CATCH: Mechanochemistry and Regulation in Smooth muscle

CATCH：平滑肌的机械化学和调节

• 批准号: 6820934
• 负责人: THOMAS M BUTLER
• 金额: $ 56.49万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820934
• 关键词: Bivalvia; actins; adenosine diphosphate; alternatives to animals in research; animal tissue; binding sites; biomechanics; chemical binding; cyclic AMP; cytoskeletal proteins; enzyme activity; fluorescent dye /probe; mechanical stress; muscle contraction; muscle function; muscle proteins; muscle tone; myosins; phosphorylation; protein kinase; protein structure function; recombinant proteins; smooth muscle; striated muscles; tissue /cell culture

DESCRIPTION (provided by applicant): The goal of this research is to understand the mechanisms that regulate tonic force maintenance with a very low energy usage and slow crossbridge cycling rate in smooth muscle. These are characteristics of a "catch" muscle, the anterior byssus retractor (ABRM) muscle of the mussel Mytilus edulis, the prototype of the "latch" state of vertebrate smooth muscle. We showed that the phosphorylation state of the mini-titin, twitchin, located on the thick filament, is a major site of regulation of force production and maintenance. Catch force is associated with the dephosphorylation of twitchin, and release of catch, or relaxation, is associated with its protein kinase A mediated phosphorylation. We have also characterized distinct mechanical states and their regulation by twitchin phosphorylation, and determined the sequence of Mytilus twitchin. The ABRM is an ideal model because of the ease (a) by which distinct mechanical states (calcium-driven cycling, non-cycling or catch crossbridges, relaxation) can be produced in intact and permeabilized muscles, and (b) of controlling the regulatory mechanisms, i.e., the state of twitchin phosphorylation and calcium. Conserved portions of proteins such as titin, projectin, C-protein and caldesmon, implicated in the regulatory processes of contraction are similar to portions of twitchin, suggesting its role in regulatory processes in general. The SPECIFIC AIMS are: (1) Determination of the role of twitchin phosphorylation-mediated changes in ADP binding to and release from myosin in controlling the mechanical states of catch muscle. (2) Determination of the mechanism by which twitchin regulates catch through measurement of the effects of recombinant portions of the molecule on mechanical parameters in permeabilized muscle.(3) Determination of the thick and thin filament binding characteristics of twitchin and recombinant portions of twitchin. (4) Determination of the extent of involvement of myosin crossbridges in catch force maintenance by measurements of changes in orientation of fluorescent probes attached to the regulatory light chain.

描述（由申请人提供）：这项研究的目的是了解使用非常低的能量使用和缓慢的跨桥循环速率在平滑肌中进行调节的机制。 这些是“捕捉”肌肉的特征，即贻贝Mytilus Edulis的前脊椎动物（ABR）肌肉，这是脊椎动物平滑肌的“闩锁”状态的原型。 我们表明，位于厚细丝上的微调丁酸Twitchin的磷酸化状态是对力生产和维持的主要位置。 捕获力与Twitchin的去磷酸化有关，捕获或松弛的释放与其蛋白激酶A介导的磷酸化有关。 我们还通过Twitchin磷酸化表征了不同的机械状态及其调节，并确定了Mytilus Twitchin的序列。 ABR是一个理想的模型，因为（a）可以以完整和透化的肌肉（钙驱动的循环，非循环或捕获杂交桥）的不同机械状态（钙驱动的循环，非循环或捕获杂交桥）产生，并且（b）控制调节机制，即。 与收缩调节过程有关的蛋白质，Projectin，C蛋白和Caldesmon等蛋白质的保守部分类似于Twitchin的部分，这表明其在调节过程中的作用一般。 具体目的是：（1）测定Twitchin磷酸化介导的ADP结合和从肌球蛋白中释放在控制捕获肌肉机械状态的ADP结合并释放的作用。 （2）测定Twitchin通过测量分子重组部分对通透性肌肉中机械参数的影响来调节捕获的机制。（3）测定Twitchin的Twitchin和重组部分的厚和薄丝结合特性。 （4）确定肌球蛋白杂交桥在捕获力维持中的参与程度，通过测量在调节轻链上附着的荧光探针方向的变化。