Figure 3.

Activities of PRMT1 and PRMT6 with and without mCerulean or mCitrine and double-reciprocal plot of PRMT1 product inhibitor analysis. The initial velocity of reactions is determined as described in the Materials and Methods section. (A) Methylation assays using PRMT1 (•), mCit-PRMT1 (○), or mCer-PRMT1 (▾) with increasing concentrations of the H4 tail peptide are shown. (B) Methylation assays using PRMT6 (•), mCit-PRMT6 (○), or mCer-PRMT6 (▾) with increasing concentrations of the H3 tail peptide are shown. The initial rate is calculated as pmol per min per nanomole of enzyme to accommodate the differences in molecular weights between fluorescent and nonfluorescent PRMTs, and kinetic parameters are listed in Table I. (C) In the presence of a constant 80-μM H4 tail peptide concentration, variable concentrations of 1.0, 2.0, 5.0, 10, and 25 μM AdoMet are incubated in methylation buffer. These reactions are repeated in the presence of fixed AdoHcy concentrations of 0 μM (□), 0.5 μM (▴), 1.0 μM (○), 5.0 μM (▪), and 20 μM (•). The pattern of intersecting lines on the y-axis is indicative of competitive inhibition for the product inhibitor AdoHcy.