (C) UCKL-1 with cytidine as substrate; (D) UCK2 with cytidine as substrate. Kinase reactions were measured at 0, 60, 120, and 180 min. Luminescence at each time point was converted to the amount of ADP formed during the kinase reaction using an ATP-to-ADP conversion curve. ADP formation was plotted versus time. Each data point represents the mean ± SD of 3–5 replicates.