Owada M K, Hakura A, Iida K, Yahara I, Sobue K, Kakiuchi S
Proc Natl Acad Sci U S A. 1984 May;81(10):3133-7. doi: 10.1073/pnas.81.10.3133.
Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the transformed state.
钙调蛋白是一种最初从鸡砂囊平滑肌中纯化出来的钙调素结合蛋白和F-肌动蛋白结合蛋白。这种蛋白以一种Ca2+和钙调素依赖的“翻转”方式与F-肌动蛋白丝结合，从而调节肌动蛋白丝的功能。在此我们报道，各种培养细胞系中含有一种77,000道尔顿的蛋白，它能与针对鸡砂囊钙调蛋白制备的亲和纯化钙调蛋白抗体发生特异性反应。在用[35S]甲硫氨酸脉冲标记的成纤维细胞蛋白中，77,000道尔顿的蛋白是在NaDodSO4凝胶上检测到的唯一与抗钙调蛋白反应的蛋白条带。使用抗钙调蛋白通过间接免疫荧光技术研究了77,000道尔顿蛋白的亚细胞分布。在所有检测的成纤维细胞系中，免疫荧光沿着细胞应力纤维和细胞前缘定位。然而，在劳氏肉瘤病毒转化的细胞（S7-1）中，荧光分布变为弥散且模糊的外观。用正常细胞和转化细胞获得的抗钙调蛋白的这些染色模式与相应状态下抗肌动蛋白的染色模式一致，强烈提示77,000道尔顿蛋白与肌动蛋白丝之间存在功能联系。我们提议将这种77,000道尔顿的蛋白称为钙调蛋白77。转化的S7-1细胞中钙调蛋白77的细胞水平降至其正常对应物（细胞系7号）的约三分之一。用感染了劳氏肉瘤病毒温度敏感转化突变体施密特-鲁平株（68 N2克隆）的正常大鼠肾细胞也得到了基本相同的结果。在允许温度（35℃）下观察到的钙调蛋白77的细胞水平约为非允许温度（38.5℃）下的三分之一。转化细胞中钙调蛋白77的这些变化可能与转化状态下Ca2+调节的丧失有关。
