Elongated analogues of neurotoxin ODAP: Structure-activity studies

Abstract

Incubation of DSIP with an extract of mouse brain resulted in the release of Trp which indicated rapid action by a brain aminopeptidase and the release of Ser which indicated internal cleavage by a brain neutral endopeptidase at the -Ala6-Ser7-bond followed by the secondary action of an aminopeptidase. This mechanism was confirmed by detection of -Gly8-Glu9 as a digest product. The finding of Glu could be attributed to cleavage of this dipeptide or direct action of a carboxypeptidase on the nonapeptide. Other products that increased with time of incubation were Gly, Ala, and Gly-Gly, indicating secondary action of brain exopeptidases on peptide intermediates containing the Gly-Gly subunit. Mechanisms of breakdown were confirmed by the use of DSIP analogs: D-Ala in positions 3 and 4 blocked internal release of amino acids and peptides at short incubation periods but not the release of Trp. Addition of Tyr to position 1 failed to affect aminopeptidase and endopeptidase actions. The low yield of Glu in the D-Ala analogs suggest that it was derived by secondary breakdown of Gly-Glu. Thus, the nonapeptide DSIP is a substrate for brain proteolytic enzymes and is degraded with release of N-terminal Trp along with other residues and peptide intermediates.