Polymerization and structure of nucleotide-free actin filaments
Polymerization and structure of nucleotide-free actin filaments
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DOI：
10.1006/jmbi.1999.3390
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发表时间：
2000-01-21
影响因子：
5.6
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
De la Cruz, EM;Mandinova, A;Pollard, TD
有两个因素限制了对无核苷酸肌动蛋白（NFA）性质的研究。首先，缺乏结合核苷酸的肌动蛋白在没有稳定剂如蔗糖的情况下迅速变性;其次，在没有变性剂如尿素的情况下，难以除去所有结合的核苷酸。我们使用腺苷三磷酸双磷酸酶、EDTA和Dowex-I来制备在蔗糖中稳定并且类似于99%不含结合核苷酸的肌动蛋白。在NFA稳定的高浓度蔗糖中，它更有利地聚合，滞后期比ATP-肌动蛋白短，临界浓度接近于零。NFA丝是稳定的，但在低蔗糖浓度下由于亚基的变性，当它们从丝末端解离时，会分解。通过负染色标本的电子显微镜，NFA形成长的长丝，其持久长度比ADP-肌动蛋白长丝长1.5倍。NFA和ADP-肌动蛋白丝在2.5 nn分辨率的三维螺旋重建显示类似的亚基间接触沿着两个长螺距螺旋股，但统计学显着的两股NFA丝之间的质量密度较低。与ADP-肌动蛋白丝相比，NFA丝的主要差异峰在空的核苷酸结合位点附近，但不一致。这些NFA细丝中的空核苷酸结合位点对于溶液中的游离核苷酸是不可接近的。NFA丝对罗丹明鬼笔环肽的亲和力低于天然肌动蛋白丝，这是由于较低的缔合速率。这项工作证实，结合核苷酸不是肌动蛋白聚合所必需的，因此核苷酸的主要功能是稳定单体，通过ATP I水解和磷酸盐释放调节细丝的机械和动力学性质，并为细丝的年龄提供内部计时器。(C)北京大学出版社.
Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA, and Dowex-l to prepare actin that is stable in sucrose and similar to 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nn resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP I hydrolysis and phosphate release, and to provide an internal timer for the age of the filament. (C) 2000 Academic Press.