Studies of proteolytic breakdown of fish muscle collagen

为阐明死后鱼肌肉嫩化的生化机制，对沙丁鱼和比目鱼肌肉提取液中的游离氨基酸和多肽进行了研究。羟脯氨酸和含有该氨基酸的多肽的逐渐增加表明，在贮藏过程中，胶原蛋白发生了降解。明胶酶谱显示50K~200K的几条裂解带。为了确定相关的蛋白水解酶的性质，我试图从沙丁鱼和比目鱼肌肉中提纯该酶，但没有成功。通过这些研究，在比目鱼皮肤提取液中观察到了类似肌肉蛋白水解酶的较强的胶原酶分解活性。由于肌肉蛋白水解酶的酶谱显示相同的裂解条带，因此明胶的水解酶也参与了该酶的降解。DEAE-纤维素洗脱物的酶谱显示单一的50K裂解带，粗提物中有几条裂解带是由单一的蛋白酶引起的。再用ConA-琼脂糖凝胶和反应红色琼脂糖凝胶进行纯化，得到均一的酶制剂。为了解该酶在肌肉组织中的细胞分布情况，制备了该酶的多克隆抗体。

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.