IDENTIFICATION OF A DISTINCT SOLUBLE SUBUNIT OF AN INTERMEDIATE FILAMENT PROTEIN - TETRAMERIC VIMENTIN FROM LIVING CELLS
IDENTIFICATION OF A DISTINCT SOLUBLE SUBUNIT OF AN INTERMEDIATE FILAMENT PROTEIN - TETRAMERIC VIMENTIN FROM LIVING CELLS
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DOI：
10.1073/pnas.82.23.7929
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
FRANKE, WW
中科院分区：
文献类型：
--
作者：
SOELLNER, P;QUINLAN, RA;FRANKE, WW
中等大小的丝 (IF) 是最难溶的细胞内蛋白质聚合物结构之一。我们分析了从培养细胞[大鼠RVF-SM细胞、猿猴病毒40转化的人成纤维细胞和人横纹肌肉瘤（RD系）细胞]裂解获得的胞质组分中存在的少量可溶性波形蛋白（一种IF蛋白）。这种可溶性波形蛋白的分子形式通过蔗糖密度梯度离心确定，并使用波形蛋白特异性抗体进行后续 ELISA 和免疫印迹分析。大多数可溶性波形蛋白以独特的形式出现，其沉降行为与在低离子强度下停滞的纯化波形蛋白的重构四聚体亚基无法区分。可溶形式波形蛋白的四聚卷曲螺旋性质通过胰凝乳蛋白酶的消化模式以及通过铜-1,10-菲咯啉和二甲基亚胺酸的化学交联来表明。通过电子显微镜证明了这种可溶性波形蛋白在较高盐浓度下组装成 IF 的能力。脉冲追踪实验表明，可溶形式并不是唯一的翻译后中间体。我们提出，在活细胞中存在一小部分独特的可溶性四聚体形式的波形蛋白，其可以与聚合的 IF 波形蛋白交换。
Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.