The broad objectives of this research project are to study the metabolism of lysine in the rat brain, to examine the regional and subcellular distribution of lysine metabolizing enzymes in the brain, and to synthesize the non-commercially available lysine metabolites for research use. Attempts will be made to elucidate the reaction mechanism of each metabolic step for lysine in the brain, and to identify the metabolic intermediates involved in each enzymatic reaction. Isotope-labeling, flourometric and spectrophotometric techniques will be used for these studies. A study is also proposed to test the hypothesis that piperidine is formed from L-lysine via delta1-piperidine by """"""""abortive transamination."""""""" Experiments are also proposed to examine the possibility of enzyme-bound intermediates in the metabolism of L-lysine to L-pipecolate. Properties of the enzymes involved in the metabolism of L-lysine in the brain will be studied with the design of new and sensitive assay methods. Affinity chromatography and affinity precipitation will be used to purify lysine oxidase, delta1-piperideine-2-carboxylate reductase and pipecolate oxidase. The metabolizing enzymes will be studied in the subcellular cortical fractions. Examination of the possible association of these enzymes with the nerve endings and other subcellular organelles will be performed with the technique of density gradient centrifugation. Synthesis of lysine metabolites, both labeled and unlabeled, will utilize microbial, enzymatic as well as organic synthesis techniques. It is hoped that the knowledge obtained in this work will help understand the etiology of human genetic disorders related to brain lysine metabolism, and the role of lysine and its metabolites play in sedation, sleep, convulsion and other neuronal function related to neurological disorders.
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