Enzymatic methylation at specific altered aspartyl and asparaginyl residues in glucagon

Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoasparty 1 and/or D-aspartyl residues. In order to identify the origin of these unusual residues, they studied the methylation of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. They found that glucagon could be methylated with the erythrocyte enzyme and S-adenosyl[methyl-3H]methionine to a maximum extent of 0.004 mol methyl groups/mol glucagon. After digestion with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg c, the labelled fragments were separated by HPLC and identified. Additionally, peptides produced by protease digestions were assayed directly for methyl-acceptor activity. They found that the major site of methylation, accounting for 60% of the total, was at Asp-9. Further analysis indicated that this site probably represents an L-isoaspartyl residue. A second site of methylation, representing 23% of the total, was detected at Asn-28. Neither Asp-15 nor Asp-21 could be identified as a methyl-acceptor site. Base treatment of glucagon (0.1 M NH4OH, 3 h, 370C) increased methylation at the Asn-28 site by 4 to 8 fold while methylation at the Asp-9 site remained unchanged. These studies suggest that base treatment enhances methylation at asparagine residues but not at aspartyl residues