Fig. 4.
Enzymatic properties of the F379M mutant. (A) Top box: coomassie-stained gel showing varying amounts of the wide-type and the F379M mutant of PRMT5, and a constant amount of histone H4 (5.0 μg) used for enzymatic assay. “C” indicates no enzyme added. Bottom box: autoradiography detection with 0.25 mCi of [3H]-SAM used in each reaction. (B) Western blot detection of asymmetric (middle box) and symmetric (bottom box) dimethylation of histone H4R3. Top box is a coomassie-blue stained gel showing proteins used in the activity assays. Please note, for a comparable level of Werstern blot signal, the amount of the F379M protein is adjusted to ∼1/10 of the wild-type protein, and the amount of PRMT1 is even smaller. (C) Double reciprocal plot analysis of the wild-type and F379 mutant of PRMT5. Derived kinetic parameters are tabulated. (D) Conserved property of the Phe-to-Met mutants of human and nematode PRMT5s. Top two boxes: coomassie staining of the wild-type and mutant enzymes and the substrate used. The right pointing arrow indicates the position of human PRMT5 proteins; an asterisk indicates the position of rat PRMT1; and the left pointing arrow marks the position of C. elegans PRMT5. Third box: Western blot detection of the flag-tagged wild-type and mutant human PRMT5. Fourth box: Western blot detection of asymmetrically dimethylated H4R3. Bottom box: Western blot detection of symmetrically dimethylated H4R3.