Immunohistochemical Study Of Cathepsin L In Octopus(Octopus Ocellatus)

Octopus is an important economic aquatic animal and contains strong endogenous protease activity.The endogenous proteases in octopus will be out of control after die and degrade its body.The phenomenon,termed autolysis,will lead to softening of the body,which deteriorates the eating quality and causes economic losses.In the present study,octopus(Octopus ocellatus)was taken as a research object to reveal the phenomenon and mechanism of autolysis.The autolysis of octopus was induced by UV irradiation.The changes in microscopic structure of octopus during autolysis were observed by optical microscope.The changes in distribution of cathepsin L in fresh and autolytic octopus was investigated by optical microscope and immunohistochemial method.The changes of activity of cathepsin L and of quantity of soluble protein were also tested.Through the project,the role of cathepsin L in autolysis of octopus will be revealed.Fresh octopus was in active state with uniform color skin.After UV irradiation,octopus became inactive gradually,and the skin appeared darker with some white burst.The cuticle of fresh octopus was intact;the epithelial cells arranged orderly;the connective tissues seemed orderly and uniform;the muscle fibers distributed orderly and uniform.After autolysis,the cuticle was damaged gradually,or even disappeared;the epithelial cells distributed irregularly and even raptured;the muscle trabeculars were broken;the muscle fibers were swelling.Fresh liver tissue was intact and connected each other to form a network.After autolysis,liver cells were broken,which resulted in a large number of empty spaces in liver tissue.Immunohistochemical study showed that cathepsin L was distributed asymmetrically.The staining spots of cathepsin L distributed densely in epithelial and loose connective tissue,while distributed sparsely in dense connective tissue.In muscle tissue,the staining spots of cathepsin L distributed densely in connective tissue,while distributed sparsely in dense connective tissue and muscle fibers.The staining spots of cathepsin L disappeared gradually during autolysis.By comparing the distribution of cathepsin L with the degree of autolysis,it was found that there was a positive relationship between them.This result indicated that cathepsin L maybe take part in the autolysis process of octopus.The activity of cathepsin L and total proteases,and the quantity of soluble protein and soluble collagen in octopus tissue increased with autolysis time.The results proved that the degradation of protein caused by endogenous proteases resulted in autolysis of octopus.The crude protease were prepared form the arm of octopus and the properties of it was investigated.The optimal temperature was 60?.The optimal pH was 5.5.The enzyme activity was inhibited moderately by Cu2+,Zn2+ and Fe2+,and was enhanced by Ca2+,Mg2+and Mn2+.The enzyme activity was inhibited completely by E-64,leupeptin and iodoacetic acid,and was enhanced by DTT and Cys.The enzyme had strong hydrolytic activity toward Z-Phe-Arg-MCA,but had light proteolytic activity toward the substrates of cathepsin B,H and K.The enzyme properties were similar with that of cathepsin L,indicating the existence of cathepsin L in octopus(Octopus ocellatus).