FIGURE 3.

High resolution ion exchange chromatography analysis of methylarginine derivatives catalyzed by TbPRMT7. A, three micrograms of GST-TbPRMT7 in the absence of additional substrate, was incubated in the presence of [³H]AdoMet in PBS for 14 h at 22 °C. Protein was precipitated with 50% trichloroacetic acid and digested into amino acids by acid hydrolysis. Amino acids were analyzed by cation exchange chromatography in the presence of unlabeled ADMA, SDMA, and MMA standards.200 μl of each fraction(1/5 of the total fraction) was removed for radioactivity analysis and 100 μl was removed for ninhydrin analysis, and the fractions were counted three times for 3 min each. B, 10 μg of bovine histones were incubated with 3 μg of GST-TbPRMT7 in the presence of [³H]AdoMet as in A, and reactions were analyzed as in A. C, 3 μg of RBP16 were incubated with 3 μg of GST-TbPRMT7 in the presence of [³H]AdoMet as in A, and reactions were analyzed as in A. D-F, in vitro reactions were carried out as in A, B, and C using 3 μg of TbPRMT7 that was treated with thrombin to remove the GST tag.