Study On Prolyl Endopeptidases In Skeletal Muscle Of Tilapia(Oreochromis Spp) And Sea Bass(Lateolabrax Japonicus)

Prolyl endopeptidase?PEP,EC 3.4.21.26?which specifically cleaves peptide bonds on the carboxyl side of proline residues is a special serine protease.Although some physiological roles of the enzyme have been elucidated,including their involvements in senile memory disorder,cell-mediated immune responses,autoimmunity,and metabolic responses.few reports about PEP in fish,have been documented,especially the functional characteristics of PEP in fish muscle is not clear.In the present study,two prolyl endopeptidases were purified to homogeneity from the skeletal muscle of tilapia and sea bass.The aim of this study was to provide experimental basis and theoretical reference for elucidating the functional properties of PEP in fish muscle.The main works are as following:1.Purification of PEP from tilapia and sea bassIn the present study,PEPs were purified from the skeletal muscle of tilapia?Oreochromis spp?and sea bass?Lateolabrax japonicus?,respectively by ammonium sulfate fractionation and a series of column chromatographies.The molecular weight of tilapia PEP was 85 kDa,and the sea bass was about 84 kDa.Using Suc-Gly-Pro-MCA as substrate,the optimum temperature of PEPs were 35 �C,the optimum pH of tilapia PEP and sea bass PEP were 6.0 and 6.5,respectively,belong to neutral weak acid enzymes and the thermal stability of the enzymes was poor.Substrate specificity of PEPs showed that it highly specific towards the carboxyl sides of proline residue of fluorescent substrates,such as Suc-Gly-Pro-MCA and Suc-Gly-Pro-Leu-Gly-Pro-MCA.The effect of proteinase inhibitors showed SUAM-14746?a specific inhibitor of PEP?and PMSF could strongly inhibit the activity of PEPs,other proteinase inhibitors such as Leupeptin and LBTI did not reveal obviously effect.Thus,PEP was regarded as a special serine protease.Metal ions of Cu²⁺ and Zn²⁺ strongly inhibited the activity of two PEPs,Ca2+ and Co2+ showed partial inhibitory effect.Ba2+,Mn2+,Mg2+,Fe2+ did not exhibit any inhibitory impact on the PEP of tilapia,while Mn2+ and Mg2+ revealed some inhibitory effect on sea bass PEP.The activation energy?Ea?of tilapia PEP was 47.42 KJ/mole and that of sea bass PEP was 40.02 KJ/mole.SUAM-14746 showed competitive inhibition to tilapia PEP,Zn²⁺ and Cu²⁺ both revealed mixed inhibition to tilapia PEP.2.Degradation of collagen peptides by tilapia PEPThe effect of purified tilapia PEP on collagen peptides was investigated by HPLC and ESI/MS.the results showed that PEP could hydrolyze collagen peptides and showed that the preferred sites of cleavage by PEP were at the carboxyl side of prolyl residue,suggesting that PEP may be play its important role after hydrolyze peptide bonds at prolyl residues of small peptides by metalloproteinase during collagen metabolism.3.Cloning of sea bass PEP and its expression in different tissuesWith RT-PCR and Rapid Amplification of cDNA Ends?RACE?,a full length cDNA with 3029 bp was obtained,which included an open reading frame?ORF?coding for 741 amino acid residues.The encoding protein showed 74.58% similarity to human endogenous PEP?PDBID:3DDU?using Swiss-Model.Fluorescence quantitative PCR technique showed that the expression of PEP in heart was the highest.The expression of PEP in other five tissues of gill,brain,liver,muscle,skin were in descending order,while the expression in intestine was the lowest.4.Inhibiton of PEP by unsaturated fatty acidsThe inhibitory effects of unsaturated fatty acids on PEP were studied,The result showed that oleic acid,linoleic acid and DHA are noncompetitive inhibitors to sea bass PEP.The IC50 values of oleic,linoleic,and DHA were 139.34 ?mol/L,145.40 ?mol/L and 158.17 ?mol/L,respectively.Their inhibition constants?KI?were determined to be 102.31 ?mol/L,113.81 ?mol/L and 140.52 ?mol/L,respectively.Oleic acid showed the most effective inhibition while linoleic acid showed lower inhibition than that of oleic acid.while DHA was the weakest.Fluorescence spectroscopy and circular chromatography scanning analysis of PEP indicated that the conformation of the protein changes during inhibition by unsaturated fatty acids.In conclusion,comparative study on the properties of PEP in tilapia and sea bass provided a reference for revealing the function of PEP.The inhibitory effect of unsaturate fatty acids on PEP provided a theoretical basis for the potential use of unsaturate fatty acids in the inhibition of PEP and further for the prevention of memory loss.