Studies of proteolytic breakdown of fish muscle collagen

鱼肌肉胶原蛋白水解分解的研究

• 批准号: 04660224
• 负责人: TOYOHARA Haruhiko
• 金额: $ 1.34万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660224/
• 关键词: collagen; collagenase; protease; マイワシ; 筋肉; 軟化; 冷蔵

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.

探讨沙丁鱼和比目鱼肌肉嫩化的生化机制，并对肌肉提取液中的游离氨基酸和多肽进行分析。羟脯氨酸和含有这种氨基酸的肽的逐渐增加表明在储存过程中胶原蛋白的蛋白水解分解。明胶酶谱分析显示，在50 ~ 200 K范围内有多条酶带。为了研究蛋白酶的性质，我尝试从沙丁鱼和比目鱼肌肉中纯化蛋白酶，但没有成功。通过这些研究，在比目鱼皮提取物中观察到类似于肌肉蛋白酶的强胶原溶解活性。由于肌肉蛋白酶在酶谱上显示出相同的裂解条带，因此相同的蛋白酶参与明胶的水解。从DEAE-纤维素洗脱的组分在酶谱上观察到单一的50 k-裂解带，在粗提物的情况下观察到的几个裂解带是由于单一的蛋白酶。随后是Con A-Sepharose和REACTIVE=RED琼脂糖，获得均质蛋白酶制剂。为了解该蛋白酶在肌肉组织中的细胞分布，制备了该蛋白酶的多克隆抗体。

项目成果

M.Ando,H.Toyohara and H.Sakaguchi: "Post-mortem tenderization of rainbow trout muscle caused by the disintegation of collagen fibers in the pericellular connective tissues" Nippon Suisan Gakkaishi. 58. 567-570 (1992)

M.Ando、H.Toyohara 和 H.Sakaguchi：“由于细胞周结缔组织中的胶原纤维解体而导致虹鳟鱼肌肉死后嫩化”Nippon Suisan Gakkaishi。

H.Toyohara,M.Kinoshita,K.Sasaki,S.Yamaguchi,Y.Shimizu,and M.Sakaguchi: "Occurrence of a modori-inhibitor in spinach leaves" Nippon Suisan Gakkaishi.58. 1705-1710 (1992)

H.Toyohara、M.Kinoshita、K.Sasaki、S.Yamaguchi、Y.Shimizu 和 M.Sakaguchi：“菠菜叶中莫多里抑制剂的出现”Nippon Suisan Gakkaishi.58。

H.Toyohara et al.,: "Elevated activity of cathepsin L-like protease in the jellied meat of Japanese flounder" Nippon Suisan Gakkaishi. 59. 1909-1914 (1993)

H.Toyohara 等人：“日本比目鱼冻肉中组织蛋白酶 L 样蛋白酶的活性升高”Nippon Suisan Gakkaishi。

M.Ando,H.Toyohara,and M.Sakaguchi: "Post-mortem tenderization of rainbow trout muscle caused by the distintegration of collagen fibers in the pericelluar connective tissues" Nippon Suisan Gakkaishi.58. 567-570 (1992)

M.Ando、H.Toyohara 和 M.Sakaguchi：“由于细胞周结缔组织中胶原纤维的崩解而导致虹鳟鱼肌肉死后嫩化”Nippon Suisan Gakkaishi.58。

M.Ando,H.Toyohara,and M.Sakaguchi: "Three-dimentional stracture of collagen network of pericellular connective tissue in association with firmness of fish muscle" Nippon Suisan Gakkaishi.58. 1361-1364 (1992)

M.Ando、H.Toyohara 和 M.Sakaguchi：“细胞周围结缔组织胶原蛋白网络的三维结构与鱼肌肉硬度的关系”Nippon Suisan Gakkaishi.58。