Protease inhibitors in puffer fish skin secretions

河豚鱼皮肤分泌物中的蛋白酶抑制剂

基本信息

批准号：
09660221
负责人：
NAGASHIMA Yuji
金额：
$ 2.11万
依托单位：
Tokyo University of Fisheries
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Skin secretion of fish functions as a mechanical protection and serves as a lubricant in locomotion machines. In addition, natural bioactive substances in skin mucus are considered to have chemically protective functions. In this research project, we focused on protease inhibitors as a bioactive substance and examined protease inhibitory activity in the epidermal secretions from 28 species of fish. Among them, the puffer fishes showed a higher level of inhibitory activity (the highest score 18.8 IU/ml) than other species and contained multiple inhibitors against trypsin, alpha -chymotypsin, elastase, kallikrein, and papain. Trypsin inhibitory activity in skin secretions from the puffer fish was labile under acidic conditions below pH 5, decreased after heating at over 50ﾟC for 10min, and completely inactivated by heating at 80ﾟC for 10min.Trypsin inhibitor was purified from skin secretions of Takifugu xanthopterus by gel filtration on Sephacryl S-200, anion-exchange chromatography on M … More onoQ HR5/5, and affinity chromatography coupling trypsin as ligand with TSKgel AF-tresyl TOYOREARL 650M.The molecular weight of the purified trypsin inhibitor was estimated to be 51k by SDS-PAGE.In contrast to T.xanthopterus, T.pardalis contained multiple trypsin inhibitors ; a specimen of Takifugu pardalis was found to have trypsin inhibitor with a molecular weight of about 50k, similar to that from T.xanihopterus, and the other contained two trypsin inhibitors with about 10k and about 7k, remarkably differing from the above trypsin inhibitor(s) in a molecular weight. On the other hand, papain inhibitor was purified from the T.porphyreus skin mucus specimen and was a proteinic substance with a molecular weight of about 10k.None of skin secretion specimens from puffer fishes showed antibacterial activity against Aeromonas hydrophila IAM12337(ATCC7966), Aeromonas salmonicida NCMB1102, Edwardsiela tarda E381, Lactococcus garviece H9507, Pasteurella piscicida Sp9587, Streptococcus sp., Vibrio anguillarum NCMB829, Salmonella typhimurium SH-1, Vibrio parahaemolyticus Vp+1, Staphylococcus aureus IAM101 1, Pseudomonas fluorescens IAM 12022, Shewanella put refaciens IAM1509, Bacillus subtilis IAM 1026, Micrococcus luteus IAM 1056, and Escherichia coli JCM1649. Therefore, it is not suggested that protease inhibitors in skin mucus directly play a role in defense mechanism against bacteria tested here. Less

鱼的皮肤分泌是机械保护的，并用作机车机器的润滑剂。此外，皮肤粘液中的天然生物活性物质被认为具有化学保护功能。在该研究项目中，我们专注于蛋白酶抑制剂作为生物活性物质，并检查了28种鱼类表皮分泌中的蛋白酶抑制剂活性。其中，河豚的抑制活性水平（最高得分18.8 IU/mL）比其他物种更高，并且包含针对胰蛋白酶，α-偶联蛋白，弹性蛋白，弹性蛋白酶，kallikrein和木瓜的多种抑制剂。在酸性条件下，胰蛋白酶抑制剂在pH 5以下的酸性条件下不稳定，在pH 5以上的酸性条件下不稳定，在超过50°C下加热后减少了10分钟，并通过在80°C下在80°C下完全灭活10分钟。 Trypsin inhibitor was purified from skin secretions of Takifugu xanthopterus by gel filtration on Sephacryl S-200, anion-exchange chromatography on M … More onoQ HR5/5, and affinity chromatography coupling trypsin as ligand with TSKgel AF-tresyl TOYOREARL 650M.The molecular weight of the purified trypsin inhibitor was estimated to be 51K由SDS-PAGE.在T.Xanthopterus中，T.Pardalis包含多种胰蛋白酶抑制剂；发现一个标本的takifugu pardalis标本具有胰蛋白酶抑制剂，其分子量约为50k，类似于T.xanihopterus的标本，另一个类似于T. Xanihopterus，而另一个则包含两个胰蛋白酶抑制剂，其大约10K和约7K，与上述分子量的胰蛋白酶抑制剂大不相同。另一方面，木瓜蛋白酶抑制剂是从孢子皮肤粘液样品中纯化的，是一种蛋白质物质，分子量约为10k。来自河豚鱼的皮肤分泌物的任何一种抗菌活性显示出抗肉细胞的活性，抗疏水液含量的抗生素活性 E381, Lactococcus garviece H9507, Pasteurella piscicida Sp9587, Streptococcus sp., Vibrio anguillarum NCMB829, Salmonella typhimurium SH-1, Vibrio parahaemolyticus Vp+1, Staphylococcus aureus IAM101 1, Pseudomonas fluorescens IAM 12022, Shewanella put RefaCiens IAM1509，枯草芽孢杆菌IAM 1026，Micrococcus Luteus IAM 1056和Escherichia Coli JCM1649。因此，不建议皮肤粘液中的蛋白酶抑制剂直接在针对此处测试的细菌的防御机制中发挥作用。较少的