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

我们认为，肌原纤维蛋白，即帕托普肌球蛋白，与肌小节的A-I连接区的连接素结合，有助于在死后的陈化过程中促进肉的嫩化。本研究测定了鸡活体和死后肌肉中连接蛋白的结合部位，以确定连接蛋白细丝上的连接蛋白的定位，并阐明了高浓度Ca~(2+)从连接蛋白结合部位释放的机制。β-连接蛋白是从鸡胸肌中提纯的一种天然形式的连接素，并在不同条件下用酶消化。用双向聚丙烯酰胺凝胶电泳法分离β连接蛋白片段。用生物素化的副肌球蛋白和抗连接蛋白单抗检测到43 kDa片段，其N端序列为YQFRVYAVNK，与人心肌肌钙蛋白/连接蛋白A-I连接区151个纤维连接蛋白3型结构域的7556-7565个氨基酸序列相似。因此，我们推测，在活体肌肉和死后肌肉中，副肌球蛋白与J3-Connectin的43-kDa片段在死后即刻结合在A-I连接区，43-kDa片段的N-末端定位在151区。此外，我们还从鸡肌原纤维中纯化了400 kDa的片段，它是p-连接蛋白N-末端的一部分，其结构中含有43 kDa的片段。对400 kDa片段的一级结构的分析正在进行中，以确定连接蛋白上的甲状旁腺肌球蛋白结合位点。

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.