黄牛肉中钙激活酶系统的动力学性质

Kinetic Characteristics of Calpain System from Beef of Chinese Yellow Cattle

【目的】阐明牛肉中钙激活酶系统的主要动力学特征。【方法】通过温度、时间、Ca2+浓度、反应时间、酶量、底物浓度等的变化,分析μ-钙激活酶,m-钙激活酶和钙激活酶抑制蛋白的动力学特性。【结果】随冷冻温度的升高和贮存时间的延长,钙激活酶系统活性都逐步降低,但冷冻处理可以显著降低钙激活酶抑制蛋白的活性,而钙激活酶对冷冻处理不太敏感;μ-钙激活酶达到最大活性一半时所需的Ca2+浓度为50μmol·L-1,m-钙激活酶为320μmol·L-1;在测定钙激活酶活性时,反应时间应控制在60min以内,酶活单位应小于0.45;在以酪蛋白为底物时,m-钙激活酶的Km值为3.185mg·mL-1,Vmax为0.015U·min-1,而μ-钙激活酶的Km值为5.320mg·mL-1,Vmax为0.017U·min-1。【结论】钙激活酶系统活性受贮藏温度和时间、Ca2+浓度、酶促反应时间、酶量、底物浓度影响明显。

[Objective ] The objective of this experiment was to demonstrate the kinetic characteristics of calpain system from beef. [Method] The kinetics of purified calpain system including μ-calpain, m-calpain and calpastatin were investigated by changes of some parameters such as storage temperature and time, calcium concentration, reaction time, enzyme amount and substrate concentration. [Result] When frozen, calpain system activity decreased with storage time, calpastatin, however, was far more sensitive to freezing than calpains. The calcium concentrations required for half maximal activity of μ-calpain and m-calpain were 50 μmol·L^-1 and 320μmol·L^-1, respectively. To assay calpain activity accurately, the reaction time should be within 60 rnin, and activity less than 0.45. When casein was used as substrate, the Km and Vmax of m-calpain was 3.185 mg·mL^-1 and 0.015 U·min^-1,μ-calpain was 5.320 mg·mL^-1, and 0.017 U·min^-1, respectively. [Conclusion] Storage temperature and time, calcium concentration, reaction time, enzyme amount and substrate concentration had great influence on the activity of calpain system.