Trybus K M
Department of Molecular Physiology and Biophysics, Given E205, University of Vermont, Burlington, Vermont 05405, USA.
Methods. 2000 Dec;22(4):327-35. doi: 10.1006/meth.2000.1085.
This article describes methods for expressing and obtaining purified smooth muscle myosin subfragments using the baculovirus/insect cell expression system, as well as methods for purifying whole myosin from tissue. Protocols for several gel assays that are routinely used with myosin are given, including gels to monitor light chain phosphorylation state and native gels to determine protein homogeneity. Steady-state myosin ATPase and actin-activated ATPase determinations are described, as are some of the more basic transient-state kinetic parameters that can be measured. The in vitro motility assay, in which the rate of actin movement over myosin or its subfragments is quantified, is also presented.
本文介绍了使用杆状病毒/昆虫细胞表达系统表达和获得纯化的平滑肌肌球蛋白亚片段的方法，以及从组织中纯化全肌球蛋白的方法。给出了几种常用于肌球蛋白的凝胶分析方法，包括监测轻链磷酸化状态的凝胶和用于确定蛋白质同质性的天然凝胶。描述了稳态肌球蛋白ATP酶和肌动蛋白激活的ATP酶的测定方法，以及一些可以测量的更基本的瞬态动力学参数。还介绍了体外运动分析，其中量化了肌动蛋白在肌球蛋白或其亚片段上的移动速率。
