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

Caldesmon是一种专门在平滑肌细胞中的纤维蛋白。我以前已经报道了该分子的一级氨基酸序列中的功能域组织。它的C末端和N末端部分分别与肌球蛋白的F-肌动蛋白/Tropomyosin和S2部分结合，并且该分子在低离子强度下可能会折叠成一半的长度。我通过电子显微镜检查了简短化学固定后的重构和天然细丝的结构，使用负染色和云母液片技术与快速冻结深蚀刻复制方法结合。重建的薄丝丝显示纤维分子与肌动蛋白的周期性相结合。如果未磷酸化，则重毛霉素（HMM）头通常不会与ATP存在的肌动蛋白相关。然而，发现未磷酸化的-HMM分子的簇结合到具有与Caldesmon蛋白质相同的周期性的重构薄膜。在类似条件下，未磷酸化的肌球蛋白也与薄膜结合。尽管天然薄丝在接近生理的离子状况下没有蛋白质显示出蛋白质，但肌动蛋白螺旋尚不清楚，表明薄膜表面上存在一些其他分子。当它们固定在较低的离子强度下时，观察到与重构薄丝的图像相似。这些结构性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是不稳定的，并且在添加ATP时很容易溶解。但是，Caldesmon的共存性可以使这种细胞的稳定性使这些细胞稳定，并在周期性的方式中添加了这种质量，以进一步添加。报道的证据表明，在松弛的平滑肌组织中存在未磷酸化的肌球蛋白丝与上述体外生化结果相抵触。现在，可以通过Caldesmon分子的这种特征特性来合理地解释生理体内情况。较少的