| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The isoform-specific assembly of troponin I isoforms of cardiac and fast skeletal muscles (CTnI and FTnI, respectively) to myofibrils (MFs) of both muscles was investigated. Epitope tagging was used to monitor the intracellular localization of exogenously introduced constructs to myofibrillar structures in cultured chicken cardiac and breast (fast) muscle cells. Exogenous CTnI and FTnI were incorporated into endogenous MFs of cardiac and breast muscle cells with high affinity, respectively. In the case of CTnI and FTnI with breast and cardiac muscle cells respectively, CTnI was not incorporated into breast MFs but FTnI was assembled onto cardiac MFs. To determine which portion of TnIs is responsible for incorporation into these MFs, we constructed chimeric TnIs with the head and tail of CTnI replaced by those of FTnI.The behavior of these chimeras depends on the tail of TnIs. These results suggest that the tail regions of TnIs bind to cardiac and breast MFs and that these affinities of
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TnI tails are responsible for the assembly of FTnI onto cardiac MFs. Further we examined the binding domains with truncated CTnI and FTnI.Deletion mutants containing CTnI amino acid residues 1-79, 43-207 and 80-207 (CTnI-head, CTnI-tail-1 and CTnI-tail-2, respectively) and FTnI amino acid residues 1-54 and 55-182 (FTnI-head and FTnI-tail, respectively) were transiently expressed in cardiac and fast skeletal muscle cells. CTnI-tail-1 was incorporated into cardiac MFs specifically, but CTnI-tail-2 was not assembled onto any MFs examined. This suggests that there is no potent actin filament binding site in CTnI-tail-2. Since CTnI-tail-1 has an amino acid extension (CTnI residues 43-79) of which the sequence is longer than that of CTnI-head-2, it appears that this sequence extension is important in binding to cardiac MFs. FTnI-tail containing the inhibitory domain of actomyosin ATPase, showed intensive and specific incorporation into fast MFs. FTnI-tail was a homologous fragment of CTnI-tail-2, but the binding patterns of these two domains differed greatly from each other. It is possible that the absence of potent binding affinity of CTnI-tail-2 corresponding to the inhibitory domain of actomyosin ATPase is advantageous for continuous cardiac muscle contraction, since a potent inhibitory activity causes a serious obstacle for cardiac muscle contraction. It can be assumed that distinctive binding ability of functional domains of TnI-tails reflect unique adaptations to muscles with different physiological properties. Less
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