REGULATION OF MITOCHONDRIAL ATP SYNTHASE

主要目的是确定在没有或存在预适应的情况下，当心脏受到全脑缺血和再灌流时，线粒体ATP合成酶所经历的变化。线粒体ATP合成酶与磷酸载体(PIC)和腺嘌呤核苷酸(ANC)一起是正常心脏功能的核心角色，必须密切同步地工作，以确保在动物或人类生命的每一秒都产生足够的ATP来满足能量需求。哺乳动物的三磷酸腺苷合成酶，又称F0F1，是一个由15种亚基类型和30多个亚基组成的复合体。它们包括两个马达，一个是包含在F0中的一个电化学驱动的马达，它迫使一个由ATP水解酶驱动的马达(F1)反向工作来制造ATP。除了这些工作部分外，根据情况，还有两种小蛋白与ATP合成酶结合。其中一种名为“IF1”，是一种ATP水解酶的抑制剂，另一种名为“B因子”，是 三磷酸腺苷合成。当没有质子的电化学梯度来驱动ATP合成时，IF1与ATP合成酶结合并阻止ATP水解酶，而当存在梯度时，因子B结合以促进ATP合成。为了达到上述项目的主要目标，将采用对照兔心脏、缺血/再灌流和预适应/缺血/再灌流的心脏。具体目标将是： 1.在每种情况下，评估ATP合成酶在分离的线粒体、内膜小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的合酶在有限蛋白分解前后的亚基带型。 2.在每种情况下，量化ATP合成酶的两个主要调节因子IF1和B因子在分离的线粒体中的相对数量，并将它们的相对结合程度与同一线粒体中的合成酶催化ATP合成和ATP水解的能力相关联。 3.还要确定在每种情况下，ATP合成酶与其近邻、磷酸盐载体(PIC)和腺核苷酸载体(ANC)相互作用的程度，以形成最近从线粒体中纯化和鉴定的ATP合成酶/PIC/ANC复合体或“ATP合成体”。

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.