BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌对于生理功能是至关重要的， 未来的临床治疗，以了解潜在的分子基础 它的收缩机制。 平滑肌的一种主要调节机制 涉及肌球蛋白轻链的磷酸化。 两种关键酶是 涉及：肌球蛋白轻链激酶（MLCK），其活性与 通过钙调蛋白和肌球蛋白轻链磷酸酶的钙瞬变 （MLCP）。 平滑肌的收缩活动水平反映了 肌球蛋白磷酸化的程度，这取决于MLCK的平衡 和MLCP活动。 这表明，钙依赖性的这一点， 平衡可能会发生变化，这解释了 平滑肌的生理反应。 研究概述如下： 检查激酶和磷酸酶组分。 知之甚少 关于肌球蛋白去磷酸化的机制， 这一建议的重点。 1型至少存在四种亚型 （PP-1）催化亚基，并且每个同种型具有不同的性质。 我们 最初的目标是鉴定与肌球蛋白相关的同种型 去磷酸化，并在原核细胞或 真核系统 PP-1亚型之间的主要差异在于 它们的C末端序列 为了验证这部分的假设， 分子表示同种型的区别特征，各种突变体 将被设计和表达，其中该区域被删除或 修改. 这些研究将侧重于MLCP，但也将澄清 其他PP-1亚型的独特性质的基础。 调控 磷酸酶活性涉及催化亚基的相互作用 与其他组件。 初步研究表明，来自砂囊的MLCP 由催化亚基加上58 kD组分组成。 后者 与肌球蛋白结合并可作为靶向亚基。对此的研究 亚基被设计用于通过筛选天然成分 cDNA文库，以表达天然形式并表征其 性质，重点是其与肌球蛋白和 催化亚单位 抑制因子-1和-2在细胞凋亡中的生理作用 没有建立平滑肌，并且提出实验以 评估其功能。 用MLCK进行结构-功能研究 关系将继续，重点是肌动蛋白结合位点 以及催化域的所需边界。 每个人的极限 通过诱变建立功能结构域。 最后一个主题 提出了第二种激酶可以磷酸化肌球蛋白的可能性 在没有Ca 2+的情况下。我们将尝试描述这一点 激酶，如果与MLCK不同，以确定其在 平滑肌的生理行为。

Smooth muscle is vital for physiological function and it is essential for future clinical treatment to understand the molecular basis underlying its contractile mechanism. A major regulatory mechanism in smooth muscle involves phosphorylation of the myosin light chains. Two key enzymes are involved: myosin light chain kinase (MLCK) whose activity is linked to Ca2+ transients via calmodulin, and a myosin light chain phosphatase (MLCP). The level of contractile activity in smooth muscle reflects the extent of myosin phosphorylation and this depends on the balance of MLCK and MLCP activities. It was suggested that the Ca2+-dependence of this balance can shift and that this explains much of the variability in physiological responses of smooth muscles. Studies are outlined to examine both the kinase and phosphatase components. Little is known about the mechanism of myosin dephosphorylation and this forms the emphasis of this proposal. At least four isoforms exist of the type 1 (PP-1) catalytic subunit and each isoform has distinct properties. Our initial objective is to identify the isoform associated with myosin dephosphorylation and to express this in either a procaryotic or eucaryotic system. The major difference between PP-1 isoforms is in their C-terminal sequence. To test the hypothesis that this part of the molecule denotes distinguishing features of the isoforms, various mutants will be designed and expressed in which this region is deleted or modified. These studies will focus on MLCP, but will also clarify the basis for distinctive properties of other PP-1 isoforms. Regulation of phosphatase activity involves interaction(s) of the catalytic subunit with other components. Preliminary studies show that MLCP from gizzard consists of the catalytic subunit plus a 58 kD component. The latter binds to myosin and may act as a targeting subunit. Studies on this subunit are designed to identify the native component, via screening of a cDNA library, to express the native form and to characterize its properties, with emphasis on its interaction with myosin and the catalytic subunit. The physiological roles of inhibitor-1 And -2 in smooth muscle are not established and experiments are proposed to evaluate their function. With MLCK, studies on structure-function relationships will be continued, with emphasis on the actin-binding site and the required boundaries of the catalytic domain. The limits of each functional domain will be established by mutagenesis. The final topic addresses the possibility that a second kinase can phosphorylate myosin in the absence of Ca2+ . Attempts will be made to characterize this kinase and if distinct from MLCK to determine its role in the physiological behavior of smooth muscle.