BIOCHEMICAL MECHANISMS OF SMOOTH MUSCLE CONTRACTION

尽管人们普遍认为肌球蛋白轻链磷酸化 钙/钙调蛋白依赖的肌球蛋白轻链激酶是一种主要的 事件在引发平滑肌收缩时，有几个方面 这一过程在细胞和分子水平上还没有被很好地理解 级别。本申请中描述的研究项目针对的是 关于平滑肌调节的几个重要问题 血管和呼吸道平滑肌中的收缩成分。分子内 (钙调蛋白结合区-假底物抑制区-催化 结构域)和分子间(催化结构域-蛋白质底物结合和 催化)将检查与激活和催化相关的事件 并且与MLCK与肌动蛋白和/或肌球蛋白细丝的结合有关。一个 涉及生物化学和分子的实验方法的结合 将用生物学来分析平滑肌肌球蛋白的这些特性 轻链激酶。新信息表明肌球蛋白轻链 可以调节激酶和磷酸酶的活性，从而使 这些活性不是在所有条件下都简单地由[Ca~(2+)]i决定的 适用于紧张性和相位性平滑肌肉。对蛋白激酶和蛋白水解酶的调节 报告的钙离子变化与磷酸酶活性有关。 收缩元件的灵敏度。肌球蛋白轻链激酶是 收缩时气管平滑肌中的磷酸化。这个 这种磷酸化的功能后果和鉴定 催化这一反应的激酶将被确定。这种蛋白质 使肌球蛋白轻链和肌球蛋白轻链去磷酸化的磷酸酶 生理学上也将鉴定激酶，并探讨其可能的机制。 监管得到澄清。本申请中提出的研究使用 综合实验方法从分子生物学、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 提供对直接涉及 收缩元素。这些信息将对理解 疾病过程引起的平滑肌功能紊乱，如 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.