Nucleotide Binding and Functions of the Subunits of the Chloroplast ATP Synthase

将研究叶绿体ATP合酶的几个方面。 这些包括核苷酸与酶（CF 1）的催化部分的相互作用以及CF 1的y和e亚基和CF 0的III亚基的功能。 将在广泛的ATP浓度范围内以及在周转条件下测量与CF 1结合的核苷酸与中等核苷酸的交换动力学。 可以确定促进交换的位点的性质和交换的动力学能力。 核苷酸（2 '，3'-三硝基苯基-ADP或-ATP）与CF 1结合后将出现停流荧光。 已经开发了一种简单的程序，可以有效地耗尽几乎所有结合核苷酸的CF 1。 将检查将核苷酸重新加载到耗尽的酶上的位点。 差示扫描量热法将用于确定核苷酸耗尽的酶的稳定性。 等温滴定量热法将用于研究核苷酸结合的热力学。 结合生化分子生物学方法将被用来检查CF 1的y和e亚基的功能。 y亚基与e相互作用的识别区域，以及快速ATP水解所需的识别区域，将继续研究。 诱变实验，与马克L。里希特，也将进行。 使用分子生物学方法和重构，将确定e亚基的截短和/或突变形式在用缺乏e的CF 1重构时抑制ATP酶活性和阻断质子传导的能力。 将通过生物化学手段研究CF 0和CF 1的亚基III之间的相互作用。 将测试CF 1和e-耗尽的CF 1与含有亚基III的囊泡的结合，并进行CF 1-亚基III制剂的化学交联。 绿色植物和藻类的光合作用提供氧气，是几乎所有生物的最终能量来源。 在光合作用过程中，来自太阳的光能转化为化学物质， 能量以还原的有机分子的形式存在，其中许多是食物。 三磷酸腺苷（ATP）的合成是光合作用的重要组成部分，部分光能用于为ATP合成提供动力。 这项提案的重点是酶，使ATP，ATP合酶。 ATP合成酶是光合作用细胞器（叶绿体）的绿色能量转换膜的一部分。 提出了几种不同的研究途径，包括研究ATP合酶与ATP和其他类似分子的相互作用以及酶的各种组分如何相互作用。 这些实验将提供关于ATP合酶如何工作以及其活性如何调节的有价值的信息。 动物细胞中的大多数ATP是在线粒体中通过类似于叶绿体ATP合成的ATP合酶产生的。 叶绿体和线粒体酶的活性调节方式不同，但可能具有相似的机制。 ***

McCarty 9405713 Several aspects of the chloroplast ATP synthase will be studied. These include the interactions of nucleotides with the catalytic portion of the enzyme (CF1) and the functions of the y and e subunits of CF1 and subunit III of CF0. The kinetics of the exchange of nucleotide bound to CF1 with medium nucleotide will be measured over a wide range of ATP concentrations, as well as under turnover conditions. The nature of the sites that promote exchange and the kinetic competence of exchange can be established. The binding of nucleotide (2',3'-trinitrophenyl-ADP or -ATP) to CF1 will be followed by stopped-flow fluorescence. A simple procedure has been developed that effectively depletes CF1 of nearly all of its bound nucleotide. The reloading of nucleotides to sites on the depleted enzyme will be examined. Differential scanning calorimetry will used to determine the stability of the nucleotide- depleted enzyme. Isothermal titration calorimetry will be used to study the thermodynamics of nucleotide binding. Combined biochemical molecular biological approaches will be utilized to examine functions of the y and e subunits of CF1. The identification regions of the y subunit that interact with e, as well as those needed for rapid ATP hydrolysis, will be pursued. Mutagenesis experiments, in collaboration with Dr. Mark L. Richter, will also be carried out. Using molecular biological methods and reconstitution, the ability of truncated and/or mutated forms of the e subunit to inhibit ATPase activity and to block proton conductance when reconstituted with CF1 lacking e will be determined. Interactions between subunit III of CF0 and CF1 will be studied by biochemical means. The binding of CF1 and e-depleted CF1 to vesicles that contain subunit III will be tested and chemical cross-linking of CF1-subunit III preparations carried out. %%% Photosynthesis by green plants and algae provides oxygen and is the ultimate source of energy for almost all orga nisms. During photosynthesis, light energy from the sun is converted to chemical energy in the form of reduced organic molecules, many of which are foodstuffs. The synthesis of adenosine triphosphate (ATP) is an essential part of photosynthesis and part of the energy of light is used to power ATP synthesis. This proposal focuses on the enzyme that makes ATP, ATP synthase. The ATP synthase is a part of green, energy-converting membranes of the photosynthetic organelle known as the chloroplast. Several different avenues of research are proposed, including studies on the interaction of the ATP synthase with ATP and other similar molecules and how various components of the enzyme interact. These experiments will give valuable information about how the ATP synthase works and how its activity is regulated. Most of the ATP in animal cells is made in mitochondria by an ATP synthase similar to the chloroplast ATP synthesis. The chloroplast and mitochondrial enzymes differ in their modes of regulation of activity, but probably have a similar mechanism. ***