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Analysis on Ayu. Muscle collagenases induced in the spawning stage

对香鱼的分析。

基本信息

批准号：
09660226
负责人：
TOYOHARA Haruhiko
金额：
$ 2.18万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660226/
关键词：
Ayu collagen collagenase gelatinase protease spawning matuvation セラチナーゼ

项目摘要

Meat texture of ayu deteriorates remarkably in the spawning season. Our recent studies shows that the enzymatic breakdown of collagen in the pericellular connective tissues is responsible for the deterioration, but the protease(s) involved in the breakdown has not yet been identified. Our primary goal in the present study was the identification of the protease(s).In the first year (1997), we made an attempt to purify the enzymes from cultured ayu muscle by DEAE or CM cellulose chromatography followed by gelatin affinity chromatography which was prepared by ayu type I collagen. By CM cellulose chromatography, we could detect a specific 70kDa activity in the extracts from the muscle of the spawning season in the gelatin zymographic analysis. Further purification of the activity fraction by a gelatin affinity column, 70kDa serine-type activity and 68kDa metallo-type activity were separately eluted in the non-adsorbed and adsorbed fractions, respectively. These results suggested that the 60-70kDa activities usually detected in ayu muscle of the spawning stage are composed of two distinct protease activities of serine-type and metallo-type.In the second year (1998), we examined the relationship between the activity level of the proteases and the degree of maturation by using ayu caught in the Kuzuryu River. As a result, 68kDa and 62kDa activities were specifically induced in the fish with the gonado-somatic index of 4.7 and 6.0 (male) and 11.1 and 14.3 (female). These results suggested that the protease activities derived purified in the first year actually expressed in the muscle of wild ayu.Through these studies, we could identify the proteases possibly involved in the deterioration of collagen in the spawning season of ayu. As a next step, it is required to reveal the mechanism of the induction of these activities.

在产卵季节，香鱼的肉质会显著恶化。我们最近的研究表明，细胞周围结缔组织中胶原蛋白的酶促分解是导致恶化的原因，但参与分解的蛋白酶尚未被鉴定。本研究的主要目的是对蛋白酶进行鉴定，在第一年（1997年），我们尝试用DEAE或CM纤维素层析，然后用香鱼I型胶原制备的明胶亲和层析，从培养的香鱼肌肉中纯化酶。通过CM纤维素层析，在明胶酶谱分析中，我们可以检测到产卵季节肌肉提取物中的特异性70 kDa活性。活性组分经明胶亲和柱进一步纯化后，未吸附组分和吸附组分中分别洗脱出70 kDa丝氨酸型活性和68 kDa金属型活性。第二年（1998年），我们以产自九津川的香鱼为材料，研究了蛋白酶活性水平与成熟度的关系。结果表明，68 kDa和62 kDa的活性在性腺-体细胞指数分别为4.7和6.0（雄性）和11.1和14.3（雌性）的鱼中被特异性诱导。这些结果表明，第一年纯化的蛋白酶活性在野生香鱼肌肉中确实有表达，通过这些研究，我们可以确定可能参与香鱼产卵季节胶原蛋白降解的蛋白酶。作为下一步，需要揭示这些活动的诱导机制。