Abstract: 【Aim】 This study aims to optimize the combination conditions in Grossowicz colorimetry for determining the activity of Mythimna separata transglutaminase (MsTGase), and to analyze the distribution of MsTGase in various larval instars based on its activity 【Methods】 MsTGase was prepared from the 4th instar larvae of M. separata, purified by tissue homogenate and precipitation, and assayed by Grossowicz colorimetry. The multiple experimental factors of Grossowicz colorimetric method were optimized by orthogonal experiment. The MsTGase activities in larvae of different instars and subcellular fractions including cell nucleus and debris, mitochondria, microsomes and cytosol, which were separated by differential centrifugation, were further determined. 【Results】 The experimental factors such as enzyme concentration, substrate concentration, pH value of the reaction system, temperature and calcium ion concentration had significant influences on the MsTGase activity, and the effect of the experimental factors was ranked in the descending order as enzyme concentration>temperature>pH value>substrate concentration>Ca²⁺ concentration. The optimum conditions for MsTGase activity determination included 20 mg/mL  of enzyme and 0.04 mol/L of substrate in a reaction system of pH 6.5, temperature 37℃, and no Ca²⁺ added. In the 1st-5th instar larvae of M. separata, the MsTGase activity in the 4th instar larvae was the highest and its specific activity was also significantly higher than those in other larval stages. The MsTGase activities in the cytosol of the 1st-5th instar larvae of M. separata accounted for 39%, 25%, 48%, 60% and 61% of the total enzyme activity of each subcellular fraction, respectively. 【Conclusion】 The optimum conditions obtained are applicable for determining the MsTGase activity. MsTGase shows a significant instar-dependent accumulation and subcellular distribution in M. separata larvae.

Key words: Mythimna separata, transglutaminase, enzyme activity, Grossowicz colorimetry, orthogonal optimization, in vivo distribution