REGULATION OF MITOCHONDRIAL ATP SYNTHASE

主要目的是确定在没有或存在预处理的情况下，心脏遭受全局缺血和再灌注时线粒体ATP合酶所经历的变化。线粒体ATP合酶与磷酸载体（PIC）和腺嘌呤核苷酸（ANC）在正常心脏功能中起着核心作用，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来满足能量需求。哺乳动物ATP合酶，也被称为“F0F1”，是一个由15个亚基类型和30多个亚基组成的复合物。这包括两个马达，一个是包含在F0中的电化学驱动马达，它迫使ATP水解驱动马达（F1）反向工作以制造ATP。除了这些工作部分，还有两种小蛋白质根据条件与ATP合酶结合。一种名为“IF1”是ATP水解的抑制剂，另一种名为“因子B”是ATP水解所必需的

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.