Protease inhibitors in puffer fish skin secretions

鱼的皮肤分泌物起着机械保护的作用，并在运动机器中充当润滑剂。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。本研究以蛋白酶抑制剂作为生物活性物质，检测了28种鱼类表皮分泌物的蛋白酶抑制活性。其中，河豚鱼显示出比其他物种更高水平的抑制活性（最高得分18.8 IU/ml），并且含有针对胰蛋白酶、α-糜蛋白酶、弹性蛋白酶、激肽释放酶和木瓜蛋白酶的多种抑制剂。黄鳍东方鲀（Takifugu xanthopterus）皮肤分泌物经Sephacryl S-200凝胶过滤、M柱阴离子交换层析、Sephacryl S-200凝胶过滤、Sephacryl S-200柱阴离子交换层析、Sephacryl S 关于我们 经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，纯化的胰蛋白酶抑制剂的分子量为51 k。发现太平洋东方鲀（Takifugu pardalis）的样本具有分子量约为50 K的胰蛋白酶抑制剂，类似于来自黄鳍东方鲀（T.xanihopterus）的胰蛋白酶抑制剂，另一种含有两种胰蛋白酶抑制剂，其分子量分别为约10 K和约7 K，与上述胰蛋白酶抑制剂明显不同。另外，从紫鳍金枪鱼皮肤分泌物中分离纯化出木瓜蛋白酶抑制剂，其分子量约为10 k，为蛋白质类物质，而河豚鱼皮肤分泌物对嗜水气单胞菌（AeromonasatriaIAM 12337）均无抗菌活性（ATCC 7966）、杀鲑气单胞菌NCMB 1102、迟缓爱德华氏菌E381、加氏乳球菌H9507、杀鱼巴斯德氏菌Sp 9587、链球菌属，鳗弧菌NCMB 829、鼠伤寒沙门氏菌SH-1、副溶血弧菌Vp+1、金黄色葡萄球菌IAM 1011、荧光假单胞菌IAM 12022、变形希瓦氏菌IAM 1509、枯草芽孢杆菌IAM 1026、藤黄微球菌IAM 1056和大肠杆菌JCM 1649。因此，不建议皮肤粘液中的蛋白酶抑制剂直接在针对本文测试的细菌的防御机制中发挥作用。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