BIOCHEMISTRY OF CONTRACTILE PROTEINS

我们的长期目标是更好地了解监管 在平滑肌肉中的机制。平滑的肌肉对于正常的身体是必不可少的 是动脉、静脉、肠道的收缩元素， 子宫等。众所周知，细胞内的增加 CA浓度可引起血管收缩。这一机制 它通过收缩来检测CA浓度的变化 装置，然后处理以产生收缩或松弛 仍然存在争议。有两种基本理论：最流行的 假说是肌球蛋白轻链激酶使肌球蛋白磷酸化 (MLCK)激活收缩装置，导致收缩， 去激活(松弛)是通过肌球蛋白去磷酸化来实现的。 肌球蛋白轻链磷酸酶(MLCP)；另一种理论是 调节是通过一种称为Leiottin的机制来实现的，并不涉及 肌球蛋白磷酸化。有相当多的证据表明 磷酸化理论至少构成了部分调控机制。 而这项提案的重点是详细审查不同方面的 这个理论。MLCK与收缩元素的联系 将会被研究。有证据表明MLCK与肌动蛋白结合。这将是 确认并确立了约束条件。如果MLCK是 仅限于细丝，细丝重叠的程度将决定 磷酸化水平。改进的分离程序 将对MLCK进行调查。有一些数据可以证明这一切 以前分离的MLCK可能来自更大的前体。这个 磷酸化反应也将被更详细地分析，因为它是 有可能磷酸化是顺序的而不是随机的，以及 这是肌球蛋白分子的一般性质，将通过研究来测试 肌球蛋白和肌球蛋白的磷酸化 它们的亚片段。一个重要的目标是建立起 建议进行磷酸化和几个实验，其中 磷酸化可与肌动球蛋白或肌球蛋白的ATPase活性相关 超沉淀。无论是否是非循环肌球蛋白-肌动蛋白附着 都是在体外条件下形成的，将进行研究。数据来自 上述实验将被用来评估肌球蛋白的作用 并质疑其他机制是否 已注明。后者也将使用CA不敏感的MLCK进行测试。

Our long range goal is to gain a better understanding of the regulatory mechanisms in smooth muscle. Smooth muscle is essential for normal body function and is the contractile element of arteries, veins, intestines, uterus, etc. It is known that an increase in the intracellular concentration of CA++ induces contraction of smooth muscle. The mechanism by which the changes in CA++ concentration are detected by the contractile apparatus and then processed to result in either contraction or relaxation is still controversial. There are two basic theories: the most popular hypothesis is that phosphorylation of myosin by a myosin light chain kinase (MLCK) activates the contractile apparatus and leads to contraction, deactivation (relaxation) is achieved by dephosphorylation of myosin by a myosin light chain phophatase (MLCP); the alternative theory is that regulation is achieved by a mechanism termed leiotonin and does not involve myosin phosphorylation. There is considerable evidence to indicate that the phosphorylation theory forms at least part of the regulatory mechanism and the focus of this proposal is to examine in detail different aspects of this theory. The association of the MLCK with the contractile elements will be studied. Evidence suggests that MLCK binds to actin. This will be confirmed and the conditions of binding established. If the MLCK is restricted to thin filaments the extent of filament overlap will dictate the level of phosphorylation. Improved procedures for the isolation of MLCK will be investigated. There are some data to sugget that all previously isolated MLCKs may be derived from a larger precursor. The phosphorylation reaction will also be analyzed in more detail since it is possible that phosphorylation is sequential rather than random, and whether this is a general property of myosin molecules will be tested by studying phosphorylation of smooth muscle and skeletal muscle muscle myosins and their subfragments. An important objective is to estgablish the role of phosphorylation and several experiments are suggested in which phosphorylation can be correlated to ATPase activity of actomyosin or superprecipitation. Whether or not non-cycling myosin-actin attachments are formed under in vitro conditions will be investigated. The data from the above experiments will be evaluated to assess the role of myosin phosphorylation and to question whether additional mechanisms are indicated. The latter will also be tested using CA++ insensitive MLCK.