Purification and characterization of a novel dipeptidase from carp ordinary muscle.

A novel dipeptidase was purified to homogeneity from the crude extract of carp ordinary muscle with an increase in specific activity of 4041-fold and a 4% recovery rate. The enzyme was determined to have molecular weights of 54,000 after reduction and 106,000 without reduction, indicating that it is composed of two sulfide-linking molecules of subunit peptide chains of identical molar size. The optimum hydrolysis pH and temperature of the enzyme were evaluated by l-leucine-glycine to be pH 8.5 and 40 degreesC, respectively, and it was markedly stable in the weak alkaline region and at temperatures below 30 degreesC. Dipeptide hydrolysis of the enzyme was inhibited by metalloprotease inhibitors, sulfide-specific reagents, metal chelating reagents and reductants. In addition, sulfide-affinity bivalent metals potently inactivated the enzyme, while Mg2+ and Mn2+ activated it to different extents, and Mn2+ was also effective on the restoration of the nearly completely inactivated enzyme. The enzyme had a broad range of action on dipeptides that are composed of only l-amino acids, such as l-leucine, l-methionine, l-phenylalanine, and l-valine at the C-terminal and l-alanine, l-leucine, l-methionine, and l-valine at the N-terminal, but it had no catalytic action on dipeptides containing l-proline and d-amino acids, tripeptides, and peptide derivatives.