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Role of myosin-binding poteins in myofibrillogenesis

肌球蛋白结合蛋白在肌原纤维发生中的作用

基本信息

批准号：
08044191
负责人：
OBINATA Takashi
金额：
$ 4.86万
依托单位：
DEPARTMENT OF BIOLOGY,CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

C-protein is a major myosin-binding protein characteristic of striated muscles. In order to clarity the roles of C-protein in myofibrillogenesis, we carried out the following experiments and obtained the results as described.1) Mouse C-protein cDNA sequences and C-protein isoform expressionThe cDNAs encoding mouse fast skeletal (F) and slow skeletal (S) C-proteins were cloned and their entire sequences were determined. Cardiac (C)-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, but in mouse skeletal muscle, C-isoform was not detected through developmental stages. S-isoform became detectable first, followed by appearance of F-isoform, In the degenerating mouse skeletal muscles, expression of S-isoform is increased.2) Two isoforms in chicken cardiac C-protein and their expressionWe detected two variants (type I and type II) in chicken cardiac C-protein and determined their entire sequences ; they differ only by the inclusion or exclu … More sion of a sequence of 15 amino acid residues (P-seq) which includes a phosphorylation site for A-kinase. We prepared the antibody specific for P-seq. By immunocytochemical methods with this and the other monoclonal antibody to cardiac C-protein (C-315), we found that two isoforms were coexpressed and colocalized in both atrial and ventricular muscles through development, but type I was scarcely expressed in embryonic skeletal muscle. These observations were confirmed by a RT-PCR method.3) Competition of C-protein (MyBP-C) with H-protein (MyBP-H) for binding to myosin in cardiac muscle cellsC-Protein and H-protein are myosin-binding proteins which localize in A-band regions of sarcomeres. To clarify functional relationship of these two myosin binding proteins, we introduced cDNAs encoding these proteins into primary cultures of chicken myocytes and examined their assembly into myofibrils. The results obtained indicate that overexpression of MyBP-C or MyBP-H in cultured cardiac myocytes can reduce the content of MyBP-H or MyBP-C, respectively. These data indicate that MyBP-H can compete with MyBP-C for binding to myosin in the cells. Less

C蛋白是横纹肌特有的主要肌球蛋白结合蛋白。为了阐明C蛋白在肌原纤维形成中的作用，我们进行了以下实验，并获得了如下结果：1）小鼠C蛋白cDNA序列和C蛋白亚型的表达克隆了编码小鼠快骨骼（F）和慢骨骼（S）C蛋白的cDNA，并测定了它们的全序列。已知心脏（C）亚型在鸡骨骼肌发育过程中首先短暂表达，但在小鼠骨骼肌中，C亚型在整个发育阶段均未检测到。鸡心肌C蛋白的两种异构体及其表达我们在鸡心肌C蛋白中检测到两种变异体（I型和II型），并测定了它们的全序列，它们的区别仅在于包含或不包含C蛋白的两种异构体，即I型和II型。 ...更多信息 15个氨基酸残基的序列（P-seq）的锡永，其包括A-激酶的磷酸化位点。我们制备了P-seq特异性抗体。通过免疫细胞化学方法与此和其他单克隆抗体的心脏C-蛋白（C-315），我们发现这两种异构体共表达和共定位在心房和心室肌通过发展，但I型几乎不表达在胚胎骨骼肌。C蛋白（MyBP-C）和H蛋白（MyBP-H）在心肌细胞中竞争与肌球蛋白结合C蛋白和H蛋白是肌球蛋白结合蛋白，定位于肌节A带区。为了阐明这两个肌球蛋白结合蛋白的功能关系，我们将编码这两个蛋白的cDNA导入鸡肌细胞的原代培养物中，并检测它们组装成肌原纤维的情况。结果表明，MyBP-C或MyBP-H在培养的心肌细胞中的过表达可分别降低MyBP-H或MyBP-C的含量。这些数据表明MyBP-H可以与MyBP-C竞争结合细胞中的肌球蛋白。少