Epstein H F, Berliner G C, Casey D L, Ortiz I
Department of Neurology, Baylor College of Medicine, Houston, Texas 77030.
J Cell Biol. 1988 Jun;106(6):1985-95. doi: 10.1083/jcb.106.6.1985.
The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.
线虫秀丽隐杆线虫的粗肌丝主要源自体壁肌肉，包含两种肌球蛋白同工型和副肌球蛋白作为其主要蛋白质。两种肌球蛋白位于表面的不同区域，而副肌球蛋白位于肌丝的主干内。管状结构构成极性区域的核心，电子致密物质存在于中央区域的核心中（爱泼斯坦，H.F.，D.M. 米勒，I. 奥尔蒂斯，和 G.C. 伯林纳。1985 年。《细胞生物学杂志》100:904 - 915）。生化、遗传和免疫学实验表明，这两种肌球蛋白和副肌球蛋白并非必需的核心成分（爱泼斯坦，H.F.，I. 奥尔蒂斯，和 L.A. 特拉格·麦金农。1986 年。《细胞生物学杂志》103:985 - 993）。因此，核心结构的存在表明，秀丽隐杆线虫的粗肌丝可能还与其他蛋白质相关。为了生化检测次要的相关蛋白质，已开发出一种用于分离高纯度且结构保存完好的粗肌丝的新方法。最后一步，甘油梯度离心，根据考马斯亮蓝染色和电子显微镜观察，得到的组分最多被肌动蛋白、原肌球蛋白或核糖体相关蛋白污染 1 - 2%。分别对未标记和 35S 标记蛋白质的凝胶电泳图谱进行银染和放射自显影，结果显示至少还有 10 条额外的条带与甘油梯度中的粗肌丝共同沉降。从野生型粗肌丝制备的核心结构包含至少六条这些与粗肌丝相关的蛋白质条带。这六种蛋白质也与通过梯度离心从缺乏肌球蛋白重链 B 的 CB190 突变体和缺乏副肌球蛋白的 CB1214 突变体中纯化的粗肌丝共同沉降。基于这些原因，我们提出这六种相关蛋白质是秀丽隐杆线虫体壁肌肉粗肌丝核心结构假定成分的潜在候选者。
