|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1997 : ¥2,200,000 (Direct Cost : ¥2,200,000)
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
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