An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors  

作　　者：CHEN Hong-Lin ZHAO Jing ZHANG Guan-Jun KOU Jun-Ping YU Bo-Yang 

机构地区：[1]Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University

出　　处：《Chinese Journal of Natural Medicines》2016年第6期421-426,共6页中国天然药物（英文版）

基　　金：supported by National Natural Science Foundation of China(No.81274131);the Graduate Student Innovation Plan of Jiangsu Province(CXLX11_0784);Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions;2011 Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education

摘　　要：Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method, using a known myosin Ⅱ ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate(ATP) and calcium chloride, p H, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L^(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method, using a known myosin Ⅱ ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate（ATP） and calcium chloride, p H, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L-1 and ammonium molybdate ： stannous chloride at the ratio of 15 ： 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.

关 键 词：Myosin Ⅱ ATPase activity BLEBBISTATIN BIOLUMINESCENCE Natural products Malachite green 

分 类 号：R965[医药卫生—药理学]