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MOLECULAR MECHANISM ON THE REGULATION OF SMOOTH MUSCLE CONTRACTION STUDIED BY NEW EXPERIMENTAL TECHNIQUES

新实验技术研究平滑肌收缩调节的分子机制

基本信息

批准号：
02454495
负责人：
KATAYAMA Eisaku
金额：
$ 1.98万
依托单位：
INSTITUTE OF MEDICAL SCIENCE,THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Caldesmon is a fibrous protein existing specifically in smooth muscle cells. I have previously reported about the functional domain organization in the primary amino-acid sequence of that molecule ; that its C-terminal and N-terminal portions specifically bind to F-actin/tropomyosin and S2 moiety of myosin, respectively, and that the molecule might fold into half a length under low ionic strength. I examined by electron microscopy, the structure of reconstituted and natural thin filaments after brief chemical fixation, using negative staining and mica-flake technique coupled with quick-freeze deep-etch replica method. Reconstituted thin-filaments showed the fibrous molecules protruding from actin with the periodicity common to trepomyosin. Heavy meromyosin (HMM) heads do not usually associate with actin in the presence of ATP,if unphosphorylated. Possible clusters of unphosphorylated-HMM molecules, however, were found to bind to reconstituted thin-filaments with the same periodicity as … More that of caldesmon protrusion. Unphosphorylated-myosin also bound to thin-filaments under a similar condition. Though natural thin-filament showed no protrusions under ionic conditions close to physiological, actin helices were not clear suggesting the presence of some additional molecules on the surface of thin-filaments. When they were fixed under lower ionic strength, a similar image to that of reconstituted thin-filaments was obsereved. These structural results well coincide with the above biochemical data.Filaments of unphosphorylated myosin are known to be unstable under in vitro conditions and easily dissolve upon addition of ATP.It was found, however, that the co-existence of caldesmon stabilizes such filaments, and that they were tethered in a periodic manner to actin-filaments further added. Reported evidence for the existence of unphosphorylated myosin-filaments in relaxd smooth muscle tissue had been in conflict with the above in vitro biochemical results. The physiological in vivo situation might be now reasonably explained by such a characteristic property of caldesmon molecule. Less

钙调素是一种纤维蛋白质，特别存在于平滑肌细胞。我以前曾报道过该分子的初级氨基酸序列中的功能结构域组织;其C-末端和N-末端部分分别特异性结合F-肌动蛋白/原肌球蛋白和肌球蛋白的S2部分，并且该分子在低离子强度下可能折叠成一半长度。我检查了电子显微镜，重组和天然细丝的结构后，简短的化学固定，使用负染色和云母片技术加上速冻深蚀刻复制方法。重构的细丝显示从肌动蛋白突出的纤维状分子，具有与曲肌球蛋白共同的周期性。重的裂肌球蛋白（HMM）头通常不与肌动蛋白在ATP的存在下，如果未磷酸化。然而，未磷酸化HMM分子的可能簇被发现与重构的细丝结合，其周期性与 ...更多信息 caldesmon protrusion突起。非磷酸化肌球蛋白也结合到细丝在类似的条件下。虽然天然细丝在接近生理的离子条件下没有显示出突起，但肌动蛋白螺旋不清楚，这表明细丝表面存在一些额外的分子。当它们在较低离子强度下固定时，观察到类似于重构细丝的图像。这些结构结果与上述生物化学数据很好地吻合。已知未磷酸化的肌球蛋白的纤维在体外条件下不稳定，加入ATP后容易溶解。然而，发现钙调蛋白的共存使这些纤维稳定，并且它们以周期性的方式与进一步加入的肌动蛋白纤维相连。松弛平滑肌组织中存在未磷酸化肌球蛋白丝的证据与上述体外生化结果相矛盾。这种钙调素分子的特性可以合理地解释体内的生理状况。少