Purification and characterization of an N-terminal acidic amino acid-specific aminopeptidase from soybean cotyledons (Glycine max).

A novel enzyme that catalyzes the efficient hydrolysis of Glu-Glu was isolated from soybean cotyledons by ammonium sulfate fractionation and successive column chromatographies of Q-sepharose, Phenyl sepharose, and Superdex 200. The apparent molecular mass of this enzyme was found to be 56 kDa and 510 kDa by SDS-polyacrylamide gel electrophoresis and Superdex 200 HR 10/30 column chromatography respectively. The enzyme had high activity against Glu-p-nitroanilide (pNA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, Ala-pNA, and Pro-pNA were not hydrolyzed. The synthetic dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but Xxx-Glu was not. The digestion of a Glu-rich oligopeptide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH(2)) using this purified enzyme was also investigated. Glutamic acid residues were cleaved one by one from the N-terminus. These observations indicate that the enzyme removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. It is thought that it was an N-terminal acidic amino acid-specific aminopeptidase from a plant.