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: 3073902
• 负责人: LEONARD A STEIN
• 金额: $ 5.44万
• 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/3073902
• 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.

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形式存在， 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 从肌球蛋白丝延伸出来的突起， 与肌动蛋白丝结合。 净滑动或收缩通过将 ATP水解导致肌动蛋白-肌球蛋白复合物的构象变化。 为了了解肌动蛋白-肌球蛋白的相互作用， 从分子的角度来看，生物化学家已经转向肌动蛋白的研究， 激活肌球蛋白亚片段ATP酶活性，这被认为是 与收缩过程相关。 拟议研究的主要目标是改善 了解ATP水解反应的动力学， 心肌和骨骼肌肌球蛋白。 在拟议的研究期间 只有肌球蛋白的可溶性亚片段，亚片段-1（S-1）和重链 将研究Meromyosin（HMM）。 这项工作将改善 了解完整的肌球蛋白分子如何在体内发挥作用， 可能会让我们更深入地了解肌球蛋白为什么有两个头。 肌动蛋白激活肌球蛋白详细动力学方案的一个优点 ATP酶的重要性在于，它可能会导致更好地了解病理性 并且改变的生理状态影响肌肉收缩的能力。 几个实验室已经报道，心肌异常可以 与ATP酶活性的改变有关。 看到的变化很小 建立因果关系还需要做更多的工作。 稳态前动力学对这些变化应更敏感。 为了形成像目前一样完整的动力学描述， 可能的话，有必要执行几个稳态和预稳态 国家测量。 这些状态包括：稳态和预稳态 肌动蛋白与可溶性肌球蛋白亚片段结合的测量 在ATP水解过程中，使用停流和空气离心技术; 和使用骤冷流的初始磷酸盐爆发的幅度，并停止 流动技术; ATP酶活性的稳态测量 直接Pi测定或PH stat技术;和稳态018交换 使用光谱分析进行测量。