Aguado-Velasco M, Aguado-Velasco C, Kuczmarski E R
Department of Physiology and Biophysics, Chicago Medical School, Illinois 60064.
Protein Expr Purif. 1993 Aug;4(4):328-32. doi: 10.1006/prep.1993.1042.
Dictyostelium has several isoforms of "unconventional" single-headed myosins that do not assemble into filaments (myosin I). In contrast, there is only one form of conventional myosin (myosin II) that self-assembles into bipolar thick filaments, and molecular genetic studies have shown that this myosin is an essential motor protein for several cell functions, including cytokinesis. Myosin II is phosphorylated on both the heavy chain and light chain, and these modifications regulate assembly and ATPase activity. A large fraction of the myosin II remains associated with structures that are resistant to extraction with nonionic detergents (the Triton cytoskeleton). These cytoskeletons retain the ability to contract in an ATP-dependent manner and can easily be isolated by low-speed centrifugation. A method for the rapid purification of myosin II from cytoskeletons is described. This method is simple and avoids the need for sonication, ultracentrifugation of large volumes, and overnight dialysis. The subunit composition, assembly properties, and enzyme activity are similar to those reported for myosin prepared from soluble extracts.
盘基网柄菌有几种“非常规”单头肌球蛋白的同工型，它们不会组装成丝（肌球蛋白I）。相比之下，常规肌球蛋白（肌球蛋白II）只有一种形式，它能自组装成双极粗丝，分子遗传学研究表明，这种肌球蛋白是包括胞质分裂在内的几种细胞功能所必需的运动蛋白。肌球蛋白II的重链和轻链都会发生磷酸化，这些修饰调节组装和ATP酶活性。很大一部分肌球蛋白II仍然与用非离子去污剂（Triton细胞骨架）提取时具有抗性的结构相关联。这些细胞骨架保留了以ATP依赖方式收缩的能力，并且可以通过低速离心轻松分离。本文描述了一种从细胞骨架中快速纯化肌球蛋白II的方法。该方法简单，避免了超声处理、大量超速离心和过夜透析的需要。其亚基组成、组装特性和酶活性与从可溶性提取物中制备的肌球蛋白报道的相似。
