|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1992 : ¥1,300,000 (Direct Cost : ¥1,300,000)
To clarify the biochemical mechanism of post-mortem fish muscle tenderization, free amino acids and peptides of the muscle extracts of sardine and Japanese flounder. Gradual increase of hydroxyproline and the peptides containing this amino acid suggested the proteolytic breakdown of collagen during storage. By gelatin-zymography, several lytic bands ranged from 50k to 200k were revealed. To characterize the responsible protease, I tried to purify the protease from sardine and flounder muscle, but not succeeded.
Through these studies, strong collagenolytic activity similar to muscle protease was observed in the extract of flounder skin. Since same lytic bands on zymogram were revealed in case of muscle protease, same protease was involved in gelatin hydrolysis. A single 50k-lytic band on zymogram was observed for the fraction eluted from DEAE-cellulose, several lytic bands observed in case of the crude extract was due to a single protease. Followed by Con A-Sepharose and REACTIVE=RED agarose, homogenous protease preparation was obtained. To know the cellular distribution of the protease in muscle tissue, polyclonal antibody was raised against for the protease.