PROLINE METABOLISM/REGULATION OF PURINES/ENERGY TRANSFER

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We propose to examine the possible role of the proline metabolizing system in regulating energy-generating and biosynthetic pathways and in mediating intercompartmental energy transfer. Based on studies with erythrocytes and hepatocytes, it appears that pyrroline-5-carboxylate reductase (PCR) activity may modulate pentose phosphate pathway activity and result in the stimulation of de novo purine nucleotide biosynthesis. In addition PCR and proline oxidase (ProOx) may be involved in transfer of cytoplasmic redox potential into energy- utilizing organelles. Because of the uncommonly high levels of PCR and purine biosynthetic activity in nodules, the presence of ProOx in bacteroids (energy-consuming compartments lacking glycolytic capability), and the extremely high flux of energy transfer from the cytoplasm into bacteroids, nodules of Glycine max are an excellent model system to investigate the proposed roles of PCR and ProOx in intercompartmental energy transfer and purine nucleotide biosynthesis. The experimental plan is designed to examine the two different, but not mutually exclusive, functions of PCR and ProOx (i.e. regulation of purine nucleotide biosynthesis and energy transfer between organelles) proposed above. The specific objectives are: 1) to localize and characterize activities of PCR, ProOx, and other enzymes of proline metabolism; 2) to determine the relationship between PCR activity and purine nucleotide synthesis; 3) to define the role of proline in intercompartmental transfer of redox potential; and 4) to demonstrate the physiological significance of PCR and ProOx in intact tissues. The experimental approach involves biochemical characterization of PCR and ProOx, subcellular fractionation and tracer studies. The results obtained will contribute to our understanding of general mechanisms of cellular and subcellular energy transfer and metabolic regulation in eucaryotes.