The aim of this research project is to understand the biochemical events governing metabolite transport in mammalian cells. We plan to describe the molecular properties of plasma membrane components which are required for transport and also to determine how the activity of these components is regulated in response to the metabolic needs of the cell. Our approach is to study the mechanism of purine base and nucleoside transport in Chinese hamster lung fibroblasts growing in tissue culture. Both wild-type cells and mutant clones which are resistant to 8-azaguanine (8-AG) and 5-bromo-2-deoxyuridine (BUdR) will be used in these experiments. Clones whose drug resistance is due to loss of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) will be employed to examine parameters of hypoxanthine exodus. Resistant mutants with normal phosphorylation and phosphoribosylation will be examined for defects in base and nucleoside transport. The interaction of bases and nucleosides for transport sites on the membrane will be studied in 8-AGr, BUdRr double mutants lacking both HGPRT and thymidine kinase. A rapid sampling method for transport assays using fibroblast monolayers will allow accurate determination of kinetic parameters. In addition, we plan to develop a continuous assay method for hypoxanthine transport in erythrocyte ghosts and phospholipid vesicles. This investigation depends on biochemically and genetically defined systems which should allow us to identify and study the membrane proteins that regulate purine utilization in mammalian cells. We believe that this will be a significant step towards understanding disorders which alter purine pools such as Lesch-Nyhan syndrome, gouty arthritis, and inherited immunodeficiency diseases.
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