Breckler J, Lazarides E
J Cell Biol. 1982 Mar;92(3):795-806. doi: 10.1083/jcb.92.3.795.
Filaments with a diameter of 80-120 A have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity. Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere.
已使用生理盐溶液和凝胶过滤色谱法从14日龄鸡胚胎骨骼肌中制备出直径为80 - 120埃的细丝。所获得的细丝由两种已知的肌肉中间丝蛋白，即波形蛋白和结蛋白，以及与波形蛋白和结蛋白相关的高分子量蛋白伴肌动蛋白（分子量230,000）组成。此外，它们还含有一种先前未鉴定的高分子量蛋白（分子量280,000），其在等电点、分子量和免疫反应性方面与伴肌动蛋白不同。使用针对280,000道尔顿多肽的抗血清对培养的成肌细胞进行免疫荧光检测，结果显示，在早期肌管中该蛋白与细胞质细丝相关联，在晚期肌管中与肌原纤维Z线相关联，其空间分布与结蛋白、波形蛋白和伴肌动蛋白相同。对发育中的胚胎骨骼肌冰冻切片进行免疫荧光检查发现，280,000道尔顿蛋白的含量逐渐减少。免疫荧光和免疫放射自显影显示，在成年骨骼肌和平滑肌中检测不到280,000道尔顿的蛋白。在组织培养中生长的鸡胚胎成纤维细胞中，尽管所有这些细胞都含有波形蛋白，但只有一部分细胞对280,000道尔顿蛋白的抗体有反应。在有反应的细胞中，波形蛋白和280,000道尔顿的多肽呈现出难以区分的细胞质丝状网络，当细胞暴露于秋水仙酰胺时，该网络会聚集形成丝状束。这些结果表明，这种新鉴定的高分子量蛋白与仅含有波形蛋白或同时含有波形蛋白、结蛋白和伴肌动蛋白的中间丝密切相关。该蛋白的表达似乎受发育调控，且似乎与其他三种中间丝蛋白中的任何一种的表达都不平行。成年肌肉细胞中不存在280,000道尔顿的多肽以及其在发育过程中的逐渐减少意味着，在肌节组装后维持Z盘结构可能不需要它。
