Protease inhibitors in puffer fish skin secretions

鱼类的皮肤分泌物具有机械保护作用，在运动机器中起润滑剂作用。此外，皮肤黏液中的天然生物活性物质被认为具有化学保护功能。在本研究项目中，我们将蛋白酶抑制剂作为一种生物活性物质，对28种鱼类表皮分泌物中的蛋白酶抑制活性进行了研究。其中，河豚鱼对胰蛋白酶、α -凝血酶、弹性酶、钾化酶和木瓜蛋白酶具有较高的抑制活性（最高18.8 IU/ml），含有多种抑制因子。在pH值低于5的酸性条件下，河豚皮肤分泌物胰蛋白酶抑制活性不稳定，在50℃以上加热10min后，胰蛋白酶抑制活性下降，在80℃加热10min时，胰蛋白酶抑制活性完全失活。采用Sephacryl S-200凝胶过滤、M…More onoQ HR5/5阴离子交换层析、TSKgel AF-tresyl TOYOREARL 650M亲和层析等方法，从黄鳍龙皮分泌物中纯化胰蛋白酶抑制剂。经SDS-PAGE估计纯化后的胰蛋白酶抑制剂分子量为51k。与T.xanthopterus相比，T.pardalis含有多种胰蛋白酶抑制剂；其中，一种龙目鱼标本含有分子量约为50k的胰蛋白酶抑制剂，与T.xanihopterus相似；另一种龙目鱼标本含有分子量约为10k和7k的两种胰蛋白酶抑制剂，与上述两种胰蛋白酶抑制剂在分子量上有显著差异。另一方面，从卟啉t菌皮肤黏液标本中纯化得到木瓜蛋白酶抑制剂，是一种分子量约为10k的蛋白质物质。河鲀皮肤分泌物标本对以下病原菌均无抑菌活性：嗜水气单胞菌IAM12337（ATCC7966）、沙门氏气单胞菌NCMB1102、迟发edwardsiera E381、garvi球菌H9507、淡鱼巴氏杆菌Sp9587、链球菌、鳗弧菌NCMB829、鼠伤寒沙门菌SH-1、副溶血性弧菌Vp+1、金黄色葡萄球菌IAM101 1、荧光假单胞菌IAM 12022、希瓦氏菌IAM1509、枯草芽孢杆菌IAM 1026、黄体微球菌IAM 1056和大肠杆菌JCM1649。因此，不建议皮肤黏液中的蛋白酶抑制剂直接在抵抗细菌的防御机制中起作用。少

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