牛肉成熟过程中细胞凋亡酶对钙激活酶的作用机制研究

Aging is one of the most effective methods improving eating qualities of postmortem muscle, especially of beef or sheep muscle, whereas the aging mechanisn up to date is not fully elucidated. Calpains are widely considered to be a major player during postmortem aging, and recent studies revealed that caspases were involved in meat protein limited degradation and, as such, meat tenderization. Meanwhile, these results showed a possible influence of caspase on calpain activity regulation during postmortem aging, however, the speculation is not proven by direct experimental data. The objective of this study is to investigate the influences of caspase and calpain inhibitor on the expression and activites of calpain and calpastatin, the degradation profiles of calpastatin by caspase-3 or μ-calpain and change in inhibitive activity of calpastatin fragments, influences of Ca2+, Zn2+ and EGTA on the changes in caspase and calpain activity, and apoptotic factors expression during postmortem aging of beef muscle by applying of SDS-PAGE and western blotting, co-immunoprecipitation, Fluorescence\ultraviolet absorption spectrum, and so on. All the results above will contribute to demonstrate the effect of caspase on calpain system during postmortem aging of beef muscle, which is helpful to supply newly theorical evidence for further elucidating meat aging mechanism.

成熟可以有效改善宰后肉（尤其牛、羊肉）的食用品质，但其机理尚未完全阐明。钙激活酶一直被认为是成熟过程中改善肉嫩度的主要作用者，最近的研究表明细胞凋亡酶在成熟早期也参与了部分肌原纤维蛋白的有限降解，从而改善宰后嫩度。同时，已有的结果显示成熟过程中细胞凋亡酶可能影响钙激活酶的活性调节，但目前还缺少直接的实验数据。本项目旨在借助蛋白质电泳与印迹、免疫共沉淀、荧光\紫外吸收光谱等技术，系统研究牛肉成熟过程中，凋亡诱导剂和抑制剂对钙激活酶和钙激活酶抑制蛋白（calpastatin）表达量及活性的影响；凋亡效应酶3和微摩尔钙激活酶对calpastatin的体外降解以及降解产物对钙激活酶抑制活性的变化；外源性钙离子、锌离子和EGTA对细胞凋亡酶、钙激活酶活性变化及相关凋亡因子表达的影响。以上研究可揭示出牛肉成熟过程中细胞凋亡酶对钙激活酶的作用机制，从而为进一步阐明肉的成熟机制提供新的理论依据。

成熟可以有效改善宰后肉（尤其牛、羊肉）的食用品质，但其机理尚未完全阐明。钙激活酶一直被认为是成熟过程中改善肉嫩度的主要作用者，最近的研究表明细胞凋亡酶在成熟早期也参与了部分肌原纤维蛋白的有限降解，从而改善宰后嫩度。同时，已有的结果表明细胞凋亡酶和钙激活酶之间可能存在相互作用，但缺少直接的实验数据。本项目研究结果发现，细胞凋亡酶抑制剂DEVD在宰后早期抑制了SERCA1的表达量，钙激活酶抑制剂无显著影响，钙激活酶在体外降解细胞凋亡酶3生成无活性的片段。钙离子注射能显著降低剪切力值与加速伴肌球蛋白、伴肌动蛋白、肌间线蛋白等细胞骨架蛋白的降解；而锌离子与钙离子相反，延缓了宰后肌肉嫩化过程。钙离子能够提高牛肉储存过程中细胞色素c的表达，从而通过调控凋亡通路上的凋亡因子而间接加速凋亡效应酶3的激活。锌离子对牛肉储存过程中细胞色素c的表达没有显著影响，其对凋亡效应酶3的抑制可能是通过直接作用于该酶而实现的。钙和锌离子对宰后牛肉嫩度的影响除了通过钙激活酶来实现外，也可能与细胞凋亡酶紧密相关。这些结果为进一步建立宰后肌肉嫩化技术提供理论依据。