Localization,antibacterial Activity And Development During Embryogenesis Of Phenoloxidase In The Amphioxus Branchiostoma Belcheri Tsingtaunese

The enzyme phenoloxidase (P0) has been shown to be present in both invertebrates and vertebrates, yet little is known about the P0 in Branchiostoma belcheri tsingtauense, the amphioxus, a cephalochordate transitional from invertebrates to vertebrates. It was shown in the thesis that the P0 activity existed Histochemically in the epidermal cells, the epithelial cells of gill bar and intestine of adult amphioxus, producing brown to black melanin deposit. No deposit in neural tube, notochord, muscle and fin-box. Ultrastructural examination revealed that P0 reaction products were located in the cytoplasmic granules of the epidermal cells, the epithelial cells of gill bar and intestine. Among the three tissues tested, the diameters of the melanin granules ranged from 420 to 700 nm, from 120 to 670nm, from 156 to 470nm, respectively, and most of them were electron-dense and homogenous and surrounded by a layer of unit membrane. Frequently, the melanin granules were also observed in the secondary lysosomes. In control, no melanin deposit was seen in the control samples incubated in the presence of phenylthiourea (PTU), a specific inhibitor of P0, or in PBS alone. 2 Biochemical detection demonstrated the presence of relatively low P0 activity in the mucus obtained from the skin of amphioxus. The P0 activity in the mucus of the animal was about 6.5 units/mm/mg. When the mucus was pretreated with activators such as SDS, trypsin or zymosan, its P0 activity increased significantly, reaching about two-fold of the untreated. This indicated that the P0 activity existed in the mucus predominantly as an inactive precursor, prophenoloxidase (proPO). Zymosan was the most efficient activator for proP0 in the mucus of amphioxus among the three activators tested. The optimum temperature for the P0 activity was 550C, and its optimum pH 9.0. This is the first report on the presence of the P0 activity in the mucus of not only amphioxus but also marine animals. The P0 enzyme could serve as protecting agents against enviromnental pathogens and foreign bodies. The mucus solution from the amphioxus body surface had little antibacterial activity against E. coil (JM 109) by itself, but it enhanced the antibacterial activity of L-dopa. Moreover, the enhancement of the antibacterial activity of L-dopa by the mucus solution was markedly suppressed by addition of the PTU. These results strongly suggest that it is the P0 that functions as a defense factor against invading materials in the body surface which may contain dopa. Development of phenoloxidase during the amphioxus embryogenesis was studied biochemically and histochemically. It was found that (1) P0 activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm; (2) P0 activity disappeared in the neural plate cells but remained unchanged in the epidennal cells when the neural plate became detached from the rest of the ectoderin. It is apparent that P0 could serve as a marker enzyme for differentiation of the neural ectoderm 3 from the epidermal ectoderm during embryonic development of the amphioxus.