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: 3344535
• 负责人: LEONARD A STEIN
• 金额: $ 4.6万
• 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: 1985-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3344535
• 关键词: adenosine triphosphate; adenosinetriphosphatase; 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 18O exchange measurements using spectrometric analysis.

普遍接受的肌肉收缩机制是滑动 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，排列在 高度有序的几何图案，其中肌球蛋白和肌动蛋白 细丝相互交叉。 根据滑动丝理论 从肌球蛋白丝延伸出来的突出物周期性地相互作用 与肌动蛋白丝。 通过耦合发生净滑动或收缩 ATP 水解导致肌动球蛋白复合物构象变化。 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 激活的肌球蛋白亚片段 ATP 酶活性被认为是 体外与收缩过程相关。 拟议研究的主要目标是改善 了解两者的 ATP 水解反应动力学 心肌和骨骼肌肌球蛋白。 在拟议的研究期间 仅肌球蛋白的可溶性亚片段、亚片段 1 (S-1) 和重片段 将研究 Meromyosin (HMM)。 这项工作应该改进 了解完整的肌球蛋白分子如何在体内发挥作用，以及 可能会导致深入了解为什么肌球蛋白有两个头。 肌动蛋白激活肌球蛋白的详细动力学方案的一个优点 ATPase 的优点是它可能会导致人们更好地理解病理学是如何发生的。 生理状态的改变会影响肌肉收缩的能力。 一些实验室报告说，心肌异常可以 与 ATP 酶活性改变有关。 看到的变化很小 还需要做更多的工作来确定它们的因果关系。 前稳态动力学应该对这些变化更加敏感。 为了形成目前为止完整的动力学描述 可能的话，需要进行多次稳态和预稳态 状态测量。 这些包括：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，使用停流和避气技术；率 以及使用骤冷流和停止的初始磷酸盐爆发的强度 流动技术；使用 ATP 酶活性的稳态测量 直接 Pi 测定或 PH 统计技术；和稳态 18O 交换 使用光谱分析进行测量。