Purification and characterization of phenoloxidase from Octopus ocellatus.
Phenoloxidase (PO) from ink sacs of Octopus ocellatus was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its biochemical and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase from O. ocellatus was isolated as a heterodimeric protein of 153.8 kDa, and two subunits of 75.6 and 73.0 kDa were often detected in preparations after SDS activation. The PO-like activity showed optimal pH of 7.0, optimal temperature of 40 degrees C, and an apparent Km value of 3.1 mM on L-DOPA, and 6.3 mM on catechol, respectively. The PO-like activity was extremely sensitive to 1-phenyl-2-thiourea and sodium sulfite, and very sensitive to ascorbic acid, thiourea, citric acid, and benzoic acid. Together with its specific enzyme activity on catechol and L-DOPA, it can be concluded that the Octopus PO is most probably a typical o-diphenoloxidase. The PO-like activity was also strongly inhibited by Cu(2+), Zn(2+), ethylenediaminetetraacetic acid and diethyldithiocarbamate (DETC), and the DETC-inhibited PO-like activity could be perfectly restored by Cu(2+). These results indicated that Octopus PO is most probably a copper-containing metalloenzyme. All these results implied that the PO from O. ocellatus has the properties of a catechol-type copper-containing o-diphenoloxidase which functions not only as a catalytic enzyme in melanin production in ink sacs but also as a humoral factor in host defense via melaninization as in other crustaceans.
Fan
Tingjun
T
Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao, China. tjfan@mail.ouc.edu.cn
Li
Mingyu
M
Wang
Jing
J
Yang
Lingling
L
Cong
Rishan
R
D000818:Animals; D004156:Catechol Oxidase / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000302:isolation & purification* / Q000378:metabolism; D049831:Octopodiformes / Q000201:enzymology* / Q000378:metabolism; D013447:Sulfites
D004792:Enzyme Precursors; D013447:Sulfites; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D008666:Metalloendopeptidases; C025026:sodium sulfite