Purification And Characterization Of A Prolyl Endopeptidase From The Skeletal Muscle Of Common Carp (Cyprinus Carpio)

Prolyl endopeptidase has been found to be widely distributed in mammals,micro-organisms and plants. PEP can specifically cleave the peptide bond on the carboxyl sideof proline residues. Thus, this enzyme class has been extensively investigated in the field ofmedicine and industry. According to the effective degradation of PEP to the peptides containingproline residues, therefore, we speculate that PEP was widely involved in the process ofanimal collagen degradation. However, so far there are a few reports on prolyl endopeptidasefrom fish muscle been documented.In the present study, a prolyl endopeptidase was purified to homogeneity from the skeletalmuscle of common carp by using a procedure comprising ammonium sulfate fractionation andcolumn chromatographies including DEAE-Sephacel, Phenyl-Sepharose, DEAE Sepharose FastFlow, and Hydroxyapatite. The molecular weight of the enzyme was82kDa detected bySDS-PAGE.Using Suc-Gly-Pro-MCA as substrate, the optimal pH of the purified enzyme was pH6.0,and the enzyme was stable in the range of pH5.0-8.0. The optimum temperature of the enzymewas35°C and the thermal stability of the anzyme was poor. Microscopic rate constants of freeenzyme (k+0) and enzyme-substrate complex (k+0′) were determined using the kinetic method ofthe thermal inactivation. The results showed that the value of k+0was larger than the value of k+0′,which indicated that the presence of substrate had a certain protective effect of thermalinactivation. At pH6.0, when the temperature were35°C and40°C, the half-lives of commoncarp prolyl endopeptidase were determined as105min and10min.The effect of proteinase inhibitors on prolyl endopeptidase showed that the enzyme activitywas not effected by the inhibitors of metalloproteinase (EDTA, EGTA) and cysteine proteinase(E-64). It was partially inhibited by serine proteinase inhibitors including PMSF and PefablocSC, whereas other serine proteinase inhibitors, such as LBTI, aprotinin, benzamidine andleupeptin, revealed no effects. But the activity was strongly inhibited by SUAM-14746, which isa competitive and specific inhibitor of prolyl endopeptidase. In conclusion, the prolylendopeptidase is classified as a serine proteinase.The Kmwas8.33μM, and the kcatwas1.71S-1, based on the Lineweaver-Burk plot using Suc-Gly-Pro-MCA. The substrate specificity of the purified enzyme showed PEP had highspecificity towards Suc-Gly-Pro-MCA and Suc-Gly-Pro-Leu-Gly-Pro-MCA, which haveproline residue on the carboxyl sides. Furthermore, prolyl endopeptidase effectively hydrolyzedNeurorensin and Bradykinin-Potentiator B, which have proline residue in the peptide chain,suggesting prolyl endopeptidase playded an important role during postmortem tenderization offish muscle.Peptide mass fingerprinting (PMF) study of the purified enzyme showed that, among theobtained of134amino acid residues,134residues were completely conserved in PEP fromAmphimedon queenslandica, with100%identity, and82were conserved in PEP from commoncarp muscle and Danio rerio with identity of61.2%, suggesting the present enzyme is PEP.