Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

摘要

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Cly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 C, respectively, and the Km and k_(cat) were 8.33 u,M and 1.71 S~(-1), respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amptiimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.