BIOCHEMICAL MECHANISMS OF SMOOTH MUSCLE CONTRACTION

虽然普遍认为肌球蛋白轻链磷酸化 由Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶是主要的 在平滑肌收缩的起始事件中，有以下方面： 这个过程在细胞和分子水平上还没有很好的理解， 程度. 本申请中描述的研究项目解决了 平滑肌调节的一些重要问题 血管和气道平滑肌中的收缩成分。 分子内 （钙调素结合域-假底物抑制区-催化 结构域）和分子间（催化结构域-蛋白质底物结合， 催化）与活化和催化相关的事件将被检查 并与MLCK与肌动蛋白和/或肌球蛋白丝的结合有关。 一 生物化学和分子生物学实验方法的结合 生物学将用于分析平滑肌肌球蛋白的这些特性 轻链激酶 新的信息表明肌球蛋白轻链 激酶和磷酸酶活性可以被调节， 这些活性并不是在所有条件下都由[Ca ~（2+）]i简单决定的 用于强直性和阶段性平滑肌。 调节激酶和 磷酸酶活性将有助于报道的Ca 2+的变化 收缩元件的敏感性。 肌球蛋白轻链激酶是 收缩时气管平滑肌磷酸化。 的 这种磷酸化的功能后果和鉴定 将测定催化该反应的激酶。 蛋白质 使肌球蛋白轻链和肌球蛋白轻链脱磷酸化磷酸酶 还将鉴定生理学上的激酶，以及 规则阐明。 本申请中提出的研究使用 从分子生物学，生物化学， 细胞生物学、生理学和药理学来解开这个复杂的系统 提供直接涉及监管机制的理解， 可收缩元素 这些信息对于理解 由疾病过程引起的平滑肌功能紊乱， 高血压和哮喘。

Although there is general agreement that myosin light chain phosphorylation by the Ca2+/calmodulin-dependent myosin light chain kinase is a primary event in the initiation of smooth muscle contraction, there are aspects of this process that are not well understood at the cellular and molecular levels. The research projects described in this application address a number of important issues dealing with the regulation of smooth muscle contractile elements in vascular and airway smooth muscles. Intramolecular (calmodulin binding domain-pseudosubstrate inhibitory region-catalytic domain) and intermolecular (catalytic domain-protein substrate binding and catalysis) events associated with activation and catalysis will be examined and related to the binding of MLCK to actin and/or myosin filaments. A combination of experimental approaches involving biochemistry and molecular biology will be used to analyze these properties of smooth muscle myosin light chain kinase. New information indicates that myosin light chain kinase and phosphatase activities may be regulated so that the ratio of these activities is not simply determined by [Ca2+]i under all conditions for tonic and phasic smooth muscles. Regulation of both kinase and phosphatase activities would contribute to reported changes in the Ca2+ sensitivity of the contractile elements. Myosin light chain kinase is phosphorylated in tracheal smooth muscle during contraction. The functional consequences of this phosphorylation and the identification of the kinase that catalyze this reaction will be determined. The protein phosphatase that dephosphorylate myosin light chain and myosin light chain kinase physiologically will also be identified, and potential mechanisms of regulation elucidated. The research proposed in this application uses integrated experimental approaches from molecular biology, biochemistry, cell biology, physiology, and pharmacology to unravel this complex system to provide an understanding of regulatory mechanisms directly involving the contractile elements. This information will be crucial in understanding derangements of smooth muscle functions caused by disease processes such as hypertension and asthma.