BIOCHEMICAL KINETICS OF CARDIAC AND SKELETAL MYOSIN

心脏和骨骼肌球蛋白的生化动力学

• 批准号: 3073904
• 负责人: LEONARD A STEIN
• 金额: $ 5.32万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3073904
• 关键词: adenosine triphosphate; adenosinetriphosphatase; chemical reaction; muscle contraction; myocardium; nonradiation isotope effect; radiotracer; stop flow technique; striated muscles

The generally accepted mechanism of muscle contraction, the sliding filament theory, involves the interaction of two proteins: actin and myosin. These proteins, which exist in vivo as filaments, are arranged in a highly ordered geometrical pattern in which the myosin and actin filaments interdigitate. According to the sliding filament theory projections, which extend from the myosin filament, cyclically interact with the actin filament. Net sliding or contraction occurs by coupling the hydrolysis of ATP to conformational changes in the acto-myosin complexes. In order to gain an understanding of the actin-myosin interaction from a molecular point of view, biochemists have turned to the study of the actin activated myosin subfragment ATPase activity which is believed to be the in vitro correlate to the contraction process. The primary objective of the proposed research is to improve the understanding of the kinetics of the ATP hydrolysis reaction of both cardiac and skeletal muscle myosin. During the proposed research period only the soluble subfragments of myosin, Subfragment-1 (S-1) and Heavy Meromyosin (HMM) will be studied. This work should improve the understanding of how the complete myosin molecule functions in vivo, and may lead to insight into why myosin has two heads. One advantage of a detailed kinetic scheme of the actin activated myosin ATPase is that it may lead to a better understanding of how pathological and altered physiological states affect the ability of muscle to contract. Several laboratories have reported that abnormalities of cardiac muscle can be associated with altered ATPase activities. The changes seen are small and more work is needed to establish their cause and effect relationship. Presteady state kinetics should be more sensitive to these changes. In order to form as complete a kinetic description as is currently possible, it is necessary to perform several steady state and presteady state measurements. These include: steady state and presteady state measurements of the binding of Actin to the soluble myosin subfragments during ATP hydrolysis using stopped flow and airfuge techniques; the rate and magnitude of the initial phosphate burst using quench flow and stopped flow techniques; steady state measurements of the ATPase activity using direct Pi assays or PH stat techniques; and steady state 018 exchange measurements using spectrometric analysis.

被普遍接受的肌肉收缩机制，滑动 细丝理论，涉及两种蛋白质的相互作用：肌动蛋白和 肌球蛋白。这些蛋白质在体内以细丝的形式存在，排列在 一种高度有序的几何图案，其中肌球蛋白和肌动蛋白 花丝交错。根据滑丝理论 从肌球蛋白细丝延伸出来的突起周期性地相互作用 肌动蛋白细丝。净滑动或收缩是通过将 ATP的水解对肌球蛋白复合体的构象变化。 为了了解肌动蛋白-肌球蛋白之间的相互作用 从分子的角度来看，生物化学家已经转向对肌动蛋白的研究 激活的肌球蛋白亚片段ATPase活性被认为是 体外实验与收缩过程相关。 拟议研究的主要目标是改善 对两者三磷酸腺苷水解反应动力学的认识 心肌和骨骼肌肌球蛋白。在建议的研究期内 只有肌球蛋白的可溶性亚片段、亚片段-1(S-1)和重 将研究肌球蛋白(HMM)。这项工作应该会改善 了解完整的肌球蛋白分子如何在体内发挥作用，以及 可能导致对肌球蛋白为什么有两个头的洞察。 肌动蛋白激活肌球蛋白详细动力学方案的一个优点 ATPase可能会让我们更好地理解它是如何病理的 生理状态的改变会影响肌肉的收缩能力。 一些实验室已经报告说，心肌的异常可以 与ATPase活性改变有关。我们看到的变化很小 要建立它们之间的因果关系，还需要做更多的工作。 稳态前动力学应该对这些变化更加敏感。 为了形成与当前一样完整的动力学描述 有可能，需要执行几个稳态和稳态前 州政府的测量。这包括：稳定状态和稳定前状态 肌动蛋白与可溶性肌球蛋白亚段结合的测定 在使用停流和充气技术的ATP水解过程中；速率 以及使用激冷流和停止的初始磷酸盐爆炸的大小 流动技术.使用的ATPase活性的稳态测量 直接PI分析或PH值统计技术；和稳定状态018交换 使用光谱分析进行测量。