Site-specificity of histone HL methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

Abstract 1. 1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N -methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis . 2. 2. When the enzymatically [methyl- 3 H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (Hl b , Hl c , Hl d and Hl e ). Both enzymes methylated Hl c and Hl b to approximately the same extent; H1 d and Hl e were methylated preferentially by enzyme V-B and V-A, respectively. 3. 3. Histone Hl c , [methyl- 3 H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. 4. Arg C protease-digestion of [methyl- 3 H]labeled Hl c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. 5. The histone Hlc methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.