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.

主要目的是定义当心脏受到全局缺血并在缺乏或存在预处理的情况下进行全局缺血和再灌注时，线粒体ATP合酶所经历的那些变化。线粒体ATP合酶与用于磷酸盐（PIC）的载体（PIC）和腺嘌呤核苷酸（ANC）是正常心脏功能期间的核心参与者，并且必须密切同步工作，以确保在动物或人类生命的每一秒内产生足够的ATP，以满足能量需求。哺乳动物ATP合酶，也称为“ F0F1”，是一个由15种亚基类型和30多个总亚基组成的复杂。这些包含两个电动机，一个电动机是F0中包含的电化学驱动电动机，迫使ATP水解驱动电动机（F1）反向工作以使ATP起作用。除了这些工作部件外，还有两种小蛋白质，根据条件，与ATP合酶结合。一个名为“ IF1”是ATP水解的抑制剂，另一个命名为“因子B”。 ATP合成。当质子的电化学梯度无法驱动ATP合成时，IF1与ATP合酶结合并阻止ATP水解，而当梯度为梯度时，B会结合以促进ATP合成。为了满足上述项目的主要目标，将采用控制兔子的心脏，即经历缺血/再灌注的兔子心脏，以及受到预处理/缺血/再灌注的兔子心脏。具体目标是： 1。在每种情况下评估ATP合酶的相对能力在孤立的线粒体，内膜囊泡以及重构的脂质体系统中起作用，并在有限的蛋白质分析之前和之后，在每种情况下都确定纯化合成酶的亚基带模式。 2。在每种情况下量化ATP合酶的两个主要调节剂的分离线粒体中的相对量，即IF1和因子B，并将其相对的结合程度与同一线粒体中合成酶的能力相关，以催化ATP ATP合成和ATP水解。 3。另外，在每种情况下，ATP合酶与其近邻居，磷酸盐载体（PIC）和腺嘌呤核苷酸载体（ANC）形成ATP合酶/PIC/ANC复合物或最近已从Mitochonondria纯化并表征的ATP合酶/PIC/ANC复合体或“ ATP Synthasosome”的程度。