REGULATION OF MITOCHONDRIAL ATP SYNTHASE

线粒体 ATP 合成酶的调节

• 批准号: 7114082
• 负责人: PETER L PEDERSEN
• 金额: $ 39.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-10 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7114082
• 关键词: adenine nucleotides; adenosine triphosphate; adenosinetriphosphatase; enzyme activity; enzyme complex; heart function; hydrolysis; ischemic preconditioning; laboratory rabbit; liposomes; mitochondria; myocardial ischemia /hypoxia; phosphates; protein binding; protein protein interaction; protein purification; protein quantitation /detection; protein structure function; proteolysis; reperfusion; transport proteins; vesicle /vacuole

The primary objective is to define those changes experienced by the mitochondrial ATP synthase when the heart is subjected to global ischemia and reperfusion in the absence or presence of preconditioning. The mitochondrial ATP synthase together with carriers for phosphate (PIC) and adenine nucleotides (ANC) are central players during normal heart function and must work in close synchrony to assure that sufficient ATP is produced during every second of an animal or human life to meet the energy demands. The mammalian ATP synthase, also called "F0F1", is a complex consisting of 15 subunit types and over 30 total subunits. These comprise two motors, one an electrochemically driven motor contained within F0 that forces an ATP hydrolysis driven motor (F1) to work in reverse to make ATP. In addition to these working parts, there are two small proteins that bind to the ATP synthase depending on conditions. One named "IF1" is an inhibitor of ATP hydrolysis, and the other named "Factor B" is required for ATP synthesis. When an electrochemical gradient of protons is not available to drive ATP synthesis, IF1 binds to the ATP synthase and blocks ATP hydrolysis, whereas when the gradient is present Factor B binds to facilitate ATP synthesis. To meet the primary objective of the project as defined above, control rabbit hearts, those subjected to ischemia/reperfusion, and those subjected to preconditioning/ischemia/reperfusion will be employed. Specific Aims will be to: 1. Assess in each case the relative capacities of the ATP synthase to work in the forward and reverse directions in isolated mitochondria, inner membrane vesicles, and in a reconstituted liposomal system, and also determine in each case the subunit banding pattern of the purified synthase prior to and after limited proteolysis. 2. Quantify in each case the relative amounts in isolated mitochondria of the two major regulators of the ATP synthase, i.e., IF1 and Factor B, and correlate their relative extent of binding to the capacity of the synthase in the same mitochondria to catalyze ATP synthesis and ATP hydrolysis. 3. Determine also in each case the extent to which the ATP synthase interacts with its close neighbors, the phosphate carrier (PIC) and the adenine nucleotide carrier (ANC) to form an ATP synthase/PIC/ANC complex or "ATP synthasome" that has been purified and characterized recently from mitochondria.

主要目标是确定当心脏在不存在或存在预处理的情况下遭受整体缺血和再灌注时线粒体 ATP 合酶所经历的变化。线粒体 ATP 合酶以及磷酸盐 (PIC) 和腺嘌呤核苷酸 (ANC) 载体是正常心脏功能的核心参与者，必须密切同步工作，以确保动物或人类生命中的每一秒都产生足够的 ATP，以满足心脏功能的需要。能源需求。哺乳动物 ATP 合酶，也称为“F0F1”，是一个由 15 个亚基类型和 30 多个亚基组成的复合体。它们包括两个电机，其中一个包含在 F0 内的电化学驱动电机，迫使 ATP 水解驱动电机 (F1) 反向工作以产生 ATP。除了这些工作部分之外，还有两种小蛋白质根据条件与 ATP 合酶结合。一种称为“IF1”，是 ATP 水解的抑制剂，另一种称为“因子 B”，是 ATP 水解所必需的。 ATP 合成。当质子电化学梯度无法驱动 ATP 合成时，IF1 会与 ATP 合酶结合并阻止 ATP 水解，而当存在梯度时，因子 B 会结合以促进 ATP 合成。为了实现上述项目的主要目标，将使用对照兔心脏、那些遭受缺血/再灌注的心脏以及那些遭受预处理/缺血/再灌注的心脏。具体目标是： 1. 评估每种情况下 ATP 合酶在分离的线粒体、内膜囊泡和重建的脂质体系统中正向和反向工作的相对能力，并确定每种情况下纯化合酶的亚基带模式在有限的蛋白水解之前和之后。 2. 在每种情况下量化 ATP 合酶的两个主要调节因子（即 IF1 和因子 B）在分离线粒体中的相对量，并将它们的相对结合程度与同一线粒体中合酶催化 ATP 合成的能力相关联和ATP水解。 3. 在每种情况下还确定 ATP 合酶与其近邻磷酸盐载体 (PIC) 和腺嘌呤核苷酸载体 (ANC) 相互作用以形成 ATP 合酶/PIC/ANC 复合物或“ATP 合体”的程度，最近从线粒体中纯化并表征。