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

Caldesmon是一种纤维蛋白，特异存在于平滑肌细胞中。我之前已经报道过该分子一级氨基酸序列的功能域组织；它的c端和n端分别特异性结合F-actin/原肌凝蛋白和肌凝蛋白的S2部分，并且在低离子强度下分子可以折叠成半长度。采用负染色和云母片技术结合速冻深蚀刻复制法，在电子显微镜下观察了经短暂化学固定后重组的天然细丝的结构。重组的细丝显示纤维分子从肌动蛋白中突出，具有与水凝蛋白相同的周期性。如果未磷酸化，在ATP存在的情况下，重肌凝蛋白（HMM）头部通常不与肌动蛋白结合。然而，发现可能的未磷酸化的hmm分子簇与重构的细丝结合，具有与caldesmon突出相同的周期性。在类似的条件下，未磷酸化的肌球蛋白也与细丝结合。虽然在接近生理的离子条件下，天然细丝没有出现突出，但肌动蛋白螺旋并不清晰，表明在细丝表面存在一些附加分子。当它们在较低的离子强度下固定时，观察到与重构的薄细丝相似的图像。这些结构结果与上述生化数据吻合得很好。已知未磷酸化肌球蛋白的细丝在体外条件下是不稳定的，并且在加入ATP后容易溶解。然而，人们发现，caldesmon的共存稳定了这些细丝，并且它们以一种周期性的方式束缚在进一步添加的行动蛋白细丝上。在松弛的平滑肌组织中存在未磷酸化的肌球蛋白丝的报道证据与上述体外生化结果相冲突。caldesmon分子的这种特性可以合理地解释体内的生理情况。少

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