BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌对于生理功能是至关重要的，对于 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。两种关键的酶是 涉及：肌球蛋白轻链激酶(MLCK)，其活性与 钙离子通过钙调蛋白和肌球蛋白轻链磷酸酶进行瞬变 (MLCP)。平滑肌收缩活动的水平反映了 肌球蛋白磷酸化的程度，这取决于MLCK的平衡 和MLCP活性。提示该细胞对钙离子的依赖性 平衡可能会发生变化，这解释了 平滑肌肉的生理反应。研究概述如下： 同时检查激酶和磷酸酶组分。鲜为人知 关于肌球蛋白去磷酸化的机制，这形成了 这项建议的重点。至少存在四种类型1的异构体 (PP-1)催化亚基和每种异构体都有不同的性质。我们的 最初的目标是鉴定与肌球蛋白相关的亚型。 去磷酸化，并在原核生物或 真核系统。PP-1亚型之间的主要区别是 它们的C-末端序列。来检验这样一个假设，即 分子表示异构体的不同特征，各种突变体 将在设计和表达中删除该区域或 修改过的。这些研究将侧重于MLCP，但也将澄清 为其他PP-1亚型的独特性质奠定了基础。规管 磷酸酶活性涉及催化亚基的相互作用(S) 与其他组件一起。初步研究表明，来自肌胃的MLCP 由催化亚基和58kD组分组成。后者 与肌球蛋白结合，并可作为靶向亚基。关于这一点的研究 亚基被设计成通过筛选一种 C DNA文库，表达其天然形式并对其进行鉴定 性质，重点是它与肌球蛋白的相互作用和 催化亚基。抑制物-1和-2在血管紧张素转换酶中的生理作用 没有建立平滑肌，建议进行实验以 评估他们的功能。用MLCK研究结构-功能 关系将继续，重点是肌动蛋白结合部位 以及催化域的所需边界。每种方法的局限性 功能域将通过诱变来建立。最后一个话题 解决了第二个激酶可以磷酸化肌球蛋白的可能性 在没有钙离子的情况下。我们将尝试对此进行描述 如果不同于MLCK，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.