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Ultrastractural localization of dystrophin N-terminal dinding proteins and their relation to dystrophin in normal skeletal myofiber.

肌营养不良蛋白 N 末端结合蛋白的超微结构定位及其与正常骨骼肌纤维中肌营养不良蛋白的关系。

基本信息

批准号：
08670728
负责人：
WAKAYAMA Yoshihiro
金额：
$ 1.34万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670728/
关键词：
aciculin calmodulin talin dystrophin binding domain skeletal muscle ultrastructure freeze etch replica N-terminus β-spectrin skeletal muscle localization

项目摘要

Calmodulin, aciculin and talin are known to link dystrophin diochemically. Among them calmodulin is reported to bind bystrophin at N terminal doma-in and the binding sites of other two proteins, aciculin and talin, are unknown. This investigation is undertaken to demonstrate that calmodulin, aciculin and talin associate with dystrophin, and further to detect the linking point at ultrastructural level. The rabbit antibodies against sy-nthetic peptide of 12 amino acid residues of aciculin peptide D (Belkin AM et al. J Cell Sci 107 : 159-173,1994) and against purified bovine brain calmodulin are generated. The monoclonal antibody against talin was avai-lable in commercial base. Double immunoglold labeling electron microscopy with (1) rabbit antiaciculin and sheep antidystrophin antibodies, and rab-bit antiaciculin and sheep antispectrin antibodies (2) monoclonal antita-lin and sheep antidystrophin antibodies, and monoclonal antitalin and sheep antispectrin antibodies (3) rabbit anticalmodulin and sheep antidystrophin antibodies and rabbit anticalmodulin and sheep antispectrin antibodies using histochemically normal human muscle disclosed that the epitopes of aciculin, talin and calmodulin formed more frequently doublet with dystrophin epitope than with spectrin epitope. Single blind analysis of doublet formation in the antibody combination of aciculin and dystrophin, and aciculin and spectrin showed that the doublet percents were 23.5(]SY.+-。[)1.8 (SE) and 12.8(]SY.+-。[)1.1 (P<0.01), respectively. In the analysis of lin-king point of dystrophin and acicul : n, the monoelonal antibodies of dys-trophin recognizig the N and C termini were used. The association domain seemed to be at both N and C terminals of dystrophin. The freeze etch electron microscopy disclosed that two different sized gold particles indicating cytoskeletons of aciculin and dystrophin were observed.

已知钙调蛋白、针状蛋白和talin以生物化学方式连接抗肌萎缩蛋白。其中钙调素与肌萎缩蛋白的N端结构域结合，而针状蛋白和talin的结合位点尚不清楚。本研究旨在证明钙调素、针状蛋白和talin与肌营养不良蛋白的相互作用，并进一步在超微结构水平上检测其相互作用点。制备了兔抗针蛋白肽D（Belkin AM等，J Cell Sci 107：159- 173，1994）的合成肽和抗纯化的牛脑钙调素的抗体。抗talin单克隆抗体已商品化。用（1）兔抗针状蛋白和羊抗肌萎缩蛋白抗体，兔抗针状蛋白和羊抗血影蛋白抗体进行免疫胶体金双标记电镜观察;（2）单克隆抗针状蛋白和羊抗肌萎缩蛋白抗体，和单克隆抗talin和羊抗spectrin抗体（3）使用组织化学正常人肌肉的兔抗钙调蛋白和羊抗肌营养不良蛋白抗体以及兔抗钙调蛋白和羊抗血影蛋白抗体公开了，针状蛋白、talin和钙调蛋白的表位与肌营养不良蛋白表位形成双联体的频率高于与血影蛋白表位形成双联体的频率。对针状蛋白和肌养蛋白以及针状蛋白和血影蛋白的抗体组合中的双联体形成的单盲分析表明，双联体的形成率为23.5 ± 0.001。[)1.8（SE）和12.8（]SY. ±-. [)1.1（P<0.01）。在抗肌营养不良蛋白（dystrophin）与acicul：n的连接点分析中，采用了识别N端和C端的dystrophin单克隆抗体。Dystrophin的N端和C端均含有结合结构域。冷冻蚀刻电镜观察到两种不同大小的金颗粒，分别指示针状蛋白和肌营养不良蛋白的细胞骨架。