草鱼和鲢鱼肌球蛋白Ca^(2+)-ATPase热力学活化参数的季节变化

SEASONAL CHANGES IN THERMODYNAMIC ACTIVATION PARAMETERS OF MYOSIN Ca^(2+)-ATPase FROM GRASS CARP AND SILVER CARP

肌球蛋白构成了鱼肌肉肌原纤维蛋白的50%以上,它的性质主要决定了肌原纤维蛋白的性质,进而影响了鱼肌肉蛋白质的加工适性。本文对来自春季(4月份)、夏季(8月份)、秋季(11月份)和冬季(1月份)的草鱼(Ctenopharyngodonidellus Valenciennes)和鲢鱼(Hypophthalmichthys molitrix Valenciennes)骨骼肌肌球蛋白进行提取和纯化,通过测定不同季节的两种淡水鱼肌球蛋白Ca2+-ATPase的热力学活化参数,从化学反应的热力学理论上证明了淡水鱼肌球蛋白的热稳定性与季节温度变化之间的关系。研究结果表明,草鱼和鲢鱼肌球蛋白Ca2+-ATPase的热力学活化参数活化能Ea、活化焓ΔH和活化熵ΔS伴随栖息环境温度的上升而增大,而活化自由能ΔG的变化幅度很小,证明了肌球蛋白的热稳定性夏季鱼明显优于冬季鱼,而春季鱼和秋季鱼分别与夏季鱼和冬季鱼的相似。另一方面,ΔH与ΔS之间显示了良好的线性相关,证明了淡水鱼能够通过其肌肉蛋白质反应的活化焓与活化熵之间的互补效应,以应对自然界季节环境温度的变化。

Eurythermal temperate freshwater fish such as grass carp, silver carp and common carp often experience large seasonal changes in body temperature. These show a high capacity in temperature adaptation which enables them to maintain locomotory performance throughout the year. The mechanisms underlying such changes in swimming performance have been shown to involve changes in the properties of muscle protein such as the thermal stability of myofibrillar ATPase. Myosin amounts to about over 50% of myofibrillar protein in fish skeletal muscles, and its properties mainly decide properties of the myofibrillar protein. Thus, the processing adaptabilities of fish muscular proteins are virtually influenced by the properties of myosin. In this paper, fast skele- tal muscles were dissected from the dorsal epaxial myotomes of grass carp （ Ctenopharyngodon ideUus Valenciennes） and silver carp （Hypophthalmichthys molitrix Valenciennes） in spring （April）, summer （August）, autumn （November） and winter （January）, taking care to avoiding contamination with slow muscle,immediately used as preparation of myosins. The purity of myosin preparations was examined by SDS-PAGE. The results showed that the myosins did not contain any appreciable amount of actin, demonstrating their high purity. Each of the purified myosin preparations contained heavy chain having a molecular mass of about 200 kDa and three kinds of light chains of about 20 kDa including alkali light chains A1 and A2, and 5,5＇-dithio-bis-2-nitrobenzoic acid （DTNB） light chain. Thermodynamic activation parameters of myosin Ca^2 ＋ -ATPase, activation energy （Ea）, enthalpy of activation （ △H^≠）, entropy of activation （△S^≠） and free energy of activation （ △G^≠）, were determined in order to illustrate the relationship between thermal stabilities of myosin and seasonal temperature changes at thermal dynamic theory of chemical reaction. The results showed that three types of thermodynamic activation parameter, activation energy （Ea）, enthalpy of activation （△H^≠） and entropy of activation （△S^≠） were 143.9kJ/mol, 141.3kJ/mol and 409.6 e.u., respectively, for myosin from the summer grass carp,and 117. lkJ/mol, 114.6kJ/mol and 324.6 e. u., respectively,for myosin from the winter grass carp, indicating that these three parameters increased with the habitat environment temperatures increasing. Whereas, myosins from the spring and autumn grass carp showed the values comparable to those from the summer and winter grass carp, respectively, and the former was slightly lower than those of the summer fish and the latter was slightly higher than those of the winter fish. However, no significant changes in free energy of activation （△G^≠） were observed. On the other hand, changes in these thermodynamic activation parameters for myosins from silver carp in different seasons were similar to those for myosins from grass carp. These results demonstrated that thermal stabilities of myosins from freshwater fish in different seasons were different, namely the thermal stability of myosin from the summer fish was significantly higher than that from the winter fish, whereas myosins from the spring and autumn fish had thermal stabilities similar to those from the summer and winter fish, respectively. In addition, a good correlation between △H^≠and △S^≠ was found, suggesting the existence of a significant compensatory effect in these two parameters, which is necessary for freshwater fish to adapt to the changes in environment temperature.