CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

在疾病状态下，例如高血压，可以追踪到原始缺陷。 对流畅的音调维持机制的调节 肌肉。对S控制声调的基本机制还不甚了解。 所有平滑肌肉的一个独特而重要的特征是 改变部队生产和维护的能源成本的能力 在等长收缩的时间过程中。这已经变得显而易见了 机械和能量之间有许多相似之处 哺乳动物肌肉在“闩锁”状态下的特性 软体动物肌肉在“抓”的过程中，这些状态的分子基础 他们的监管也没有得到很好的理解。Catch的一个独特功能 肌肉是一种机制的运作，它似乎控制着 立交桥的脱落率。这样的机制还没有被 在哺乳动物肌肉中发现，但作为一种 调节音调的手段，从而调节生理功能，如 和血压一样。因此，在《捕获物》中对这种机制的解释 有可能提供对正常和异常的重要见解 心血管、肺、消化和生殖功能 系统。这项提案的总体目标是定义 肌球蛋白交叉桥及其在静息和时相的周转动力学 和捕捉收缩的前股后伸肌(ABRM) 并鉴定其磷酸化变化的蛋白质。 状态是调节细胞周期变化的分子基础 跨桥进入和离开接球状态。具体目标是 测定：(1)在渗透性ABRM中，核苷酸和/或无机 磷酸盐在不同的机械条件下与肌球蛋白结合， 包括休眠状态(交叉桥分离或弱结合到 肌动蛋白)，阶段性收缩(跨桥循环和维持力) 和抓钩状态(可能连接和维护的横桥 力，但循环非常慢)；(2)核苷酸和/或 不同力学条件下肌球蛋白磷酸转化率的研究 单周转技术，其中放射性标记的核苷酸是 通过笼状化合物的光解产生，从而获得定量 关于在这些情况下肌球蛋白ATPase周期的速率的信息 非常不同的机械条件。我们还将确定是否 由cAMP发起的快速放松捕获物与快速 与肌球蛋白结合的ATP水解物的交换，以便 直接解决肌球蛋白是否改变其在 从捕获物过渡到休息。(三)在投放渔获物时， 蛋白质在一段时间内表现出磷酸化水平的变化 与肌球蛋白和/或松弛状态的变化相对应 笼中cAMP的光解形成cAMP。这些实验将 确定可能的分子调控蛋白(S) 接住。

In disease states, such as hypertension, the primary defect may be traced to the regulation of the mechanisms for maintenance of tone in smooth muscle. The basic mechanism(s) controlling tone are not well understood. A unique and important characteristic of all smooth muscles is the ability to vary the energy cost of force production and maintenance during the time course of isometric contractions. It has become obvious that there are many similarities among the mechanical and energetic properties of mammalian smooth muscles during the "latch" state and molluscan muscles during "catch," yet the molecular basis of these states and their regulation is not well understood. A unique feature of catch muscle is the operation of a mechanism that appears to control the detachment rate of the crossbridge. Such a mechanism has not yet been identified in mammalian muscle, but would be of great importance as a means of regulating tone and consequently physiological functions, such as blood pressure. The elucidation of such a mechanism in catch thus has the potential to provide important insights into normal and abnormal function of the cardiovascular, pulmonary, digestive, and reproductive systems. The overall goal of this proposal is to define the state of the myosin crossbridge and its kinetics of turnover at rest and during phasic and catch contractions in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, and to identify proteins whose change in phosphorylation state is the molecular basis for the regulation of the transition of the crossbridge into and out of the catch state. The Specific Aims are to determine: (1) in permeabilized ABRM, the nucleotide and/or inorganic phosphate bound to myosin under different mechanical conditions, including the resting state (crossbridges detached or weakly bound to actin), phasic contraction (crossbridges cycling and maintaining force) and the catch state (crossbridges likely to be attached and maintaining force, but cycling very slowly); (2) the rate of nucleotide and/or phosphate turnover on myosin under different mechanical conditions using single turnover techniques in which radiolabeled nucleotides are generated by photolysis of caged compounds, and thereby gain quantitative information concerning the rate of the myosin ATPase cycle under these very different mechanical conditions. We will also determine whether the rapid relaxation from catch initiated by cAMP is associated with a rapid exchange of the products of ATP hydrolysis bound to myosin, in order to directly address the question of whether myosin changes its state in the transition from catch to rest. (3) during the release of catch, which proteins show changes in levels of phosphorylation on a time course that corresponds to the change in state of myosin and/or relaxation following cAMP formation from photolysis of caged cAMP. These experiments will identify proteins which are candidates for the molecular regulator(s) of catch.