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

叶绿体ATP合酶的几个方面将被研究。这些包括核苷酸与酶的催化部分（CF1）的相互作用以及CF1的y和e亚基以及CF0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CF1结合的核苷酸与中等核苷酸交换的动力学。可以确定促进交换的位点的性质和交换的动态能力。核苷酸（2',3'-三硝基苯基- adp或-ATP）与CF1结合后将采用停流荧光。已经开发出一种简单的程序，可以有效地耗尽CF1几乎所有的结合核苷酸。将检查核苷酸重新装载到耗尽酶的位点上。差示扫描量热法将用于测定核苷酸耗尽酶的稳定性。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的方法将被用来检查CF1的y和e亚基的功能。与e相互作用的y亚基的识别区域，以及那些需要快速ATP水解的区域，将继续进行。与Mark L. Richter博士合作的诱变实验也将进行。利用分子生物学方法和重组，将确定截断和/或突变形式的e亚基在与缺乏e的CF1重组时抑制atp酶活性和阻断质子传导的能力。CF0亚基III与CF1之间的相互作用将通过生化手段进行研究。将测试CF1和e-贫CF1与含有亚基III的囊泡的结合，并进行CF1-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，来自太阳的光能以还原有机分子的形式转化为化学能，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。这个建议关注的是产生ATP的酶，ATP合酶。ATP合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ATP合酶与ATP和其他类似分子的相互作用以及酶的各种成分如何相互作用的研究。这些实验将提供有关ATP合酶如何工作及其活性如何被调节的宝贵信息。动物细胞中的大部分ATP是在线粒体中通过ATP合成酶合成的，类似于叶绿体ATP合成。叶绿体和线粒体酶的活性调节模式不同，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. ***