Kuczmarski E R, Tafuri S R, Parysek L M
Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago, Illinois 60611.
J Cell Biol. 1987 Dec;105(6 Pt 2):2989-97. doi: 10.1083/jcb.105.6.2989.
In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.
在盘基网柄菌变形虫中，肌球蛋白似乎组装成细丝，这些细丝在细胞分裂期间以及对环磷酸腺苷（cAMP）刺激的反应中重新定位。为了更好地理解肌球蛋白组装的调控机制，我们研究了纯化的盘基网柄菌肌球蛋白的聚合特性。在150 mM氯化钾中，经100,000 g离心后，肌球蛋白仍留在上清液中。旋转阴影显示，这种可溶性肌球蛋白是单体，约80%的分子在距头尾连接处98 nm处有一个单一弯曲。在极低浓度的氯化钾（低于10 mM）中，盘基网柄菌肌球蛋白在100,000 g时也可溶。但大多数分子不是单体，而是缔合成二聚体或四聚体。在pH 7.5的50 mM氯化钾中，去磷酸化的肌球蛋白聚合成细丝，而磷酸化至0.85摩尔磷酸根/摩尔重链水平的肌球蛋白则无法形成细丝。通过降低pH值或增加镁离子浓度至10 mM，可诱导磷酸化肌球蛋白形成细丝。形成的细丝是双极的，末端钝圆，长度均匀约为0.43微米。相比之下，由完全去磷酸化的肌球蛋白形成的细丝更长，末端逐渐变细，并聚集形成非常长的丝状结构。盘基网柄菌肌球蛋白组装的临界浓度非常低，约为5微克/毫升，且该值似乎不受重链磷酸化水平的影响。然而，平衡时聚合物的浓度显著降低，表明重链磷酸化抑制了亚基之间的亲和力。详细的组装曲线显示，氯化钾、镁离子、ATP或氢离子浓度的微小变化会强烈影响组装程度。因此，细胞内环境的变化和重链磷酸化水平都可能响应生理刺激来控制肌球蛋白组装 的位置和状态。
