MOLECULAR MECHANISM OF CONTRACTILE REGULATION IN MUSCLE

在所有被研究的肌肉中，收缩是由 细胞内钙离子。在脊椎动物的横纹肌中，分子 机制涉及钙与调节性TNC亚单位的结合 肌钙蛋白位于细丝上。全钙 调控需要另外两种肌钙蛋白的协同作用 亚基(TnT和TnI)以及与原肌球蛋白和肌动蛋白的相互作用。在……里面 缺乏表面膜的细胞(所谓的“带皮”纤维)来自 哺乳动物骨骼肌和心肌，TNC可以部分去除， 使纤维松弛，对钙不敏感。然后，可以将光纤 补充TNC，允许恢复钙控制。回收利用 利用基因工程技术，TNC在探测 参与调控的TNC分子区域。不幸的是，类似的 用剥皮纤维进行耗尽/再耗尽实验研究TNT和TnI 已经完全不成功了。然而，最近我们发现， TNT(和一种功能未知的75kD蛋白质)可以从 将龙虾的快速肌肉纤维剥皮，使纤维充分呈现 即使在没有钙的情况下也会被激活。纤维可以通过以下方式松弛 添加富含龙虾TNT的分离龙虾蛋白混合物 和75kD的蛋白。这代表了唯一的蒙皮纤维 TNT提取和再补充成功的情况下的准备工作。这个 这个项目的总体目标是建立龙虾皮肌肉 纤维作为研究横纹肌收缩的模型系统。 我们希望利用这一独特的准备来探索 TNT，其方式与上述TNC实验相同。在……里面 此外，我们希望从去皮的龙虾中提取(然后补充)TnI 使用最近从龙虾肌肉中分离出的一种蛋白水解酶的纤维 这似乎是调节蛋白特有的。我们还有 发现龙虾快肌的皮肤纤维收缩 对酸中毒(即pH降低)非常不敏感，与 来自哺乳动物骨骼肌或心肌的结皮纤维。在哺乳动物中， 酸中毒被认为是肌肉萎缩的主要原因。 与疲劳和缺氧相关的收缩，尽管分子 人们对这种影响的基础知之甚少。通过比较 对哺乳动物和龙虾肌肉的研究，我们希望能阐明哪些步骤 收缩过程的一部分受pH变化的影响。

In all muscles studied, contraction is regulated by changes in intracellular calcium. In vertebrate striated muscles, the molecular mechanism involves calcium binding to the TnC subunit of the regulatory protein troponin located on the thin filament. Complete calcium regulation requires the concerted action of the two other troponin subunits (TnT and TnI), and interaction with tropomyosin and actin. In cells lacking surface membranes (so-called "skinned" fibers) from mammalian skeletal and cardiac muscle, TnC can be partially removed, rendering the fiber relaxed and calcium-insensitive. Fibers can then be repleted with TnC permitting restoration of calcium control. Repletion with genetically engineered TnC has been very useful in probing the molecular regions of TnC involved in regulation. Unfortunately, similar depletion/repletion experiments with skinned fibers to study TnT and TnI have been completely unsuccessful. Recently, however, we discovered that TnT (and a 75 kD protein of unknown function) could be extracted from skinned fast muscle fibers of the lobster, rendering the fibers fully activated even in the absence of calcium. Fibers could be relaxed by addition of a mixture of isolated lobster proteins rich in lobster TnT and the 75 kD protein. This represents the only skinned fiber preparation where TnT extraction and repletion has been successful. The overall goal of this project is to establish lobster skinned muscle fibers as a model system for the study of striated muscle contraction. We wish to capitalize on this unique preparation to probe the function of TnT in the same fashion as the Tnc experiments outlined above. In addition, we hope to extract (and then replete) TnI from skinned lobster fibers using a recently described proteinase isolated from lobster muscle which appears to be specific for the regulatory proteins. We have also discovered that contraction of skinned fibers from lobster fast muscle is remarkably insensitive to acidosis (i.e. decreased pH), as compared with skinned fibers from mammalian skeletal or cardiac muscle. In the mammal, acidosis is thought to play a major role in the decreased muscle contraction associated with fatigue and hypoxia, although the molecular basis for this influence is poorly understood. Through comparative studies of mammalian and lobster muscle, we hope to elucidate which steps of the contractile process are affected by changes in pH.