title: An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.
abstract: 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, pH, 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.
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, China.
authors.forename: Hong-Lin
authors.initials: HL
authors.lastname: Chen
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, China.
authors.forename: Guan-Jun
authors.initials: GJ
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, China. Electronic address: junpingkou@cpu.edu.cn.
authors.forename: Jun-Ping
authors.initials: JP
authors.lastname: Kou
authors.affiliation: Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, Jiangsu 211198, China.
authors.forename: Bo-Yang
authors.initials: BY
authors.lastname: Yu
mesh_terms: D000818:Animals; D001688:Biological Products / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry*; D009218:Myosins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits
chemical_list: D001688:Biological Products; D004791:Enzyme Inhibitors; C017541:molybdenum blue; D008982:Molybdenum; D009218:Myosins
keywords: Bioluminescence; Blebbistatin; Malachite green; Myosin II ATPase activity; Natural products