CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

Catch--平滑肌的机械化学和调节

• 批准号: 2082215
• 负责人: MARION Joyce SIEGMAN
• 金额: $ 23.86万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2082215
• 关键词: Bivalvia; actins; alternatives to animals in research; chemical kinetics; cyclic AMP; enzyme activity; flash photolysis; high performance liquid chromatography; mechanical stress; muscle contraction; muscle relaxation; muscle tone; myosins; nucleotides; phosphates; phosphoproteins; phosphorylation; radiotracer; smooth muscle

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.

在疾病状态下，如高血压，原发性缺陷可以追溯到 以调节维持音调平稳的机制 肌肉. 控制音调的基本机制还不清楚。 所有平滑肌的一个独特而重要的特征是 改变部队生产和维持的能源成本的能力 在等长收缩的时间过程中。 很明显 在机械和能量之间有许多相似之处， 在“闭锁”状态期间哺乳动物平滑肌的特性， 软体动物的肌肉在“捕捉”，但这些国家的分子基础， 并且它们的调节也不太清楚。 catch的一个特点 肌肉是一种机制的运作，似乎控制着 天桥的脱落率。 这样的机制还没有 在哺乳动物肌肉中发现，但作为一种 调节音调和生理功能的手段，如 就像血压一样。 因此，对捕获量中这种机制的阐明， 提供关于正常和异常的重要见解的潜力 心血管、肺、消化和生殖功能 系统. 本提案的总体目标是确定 肌球蛋白桥及其在静息和运动时的转换动力学 并捕捉前足丝牵开肌（ABRM）的收缩， 贻贝，并确定蛋白质的磷酸化变化， 状态是调节的过渡的分子基础， 过桥进入和离开捕获状态。 具体目标是 确定：（1）在透化ABRM中，核苷酸和/或无机 在不同的机械条件下与肌球蛋白结合的磷酸盐， 包括静止状态（横桥分离或弱结合到 肌动蛋白），相位收缩（横桥循环和维持力） 以及捕获状态（可能连接并维持的横桥 力，但循环非常缓慢）;（2）核苷酸和/或 在不同机械条件下肌球蛋白磷酸周转 单周转技术，其中放射性标记的核苷酸被 通过笼状化合物的光解产生，从而获得定量的 关于这些条件下肌球蛋白ATP酶循环速率的信息 不同的机械条件。 我们还将确定 由cAMP引发的捕获的快速松弛与快速的 ATP水解产物与肌球蛋白结合， 直接解决肌球蛋白是否改变其状态的问题， 从捕捉到休息的过渡。 (3)在释放渔获物期间， 蛋白质显示磷酸化水平随时间的变化， 对应于肌球蛋白状态的变化和/或以下的松弛 由笼状cAMP的光解形成cAMP。 这些实验将 鉴定作为以下分子调节剂的候选物的蛋白质 接住。