title: Characterization of phenoloxidase from the sea cucumber Apostichopus japonicus.
abstract: Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85°C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca(2+), Mg(2+) and Mn(2+), and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic-polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses.
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China. Electronic address: zunchunz@hotmail.com.
authors.forename: Zunchun
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Guan
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Bai
authors.initials: B
authors.lastname: Wang
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Bei
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Aifu
authors.initials: A
authors.lastname: Yang
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.affiliation: Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, Liaoning 116023, PR China.
authors.forename: Hongjuan
authors.initials: H
authors.lastname: Sun
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004050:Ditiocarb / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D008070:Lipopolysaccharides / Q000276:immunology; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000493:pharmacokinetics*; D011070:Poly I-C / Q000276:immunology; D020033:Protein Isoforms / Q000235:genetics*; D047528:Stichopus / Q000201:enzymology*
chemical_list: D000975:Antioxidants; D008070:Lipopolysaccharides; D020033:Protein Isoforms; D003300:Copper; D004050:Ditiocarb; D004492:Edetic Acid; D014442:Monophenol Monooxygenase; D011070:Poly I-C
keywords: Biochemical characteristics; Immune-responsive characteristics; Metalloenzyme; Phenoloxidase; Sea cucumber (Apostichopus japonicus)