青岛文昌鱼酚氧化酶的定位，功能及其在胚胎发育中的变化

IOCAS-IR > 海洋环流与波动重点实验室

	青岛文昌鱼酚氧化酶的定位，功能及其在胚胎发育中的变化
	李国荣
学位类型	博士
	2000
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
关键词	文昌鱼酚氧化酶粘液胚胎发育超微结构
摘要	本文利用生化，组化，电镜酶细胞化学技术研究了文昌鱼酚氧化酶（PO）的定位，生理功能以及在胚胎发育中的变化。文昌鱼的体表粘液中存在PO，并且主要以酚氧化酶原（proPO）的形式存在，proPO可被胰蛋白酶，SDS和酵母聚糖激活。PO的最适温度为55 ℃，最适pH为9.0。组化结果证明：PO 分布于文昌鱼的表皮，鳃上皮和肠上皮细胞中。超微结构进一步显示：在表皮细胞和肠上皮细胞中，PO 位于均质状的圆形颗粒中，电子密度较高；而在鳃上皮细胞中，PO存在于圆形颗粒和次级溶酶体中。PO能够增强多巴的抗菌活性。在文昌鱼的胚胎发育过程中，PO出现的最早时期是在早期神经胚，PO活性在孵化期达到最高，至一天幼虫时，PO活性开始略有下降。在早期神经胚中，整个外胚层包括神经外胚层和表皮外胚层细胞中均有PO 阳性产物，而中胚层和内胚层细胞中则呈阴性反应。随着神经胚的继续发充，神经外胚层细胞中PO的染色强度逐渐减弱，而表皮外胚层细胞中的PO 染色强度不发变化。当神经板分化出来时，PO 活性从神经板细胞中消失，而在表皮细胞中保持不变。PO可以作为神经处胚层从表皮外胚层中分化出来的标志酶。本文首次报道PO 存在于文昌鱼的体表粘液中，证明它可以起免疫防御作用；并首次发现PO 可以作为神经外胚层与表皮外胚层分化的标志酶。
其他摘要	The enzyme phenoloxidase (PO) has been shown to be present in both invertebrates and vertebrates, yet little is known about the PO in Branchiostoma belcheri tsingtauense, the amphioxus, a cephalochordate transitional from invertebrates to vertebrates. It was shown in the thesis that the PO 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 PO 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 470 nm, 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 PO, or in PBS alone. Biochemical detection demonstrated the presence of relatively low PO activity in the mucus obtained from the skin of amphioxus. The PO activity in the mucus of the animal was about 6.5 units/min/mg. When the mucus was pretreated with activators such as SDS, trypsin or zymosan, its PO activity increased significantly, reaching about two-fold of the untreated. This indicated that the PO activity existed in the mucus predominantly as an inactive precursor, prophenoloxidase (proPO). Zymosan was the most efficient activator for proPO in the mucus of amphioxus among the three activators tested. The optimum temperature for the PO activity was 55 ℃, and its optimum pH 9.0. This is the first report on the presence of the PO activity in the mucus of not only amphioxus but also marine animals. The PO enzyme could serve as protecting agents against environmental pathogens and foreign bodies. The mucus solution from the amphioxus body surface had little antibacterial activity against E.coli (JM109) 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 result strongly suggest that it is the PO 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) PO 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) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate became detached from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of the amphioxus.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1023
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	李国荣. 青岛文昌鱼酚氧化酶的定位，功能及其在胚胎发育中的变化[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2000.

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