Study On Cathepsin L And Its Endogenous From Abalone (Haliotis Diversicolor)

Cathepsin L （EC3.4.22.15）, a member of the papain superfamily of cysteine proteases, hasbeen found widespreadly in animals, plants, bacteria, viruses and other organisms. Cathepsin Lplays a very important role not only in the process of protein hydrolysis in lysosomes, but also inmany other important life activities. So far, cathepsin L has very extensive roles in higheranimals, which is involved in a variety of physiological and pathological processes, havingacquired much attention from scholars, but the function of cathepsin L is poorly understood incrustaceans and is worthy of further study. In view of this, cathepsin L was purified from theviscera of abalone and its enzymatic properties were also studied, in order to laying thefoundation for the further research about physicochemical and functional properties of abaloneand to provide theoretical basis for the research.In this present study, cathepsin L was purified to homogeneity from the viscera of abalone（Haliotis Diversicolor）. The purification procedure consisted of ammonium sulfate fractionationand chromatographies including SP-Sepharose Fast Flow, Sephacryl S-200and Hydroxyapatite（CHT-Cartridge） column. Experimental results showed that, the yield of cathepsin L was13.4%and the purification folds was1582.1. SDS-PAGE, Native-PAGE and gel filtration showed thatcathepsin L was a monomer and the molecular weight was approximately28kDa. Cathepsin Lwas a kind of glycoprotein by glycoprotein staining experiments. The enzymatic activity wasoptimal at37℃and pH5.5and the enzyme was relatively stable below40℃and from pH5.0to6.5. Cathepsin L hydrolysed Z-Phe-Arg-MCA effectively，which was a kind of substrate forcathepsins, However, the enzyme didn’t hydrolyse Z-Arg-Arg-MCA and other fluorescentsubstrates. The enzyme activity of cathepsin L was strongly inhibited by E-64of cystatinproteinases inhibitor, but was not obviously affected by other proteinase inhibitors. Theexperiments of substrate specificity and the inhibitors experiment showed that the enzyme was acysteine protease. Divalent cation ions such as Mn²⁺, Ba²⁺and Mg²⁺enhanced the enzymeactivities to different extents.while Co²⁺, Cu²⁺, Zn²⁺, Fe²⁺and Ca²⁺inhibited the activities. UsingZ-Phe-Arg-MCA as substrate, the kinetic constants of cathepsin L were determined with K_m1.21μmol/L and k_cat21.33s^-1and k_cat/K_m1.8x10⁴（mol/L）^-1s^-1. Using a highly specific polyclonalantibody, the location of cathepsin L was detected to distribute in different tissues of abalone,mainly in gonad, hepatopancreas and gill.An endogenous inhibitor of cathepsin L was purified to homogeneity from the viscera of abalone by ammonium sulfate fractionation, column chromatographies of DEAE-Sepharose andSephacryl S-200with a yield of6.3%and purification folds of1447.4. SDS-PAGE showed itsmolecular weight was12kDa and it was a cysteine protease inhibitor of low molecularweight.The inhibitor had good stability of temperatures and pH.Below80℃, its inhibitoryactivity was not affected by temperature. Even in high temperature such as up to100℃，itsinhibitory activity was also above80%. In the pH4.0^-11.0，inhibitory activity had little change,but below pH3.0, the stability was poor.