Phenoloxidase In The Humoral Fluids Of Amphioxus Branchiostoma Belcheri Tsingtauense: Isolation, Purification, Identification, Induction And Gene Cloning

Phenoloxidase (PO) or tyrosinase (EC1.14.18.1), is a bifunctional copper-dependent enzyme. It catalyzes the oxidation of dopa rapidly to dopaquinone, which in turn polymerize non-enzymetically into insoluble melanin. PO has been found in a variety of animals including both vertebrates and invertebrates and is involved in immune responses in invertebrate. However, phenoloxidase in amphioxus, an intermediary species from invertebrates and vertebrates, remains very much limited at present. Our previous investigation has showed that in amphioxus, PO activity is present in both epidermal cells of gill, intestine and epidermis mucus, and PO in the mucus exhibits an antibacterial activity. However, whether the enzyme PO exists in amphioxus humoral fluid is unkown.In this study, the presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma. belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30℃to 35℃, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, Zs and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.PO from the humoral fluid of amphioxus was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE SepharoseTM...