Characterization Of The Phenoloxidases From The Coelomocytes Of Sea Cucumber Apostichopus Japonicus

Phenoloxidase?PO?is an important immune-related enzyme in the innate immune system of invertebrates.In order to understand the biochemical and enzymatic characteristics of phenoloxidase in the coelomocytes of Apostichopus japonicus,the coelomocytes of females and males were used as samples,and then using the method of linear-gradient native-PAGE combined with catechol staining,the POs were purified and characterized.The results were as following:1.Four POs were detected in the coelomocytes of A.japonicus.They were named as AjPO1,AjPO2,AjPO3 and AjPO4 according to their molecular masses from high to low after reaction with catechol in polyacrylamide gel.2.AjPO4 is specifically distributed in the coelomocytes of adult female A.japonicus,and all the four POs were not identified in the juvenile.This indicated that there were significant differences in the composition of PO system between juveniles and adults,as well as females and males,in A.japonicus.3.The optimum pH for the activities of AjPO1,AjPO2,AjPO3 and AjPO4 was9.0,8.0,9.0 and 8.0,respectively.The activities of AjPO1,AjPO2 and AjPO3 in boiling water were stable,while AjPO4 had a sharp drop in activity after incubated in boiling water for 20 minutes.It showed that the heat resistance of AjPO1,AjPO2 and AjPO3 was better than that of AjPO4.4.The four POs could catalyze catechol,lL-dopa,dopamine and hydroquinone,among which the optimal substrate was catechol,but failed to catalyze tyrosine,indicating that the four POs belong to laccase-type phenoloxidase.5.The four AjPOs could be activatedby Mn²⁺and Fe²⁺,indicating that the function of PO systemin A.japonicus may be regulated by the divalent metal ions in vivo.In addition,the activities of four POs can be inhibited by citric acid,suggesting they are copper-containing enzymes.