Analysis on Ayu. Muscle collagenases induced in the spawning stage

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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.