Abstract:

【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 320 µmol•L-1, respectively. To assay calpain activity accurately, the reaction time should be within 60 min, 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.

Key words: µ')">µ, -calpain, m-calpain, calpastatin, calcium concentration, kinetics