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.

主要目的是确定当心脏在有或无预处理的情况下经受全脑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合酶与磷酸盐载体（PIC）和腺嘌呤核苷酸（ANC）一起是正常心脏功能的核心参与者，必须密切同步工作，以确保在动物或人类生命的每一秒中都产生足够的ATP以满足能量需求。哺乳动物ATP合酶（ATP synthase），也称为“F0 F1”，是由15种亚基类型和超过30种总亚基组成的复合物。这些包括两个马达，一个是包含在F0内的电化学驱动马达，其迫使ATP水解驱动马达（F1）反向工作以产生ATP。除了这些工作部分外，还有两种小蛋白质根据条件与ATP合酶结合。其中一种称为“IF 1”，是ATP水解的抑制剂，另一种称为“因子B”， ATP合成。当质子的电化学梯度不可用于驱动ATP合成时，IF 1结合ATP合酶并阻断ATP水解，而当存在梯度时，因子B结合以促进ATP合成。为了满足上述项目的主要目的，将使用对照兔心脏、缺血/再灌注兔心脏和预处理/缺血/再灌注兔心脏。具体目标是： 1.在每种情况下，评估ATP合酶在分离的线粒体、内膜囊泡和重组脂质体系统中正向和反向工作的相对能力，并在每种情况下确定纯化合酶在有限蛋白水解之前和之后的亚基带型。 2.在每种情况下定量ATP合酶的两种主要调节剂在分离的线粒体中的相对量，即，IF 1和因子B，并将它们的相对结合程度与同一线粒体中的合酶催化ATP合成和ATP水解的能力相关联。 3.还确定在每种情况下，ATP合酶与其近邻磷酸盐载体（PIC）和腺嘌呤核苷酸载体（ANC）相互作用的程度，以形成ATP合酶/PIC/ANC复合物或最近从线粒体中纯化和表征的“ATP酶体”。