The ultimate objective of the proposed research is to elucidate the molecular mechanism and regulation of the facilitated transport of nucleosides and nucleobases in cultured mammalian cells and to isolate the membrane components involved. In previous studies we have applied rapid kinetic techniques to determine detailed time courses of substrate accumulation to transmembrane equilibrium in cells in which phosphorylation was blocked genetically or by depletion of ATP (PRPP). Integrated rate analysis showed that zero-trans entry and equilibrium exchange data were consistent with the simple carrier model and that the transporters exhibit directional symmetry and no differential mobilities of loaded and empty carrier. The nucleoside carrier transports all natural nucleosides. We propose to determine the affinity of the transporter for various nuclecsides and nucleoside analogs, and explore possible overlaps with the systems transporting nucleobases. Substrate specificity will also be explored with two series of nucleoside analogs with modifications in the nucleobase and ribose moieties respectively, which we propose to synthesize. The molecular mechanism of transport will be further explored by determining the effects of sulfhydryl, oxidizing and reducing agents on nucleoside transport. Because of its great affinity for the transporter (Kd equals 0.1nM) we hope to use nitrobenzylthioinosine (NBTI) as a marker for the detection, identification and isolation of the transporter. We hope to establish that binding of (3N)NBTI to high affinity sites correlates with the inhibition of transport. We also plan to construct two related cross-linking, high affinity probes. Attempts will be made to isolate transport mutants. We plant to extract selectively from plasma membranes of transport negative and positive cells peripheral and integral membrane protein and then will be solubilized with detergents. Its function will be tested by implantation into liposomes and the plasma membrane of transport mutants. For comparative purposes we also plan to reinvestigate the kinetic properties of the nucleoside transporter of human erythrocytes which differ from those of the transports of cultured cells.
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