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.

众所周知，钙调蛋白，钩环蛋白和塔林在二杆菌上连接肌营养不良蛋白。据报道，其中的钙调蛋白在N末端结构域IN结合了Bystrophin，而其他两种蛋白质的结合位点，即痤疮蛋白和塔林，尚不清楚。这项投资是为了证明钙调蛋白，a麦和塔林与肌营养不良蛋白相关，并进一步检测到超微结构水平的连接点。对12个痤疮胡椒d的12个氨基酸残基的兔抗体（Belkin Am等人J细胞科学107：159-173,1994）和抗纯化的牛脑钙调蛋白的抗体。对塔林的单克隆抗体在商业基础上具有AVAI含量。用（1）兔抗蛋白和绵羊抗肿瘤抗体，兔抗蛋白和绵羊抗光谱抗体（2）单克隆抗抗通过抗抗蛋白和绵羊抗肿瘤抗体抗体抗体和绵羊抗体抗体抗体抗体（3）抗体抗体（3）使用组织化学上正常的人类肌肉的抗体和兔抗肿瘤抗体揭示，与谱蛋白表位相比，用肌营养不良蛋白表位更频繁地形成a粘蛋白，塔林和钙调蛋白的表位，而伴有肌营养不良蛋白表位。对双重组形成的单一盲分析在痤疮和肌营养不良蛋白的抗体组合以及a钩蛋白和光谱蛋白的抗体组合中表明，双重的percents分别为23.5（]SY。+ - 。[）1.8（se）和12.8（se）和12.8（]sy。+ - 。+ - 。[）1.1（p <0.01）。在肌营养不良蛋白和acicul的Lin-king点的分析中，使用了肌营养不良蛋白识别的单元抗体N和C末端。该关联域似乎位于肌营养不良蛋白的N和C末端。冻结蚀刻电子显微镜揭示了观察到两个不同尺寸的金颗粒，这些颗粒表明a刺蛋白和肌营养不良蛋白的细胞骨架。