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 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 transport in Chinese hamster lung fibroblasts growing in tissue culture. Both wild type cells and mutant clones which are resistant to 8-azaguanine or 2,6-diaminopurine will be used in these experiments. Clones whose resistance to these agents is due to loss of hypoxanthine-guanine or adenine phosphoribosyltransferase will be used to study hypoxanthine and adenine transport in the absence of subsequent metabolism. Resistant mutants with normal levels of phosphoribosyltransferases may contain transport defects that can be used for identification of membrane components involved in purine transport. We plan also to develop methods for isolation of plasma membrane vesicles that retain transport capacity. These preparations will be characterized as to permeability, topology, and homogeneity. Vesicles will be isolated from cells whose in vivo purine transport activity is modulated by genetic alterations, rates of cellular growth, or incubation with cyclic nucleotides. Purine transport experiments with these plasma membrane vesicles will focus on the mechanisms that control transport activity. This investigation using our biochemically and genetically defined system should allow us to identify and study the membrane proteins that regulate purine utilization in mammalian cells. We believe this will be a significant step in understanding disorders which alter purine pools such as Lesch-Nyhan syndrome or combined immunodeficiency disease.