CATCH--MECHANOCHEMISTRY AND REGULATION IN SMOOTH MUSCLE

Catch--平滑肌的机械化学和调节

• 批准号: 2853462
• 负责人: MARION Joyce SIEGMAN
• 金额: $ 35.73万
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2853462
• 关键词: Bivalvia; actins; alternatives to animals in research; biomechanics; cyclic AMP; cytoskeletal proteins; enzyme activity; gel electrophoresis; high performance liquid chromatography; mechanical stress; muscle contraction; muscle function; muscle proteins; muscle tone; myosins; phosphorylation; protein kinase; protein structure function; radiotracer; smooth muscle; striated muscles; tissue /cell culture

A unique and important characteristic of all smooth muscles (mammalian and invertebrate) is the ability to vary the energy cost of force production and maintenance during the time course of isometric contractions. The basic mechanisms controlling force maintenance, or tone in smooth muscle are not well understood. Our discovery that the phosphorylation state of twitchin (a mini-titin) regulates catch and force production in the anterior byssus retractor muscle (ABRM) of Mytilus edulis, has opened the way to new studies described here on the molecular basis of its function. Our overall goal is to determine the mechanisms whereby twitchin controls actin-myosin interaction and resulting mechanical output. The ubiquitous presence of twitchin in invertebrate striated, smooth catch and phasic muscles suggests that twitchin may serve as a regulator in all of these muscle types. The understanding of the basic mechanisms underlying this regulation may very well provide new insights on the control of other muscle types, especially mammalian smooth muscle, which shows very similar mechanical characteristics as the smooth muscles from invertebrates. In the proposed studies the smooth ABRM of M. edulis, and striated adductor of the sea scallop (P. magellanicus) will serve as experimental models. Intact and permeabilized invertebrate muscles will be used, as needed. The Specific Aims are to (1) Determine the mechanism by which twitchin phosphorylation gives rise to an increase in the detachment rate constant of the myosin crossbridge; (2) Determine the relationship between the degree of phosphorylation of twitchin and its mechanical effect in intact and permeabilized ABRM; (3) Test the hypothesis that the intrinsic rate of actin movement by myosin is attenuated by the presence of twitchin, and that the phosphorylation of twitchin restores the intrinsic fast rate, using in vitro motility studies of actin movement on native thick filaments from ABRM and the body wall of C. elegans. Nucleotide turnover on the thick filaments will be measured in order to learn how twitchin phosphorylation alters the ATPase activity when calcium concentration is varied. (4) Determine of the effect of twitchin phosphorylation on fast striated scallop adductor muscle in order to learn whether this is a generalized mechanism for the modulation of crossbridge kinetics in invertebrate muscle. (5) Biochemical studies on twitchin will characterize the twitchin molecule and determine the mechanism by which its phosphorylation by protein kinase A controls the interaction of contractile proteins.

所有平滑肌肉（哺乳动物和无脊椎动物）的独特而重要的特征是在等距收缩期间改变力产生和维护的能源成本的能力。 控制力量维护或平滑肌中音调的基本机制尚不清楚。 我们的发现，Twitchin（一种迷你丁字素）的磷酸化状态调节了Mytilus Edulis的前缩回肌肉肌肉（ABR）的捕获和迫使产生，为此处介绍的有关其功能分子基础的新研究开辟了道路。 我们的总体目标是确定Twitchin控制肌动蛋白 - 肌球蛋白相互作用和产生的机械输出的机制。 无脊椎动物横纹，光滑的捕获和阶段肌肉中Twitchin无处不在的存在表明，Twitchin可以作为所有这些肌肉类型的调节剂。 对本法规的基本机制的理解可以很好地为控制其他肌肉类型的控制提供新的见解，尤其是哺乳动物平滑肌，哺乳动物平滑肌表现出与无脊椎动物的平滑肌肉非常相似的机械特征。在拟议的研究中，Edulis M. eDulis的平滑ABR和Sea Scallop（P. magellanicus）的横纹内收肌将用作实验模型。 根据需要，将使用完整的和透化的无脊椎动物肌肉。 具体目的是（1）确定Twitchin磷酸化导致肌球蛋白Crossbridge的脱离速率常数增加的机制； （2）确定Twitchin的磷酸化程度与其在完整和通透性ABR中的机械作用之间的关系； （3）检验以下假设：肌球蛋白的肌动蛋白运动的固有速率通过Twitchin的存在减弱，而Twitchin的磷酸化恢复了固有的快速速率，使用了肌动蛋白在ABR和ABR的天然厚细胞上的肌动蛋白运动的体外运动研究。 将测量厚细丝的核苷酸更新，以了解当钙浓度变化时Twitchin磷酸化如何改变ATPase活性。 （4）确定Twitchin磷酸化对快速扇形扇贝内收肌的影响，以了解这是否是对无脊椎动物肌肉中Crossbridge动力学调节的通用机制。 （5）关于Twitchin的生化研究将表征Twitchin分子，并确定其通过蛋白激酶A磷酸化控制收缩蛋白的相互作用的机制。