Study Of A Prolyl Endopeptidase And Its Endogenous Inhibitor From Blue Scad (Decapterus Maruadsi) Muscle

Prolyl endopeptidase(PEP),a member of serine proteases,specifically cleaves peptide bonds on the carboxyl side of proline residue,and has been widely found in animals,plants and micro-organisms.PEP involves in a variety of physiological and pathological processes and has been utilized in the field of medicine.However,till now few reports about PEP from fish have been documented,and the mechanism concerning the possible PEP participating in fish muscle physiological changing process is poorly understood.In the present study,a prolyl endopeptidase was purified from the muscle of blue scad,which belongs to offshore migratory fish.Characterization on the prolyl endopeptidase was carried out in detail.The endogenous inhibitor of PEP was also investigated,in order to lay a foundation for further research about the functional properties of PEP in fish muscle.PEP was purified to homogeneity from muscle of blue scad and the purification procedure consisted of ammonium sulfate fractionation and column chromatographies including DEAE-Sephacel,Phenyl-Sepharose,and Q-Sepharose.SDS-PAGE showed that the molecular weight was approximately 82 ku.Peptide mass fingerprinting(PMF)study of the purified enzyme showed that among the obtained 16 peptide fragments(with a total of 169 amino acid residues),149 residues were conserved in PEP from blue scad and Neolamprologus brichardiand with identity of 98.8%,suggesting that the purified protein is PEP.Enzymatic properties analysis showed that the enzymatic activity was optimal at 35 ?and pH 6.0.PEP was relatively stable at low temperature and pH ranges from 5.0 to 7.5.Study on thermal inactivation of the enzyme showed that the thermal inactivation rate constant A increased with the increase of temperature.Substrate specificity of PEP showed that it highly specific towards the carboxyl sides of proline residue of fluorescent substrates.However,the enzyme could not hydrolyze other fluorescent substrates.The effect of proteinase inhibitors on PEP showed that the enzyme was strongly inhibited by SUAM-14746,which is a specific inhibitor of prolyl endopeptidase.Also,it was effectively inhibited by PMSF,a serine proteinases inhibitor,while was not obviously affected by other proteinase inhibitors.Based on these characteristics,the PEP is classified as a specific serine proteinase.To investigate the effect of the purified PEP on collagen peptides,PEP was allowed toincubate with three synthetic fish collagen peptides.By HPLC analysis of the hydrolysates,the PEP could hydrolyze all of the three collagen peptides.ESI/MS analysis on the hydrolysates showed that the preferred sites of cleavage by PEP were at the carboxyl side of prolyl residue.It was thus proposed that PEP may work together with metalloproteinase and hydrolyze peptide bonds at prolyl residues of small peptides and plays an important role during degradation of fish collagen.Circular dichroism spectroscopy analysis of the secondary structure of PEP indicated that the PEP is mainly consisted of ?-sheet in the secondary structure.Further,an endogenous inhibitor of PEP(PEPI)was purified to homogeneity from the muscle of blue scad by heat treatment,ammonium sulfate fractionation and column chromatographies of Q-Sepharose,Sephacryl S-200 gel-filtration and Capto Q ion-exchange.SDS-PAGE showed that its molecular weight was about 16 ku.Peptide mass fingerprinting study of the inhibitor showed that the sequences of tryptic digested peptide fragments of PEPI revealed high identity to that of nucleoside diphosphate kinase A(NDPKa)(100%)and nucleoside diphosphate kinase B(NDPKb)(94%)respectively,suggesting that the purified endogenous inhibitor is a protein closely related to NDPK.The PEPI revealed high thermal and pH stability.It was stable when was incubated below 90 ?.Also,even incubation at high temperature such as up to 100 ? for 30 min,approximately 70% of its inhibitory activity remained.In the pH range from 4.0 to 12.0,its inhibitory activity was not much affected.However,at pH 2.0-3.0,it lost its inhibitory activity drastically.The inhibitor revealed a specific competitive reversible inhibitory effect against prolyl endopeptidase,and it had no inhibition on other types of proteinase.This study for the first time revealed an important fact that nucleoside diphosphate kinase which is a multifunctional protein works as the PEP endogenous inhibitor.The result of our present study lays a foundation for further research on the physiological role of PEPI and the relationship between NDPK and PEPI.