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

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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. ***