Reconstitution of intermediate-sized filaments from denatured monomeric vimentin.
Reconstitution of intermediate-sized filaments from denatured monomeric vimentin.
复制标题
从变性的单体波形蛋白重建中等尺寸的丝。
DOI：
10.1016/0022-2836(81)90303-x
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发表时间：
1981
影响因子：
5.6
通讯作者：
E. Mandelkow
中科院分区：
文献类型：
--
作者：
W. Renner;Werner W. Franke;E. Schmid;Norbert Geisler;Klaus Weber;E. Mandelkow
波形蛋白是间充质组织的许多细胞以及各种培养细胞的主要细胞骨架蛋白，是一类特殊的中等大小（7至11 nm）细丝的组成蛋白。在生理相关pH范围（5·5至7·5）内，这些物质在高和低离子强度下均不溶。将来自各种细胞（猪眼透镜组织、小鼠3 T3、大鼠RVF-SMC和仓鼠BHK-21细胞）的细胞骨架的波形蛋白纯化并溶解在尿素或盐酸胍中。当变性的单体波形蛋白的溶液在含有2-巯基乙醇的各种离子强度的缓冲液中透析时，波形蛋白复性并形成长的（高达3.5 μm）中等尺寸的细丝。通过电子显微镜和X-射线衍射，重构的细丝与天然波形蛋白细丝难以区分。本机和重建的中等大小的长丝含有其他蛋白质（前角蛋白，结蛋白）进行了平行检查。波形蛋白在0.15和0.51 nm处的赤道反射和0.98和6.3 nm处的赤道反射表明，波形蛋白的α-螺旋以卷曲-卷曲的方式排列，表明波形蛋白在很宽的盐浓度范围内自发地重折叠成α-螺旋排列，并组装成长的中等大小的纤维。该过程不涉及二硫键形成或其他蛋白质的存在。各种中间丝蛋白的相似重构特性表明，这些蛋白质虽然在多肽组成和氨基酸序列上不同，但含有具有相似序列排列原则的区域，这些区域定义了α-螺旋结构域，并负责组装成形态相似的中间丝。
Vimentin, a major cytoskeletal protein of many cells of mesenchymal tissues as well as of cultured cells of various kinds, is the constituent protein of a special class of intermediate-sized (7 to 11 nm) filaments. These are insoluble at high and low ionic strength over the physiologically relevant pH range (5·5 to 7·5). Vimentin from cytoskeletons of various cells (porcine eye lens tissue, mouse 3T3, rat RVF-SMC, and hamster BHK-21 cells) was purified and solubilized in urea or guanidinium hydrochloride. When solutions of denatured, monomeric vimentin were dialyzed against buffers of various ionic strengths containing 2-mercaptoethanol, vimentin renatured and long (up to 3·5 μm) intermediate-sized filaments were formed. The reconstituted filaments were indistinguishable from native vimentin filaments by electron microscopy and X-ray diffraction. Native and reconstituted intermediate-sized filaments containing other proteins (prekeratin, desmin) were examined in parallel. The high degree of molecular order in reconstituted filaments was demonstrated by meridional reflections at 0·15 and 0·51 nm and equatorial reflections at 0·98 and 6·3 nm, indicating coiled-coil arrangements of α-helices of vimentin.The results show that vimentin spontaneously refolds into α-helical arrangements and assembles, over a broad range of salt concentrations, into long intermediate-sized filaments. The process does not involve disulfide bond formation or the presence of other proteins. The similar reconstitution properties of various intermediate filament proteins suggest that such proteins, albeit different in polypeptide composition and amino acid sequence, contain regions with similar principles of sequence arrangement that define α-helical domains and are responsible for the assembly into morphologically similar intermediate-sized filaments.
来自三种牛组织的 10 nm 细丝的比较。
影响因子：
3.7
作者：
Gipson,IK;Anderson,RA
通讯作者：
Anderson,RA