Protease inhibitors in puffer fish skin secretions

鱼的皮肤分泌物起到机械保护的作用，并在运动机械中起到润滑剂的作用。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，并检测了28种鱼类表皮分泌物中的蛋白酶抑制活性。其中，河豚对胰蛋白酶、α-凝乳酶、弹性蛋白酶、激肽释放酶和木瓜蛋白酶的抑制活性较高(最高为18.8IU/ml)，并含有多种抑制物。河豚皮肤分泌物中的胰蛋白酶抑制活性在pH 5以下的酸性条件下不稳定，在50ﾟC以上加热10min后活性下降，在80ﾟC加热10min后完全失活。黄鳍河豚皮肤分泌物经Sephacryl S-200凝胶过滤和M…阴离子交换层析纯化得到胰蛋白酶抑制物纯化的胰酶抑制物的相对分子质量约为51k。与黄鳍金龟子不同的是，金边斑潜蝇含有多种胰酶抑制物；一只小斑潜蝇含有约50k左右的胰酶抑制物，与黄鳍金龟子相似，另一份含有约10k和7k左右的两种胰酶抑制物，与上述胰酶抑制剂(S)的相对分子质量明显不同。河豚鱼皮肤分泌物对嗜水气单胞菌IAM12337(ATCC7966)、嗜盐气单胞菌NCMB1102、迟缓爱德华氏菌E381、加维氏乳杆菌H9507、黄色巴氏杆菌Sp9587、链球菌、弧菌NCMB829、副溶血性弧菌VP+1、金黄色葡萄球菌IAM101、荧光假单胞菌IAM 12022、施氏施氏杆菌1509、枯草杆菌H9507、微鲁氏杆菌1056、大肠杆菌1649均无抑菌活性。因此，我们并不认为皮肤粘液中的蛋白水解酶抑制剂直接参与了细菌的防御机制。较少

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