The ultimate objective of our research is to isolate the transporter for nucleosides and nucleobases from cultured mammalian cells and human erythrocytes, determine their amino acid sequences and configuration in the membrane, and elucidate the molecular mechanism by which they facilitate the transfer of hydrophilic substances across the lipid bilayer and how this process is regulated. In previous studies we have applied rapid kinetic techniques to determine detailed time courses of transmembrane equilibration of the substrates. Integrated rate analyses showed that zero-trans entry and equilibrium exchange data were consistent with the simple carrier model and that the nucleoside carrier transports all natural nucleosides. Two types of transporters have been identified; one binds nitrobenzylthioinosine (NBTI) with high affinity (K-D less than 1nM) which results in blockage of transport, whereas the other lacks high affinity binding sites and is not inhibited. The transporter has been identified as a 45 to 65 kd membrane protein and has been partially purified from human erythrocytes and cultured cells. We propose now to further purify the transporters for both types of cell, using affinity chromatography with monoclonal antibodies and NBTI as ligands, sequence their amino terminals and, on the basis of the coding sequences derived therefrom, synthesize oligodeoxynucleotide probes. We will also attempt to select, from cultured cells, variants with amplified nucleoside transporter genes, and construct cDNA libraries to their mRNA. These libraries and a cDNA library to human T-leukemia cells will be screened with the oligonucleotide probes, nick-translated cDNA probes to mRNA from cells with amplified genes and from a transport deficient mutant and for expression of the transporter in COS-1 and E. coli cells by an immunoassay. We will also try to transform the transport deficient mutant with DNA from a human genomic library. In other experiments, we will assess the functional properties of the isolated transporter in reconstituted liposomes and continue our studies on the substrate specificity and other properties of the transporters.
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