Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense.
Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.
Pang
Qiuxiang
Q
Department of Marine Biology, Ocean University of China, Qingdao 266003, PR China.
Zhang
Shicui
S
Shi
Xiaodong
X
Su
Feng
F
Wu
Di
D
D000645:Ammonium Sulfate; D000818:Animals; D015153:Blotting, Western; D002816:Chordata, Nonvertebrate / Q000201:enzymology*; D002845:Chromatography; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D010670:Phenylthiourea / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity
D003911:Dextrans; D007106:Immune Sera; D010670:Phenylthiourea; C025614:sephadex; D019810:Sodium Azide; D014442:Monophenol Monooxygenase; D000645:Ammonium Sulfate