Translocation of Paratropomyolsin from the A-I Junctiion Region to Contribute the Resolution of Rigor-Mortis of Postmortem Skeletal Muscles

我们已经提出，肌原纤维蛋白，副肌球蛋白，是绑定到连接蛋白在A-I连接区的肌节，有助于肉嫩化在死后老化。本研究通过测定鸡肌肉中连接蛋白上的副肌球蛋白结合位点，以确定副肌球蛋白在鸡肌肉中连接蛋白丝上的定位，并阐明高Ca^2+浓度下副肌球蛋白从该位点释放的机制。从鸡胸肉中分离纯化了一种蛋白酶，并在不同条件下进行了酶解。酶切后的β-connectin片段经双向聚丙烯酰胺凝胶电泳分离。通过生物素化的副肌球蛋白和抗连接蛋白单克隆抗体检测到在A-I连接区与副肌球蛋白结合的43-kDa片段。发现43-kDa片段的N-末端序列是YQFRVYAVNK，类似于位于人心脏肌联蛋白/连接蛋白A-I连接区的151个纤连蛋白3型结构域中的7556-7565个氨基酸的序列。因此，我们建议，副肌球蛋白结合到43-kDa的片段从j3-connectin在A-I交界处的区域，在活的肌肉和肌肉立即死后，和N-末端的43-kDa的片段是本地化的151域。此外，我们已经从鸡肌原纤维中纯化了400-kDa片段，该片段是p-连接蛋白的N-末端侧的一部分，并且在其结构中包含43-kDa片段。400 kDa片段的一级结构的分析正在进行中，以指定连接蛋白上的副肌球蛋白结合位点。

We have suggested that myofibrillar protein, paratropomyosin, is bound to connectin in the A-I junction region of a saroomere to contribute to meat tenderization in postmortem ageing. This study was conducted to determine the paratropomyosin binding site on connectin in living and immediately postmortem chicken muscles in order to confirm the localization of paratropomyosin on connectin filaments, and to clarify the mechanism for release ofparatropomyosin from the site by the high Ca^<2+> concentration.β-Connectin, which was a native form of connectin, was purified from chicken breast muscle and digested by protease under various conditions. The digested β-connectin fragments were separated by two-dimensional polyacrylamide gel electrophoresis. The 43-kDa fragment, to which paratropomyosin bound at the A-I junction region, was detected by biotinylated paratropomyosiii and anti-connectin monoclonal antibody The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the 151 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from j3-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the 151 domain. Moreover, we have purified the 400-kDa fragment from chicken myofibrils, which is a part of N-terminal side of p-connectin and contains the 43-kDa fragment in its structure. The analysis of the primary structure of the 400-kDa fragment is in progress to specify the paratropomyosin binding site on connectin.