摘要
【目的】本研究旨在优化Grossowicz氧肟酸比色法测定粘虫Mythimna separata谷氨酰胺转胺酶(Ms TGase)活力的组合条件,以Ms TGase酶活力为依据分析其在不同龄期幼虫体内的分布规律。【方法】取4龄粘虫幼虫,通过组织匀浆和沉析纯化制备Ms TGase,采用Grossowicz比色法测定Ms TGase酶活力,并对Grossowicz比色法的多重实验因素进行正交优化,进一步结合差速离心法分析不同龄期幼虫体内和亚细胞组分(细胞核和细胞碎片,线粒体,微粒体以及胞质溶胶)中Ms TGase酶活力。【结果】结果表明,酶浓度、底物浓度、反应体系pH值、反应温度及钙离子浓度等实验因素都对Ms TGase酶活力测定结果产生显著影响,其影响大小顺序为:酶浓度>温度> p H>底物浓度>Ca2+浓度。Ms TGase酶比活力测定的最优化条件:酶浓度20 mg/m L、底物浓度0. 04 mol/L、反应体系pH值6. 5、测定温度37℃,不添加钙离子。在1-5龄幼虫中以4龄幼虫的Ms TGase酶活力最高,其比活力也显著高于其他龄期的,且在1-5龄幼虫胞质溶胶中Ms TGase酶活力分别占各亚细胞组分酶活力总和的39%,25%,48%,60%和61%。【结论】所获得的最优化条件适用于粘虫Ms TGase酶活力测定。Ms TGase在粘虫体内呈显著的龄期表达特征和亚细胞分布规律。
【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 >Ca 2+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 Ca 2+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.
作者
饶文兵
徐久永
章先飞
Solange MUHAYIMANA
黄青春
RAO Wen-Bing;XU Jiu-Yong;ZHANG Xian-Fei;Solange MUHAYIMANA;HUANG Qing-Chun(Shanghai Key Laboratory of Chemical Biology,School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2019年第1期41-48,共8页
Acta Entomologica Sinica
基金
国家重点基础研究发展计划项目(2010CB126101)
上海市生物医药科技支撑计划项目(15431906800)
华东理工大学来华留学生教育教学项目(ZC1824101)
