| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
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.
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