Two purified isozymes of protein carboxyl methyltransferase from bovine brain catalyze the substoichiometric transfer of methyl groups in vitro from S-adenosyl-L-[methyl-3H]methionine to several erythrocyte membrane proteins, which include bands 2.1, 3, and 4.1, as well as several integral membrane polypeptides. D-Aspartic acid f3-[3H]methyl ester has been isolated from proteolytic digests of these methylated proteins, suggesting that protein D-aspartyl residues can serve as methyl-acceptor sites for the two brain enzymes. This formation of D-aspartic acid 3-[3H]methyl ester is competitively inhibited by the peptide L-Val-L-Tyr-L-Pro-LisoAsp-Gly-L-Ala, which contains an L-aspartyl residue in an unusual /-peptide linkage. Since this peptide is a stoichiometric substrate for the brain methyltransferases, it appears that one enzymatic activity can catalyze methyl ester formation at both D-aspartyl and L-isoaspartyl sites. In these respects, the activity of both brain isozymes closely resembles those previously described for the erythrocyte enzyme. The results are discussed in terms of a model in which derivatized aspartyl residues in proteins, arising by either racemization or isomerization, are recognized by the methyltransferase; the enzyme may function in either the metabolism or correction of the altered structures. The presence of a similar enzyme in both translationally active (brain) and inactive (erythrocyte) tissues suggests that the reactions are of general importance to cellular integrity.Protein carboxyl methyltransferases are widely distributed in both bacteria and eukaryotic tissues (1). In bacteria, very specific carboxyl methyltransferases regulate the bacterial chemotactic response by the methylation of several glutamyl residues in membrane chemoreceptor proteins (2, 3). The eukaryotic enzymes, on the other hand, have very different characteristics. In vitro, these enzymes methylate a large variety of both heterologous and endogenous proteins in a substoichiometric fashion (1, 4-6). The "nonspecific" methyltransferase is widely distributed in mammalian tissues (1) and has been purified from both mammalian brain (1, 5-7) and erythrocyte (8,9) tissues. In brain, there are at least two functionally similar isozymes that can be separated by DEAE-cellulose chromatography (5).The precise function of eukaryotic protein carboxyl methylation reactions has not been determined. It has been proposed that the brain enzyme regulates several cellular processes, including, among others, the storage and release of neuroendocrine substances (10, 11), the modulation of receptor function (12, 13), and the regulation of calmodulin activity (14, 15). However, the data supporting such roles have not been compelling (4,16,17). In particular, the broad substrate specificity and substoichiometric nature of the reactions catalyzed by the enzyme are unusual features for regulatory post-translational modifications. Alternative roles for eukaryotic methylation reactions in the repair or metabolism of damaged proteins ha...