FIGURE 5.
A, under our experimental conditions, His-PRO/ENZ complex activity progresses in a linear range at the 2-h reaction time. Five identical methylation reactions were set up. At each time point, a single reaction was quenched. After separation by SDS-PAGE, the gel was incubated with EN3HANCE solution for 1 h, dried, and exposed to film at −80 °C overnight. The density of each signal was measured and plotted (lower panel). NoS, no substrate was added to reaction. The image is representative of two technical replicates. B, high resolution ion exchange chromatography analysis of methylarginine derivatives catalyzed by His-TbPRMT1PRO/ENZ. His-TbPRMT1PRO/ENZ was incubated with substrate and TCA-precipitated. The resulting protein mixture was digested by acid hydrolysis. Amino acids were analyzed by cation exchange chromatography in the presence of unlabeled ADMA, SDMA, and MMA standards. The experiment was performed once.