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Proteinaceous toxin selected from causative dinoflagellates of paralytic shellfish poisoning and its effect on crustacean

麻痹性贝类中毒致病甲藻蛋白质毒素及其对甲壳类动物的影响

基本信息

批准号：
11660209
负责人：
OGATA Takehiko
金额：
$ 2.3万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

Toxicity of cell free media of Alexandrium tamarense to crustacean was confirmed by a bioassay using brine shrimp, Artemia salina. Similar effects were also observed in A. catenella and A. minutum. On the other hand, PSP toxicity of toxic dinoflagellate such as A. catenella decreased significantly under an optimum growth conditions, indicating that toxin productivity would be very low when they form red tide. During the course of a batch culture, toxicity to brine shrimp of cell free media did not showed a parallel relationship to the PSP toxicity. These results indicated that toxicity of eel free media to brine shrimp is possibly caused by some other compound than PSP toxins. Brine shrimp toxicity was found in both higher and lower molecular weight fractions when the cell free medium was fractionated with ultra-filtration (MW 10kda). Several components with different polarities were recognized to show the brine shrimp toxicity in the lower molecular weight fraction. Some of them were identified as polvunsaturated fatty acids and glycolipids by GC or HPLC analyses. These substances showed a hemolytic activity, suggesting that crustacean died with a cytotoxicity of these substances. High molecular weight fraction was further fractionated by gel filtration and ion exchange chromatography to isolate toxic principleas. Three toxic components were found in the this fraction. These components showed absorption maxima at 280 nm and lost their activities by protease treatment, indicating that these are composed of protein. Molecular weight of these toxic proteins was estimated to be 10, 25, and 100 Kda, respectively. Among these toxins, components with the highest and lowest molecular weight showed hemolytic activity and collapsed diatom cells. On the other hand, protein with molecular weight of 25 Kda did not show any cytotoxicity but an acute toxicity to brine shrimp, suggesting that this component possess neurotoxicity.

塔玛亚历山大藻（Alexandrium tamarense）无细胞培养液对卤虫（Artemia salina）的生物毒性试验表明，塔玛亚历山大藻无细胞培养液对甲壳类具有较强的毒性。在A. catenella和A.分钟另一方面，对甲藻A.在最适生长条件下，链状藻的产毒能力明显下降，说明链状藻在形成赤潮时产毒能力很低。在分批培养过程中，无细胞培养基对卤虫的毒性与PSP的毒性没有平行关系。这些结果表明，无鳗培养基对卤虫的毒性可能是由PSP毒素以外的其他化合物引起的。用超滤法（分子量10kda）对无细胞培养液进行分级分离，发现高分子量和低分子量组分对卤虫均有毒性。在低分子量组分中，几种具有不同极性的组分显示出对卤虫的毒性。经GC或HPLC分析，鉴定为多不饱和脂肪酸和糖脂。这些物质显示出溶血活性，表明甲壳类动物因这些物质的细胞毒性而死亡。高分子量组分进一步通过凝胶过滤和离子交换层析分离出毒性成分。在该馏分中发现了三种有毒成分。这些组分在280nm处显示出最大吸收，并且通过蛋白酶处理丧失了它们的活性，表明这些组分由蛋白质组成。这些毒性蛋白质的分子量估计分别为10、25和100Kda。在这些毒素中，分子量最高和最低的组分表现出溶血活性，并使硅藻细胞崩溃。另一方面，分子量为25Kda的蛋白对卤虫没有细胞毒性，但具有急性毒性，提示该蛋白具有神经毒性。