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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024468-09
Application #
3272317
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-12-01
Project End
1991-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
9
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Plagemann, P G (1991) Mycoplasma contamination greatly enhances the apparent transport and concentrative accumulation of formycin B by mammalian cell culture. Biochim Biophys Acta 1064:162-4
Plagemann, P G (1991) Na(+)-dependent, active nucleoside transport in S49 mouse lymphoma cells and loss in AE-1 mutant deficient in facilitated nucleoside transport. J Cell Biochem 46:54-9
Woffendin, C; Chen, Z Y; Staskus, K et al. (1991) Mammalian mRNAs encoding protein closely related to ubiquitin-conjugating enzyme encoded by yeast DNA repair gene RAD6. Biochim Biophys Acta 1090:81-5
Plagemann, P G (1991) Na(+)-dependent, concentrative nucleoside transport in rat macrophages. Specificity for natural nucleosides and nucleoside analogs, including dideoxynucleosides, and comparison of nucleoside transport in rat, mouse and human macrophages. Biochem Pharmacol 42:247-52
Plagemann, P G; Woffendin, C (1990) Mycoplasma contamination alters 2'-deoxyadenosine metabolism in deoxycoformycin-treated mouse leukemia cells. J Cell Biochem 43:161-72
Plagemann, P G; Aran, J M; Woffendin, C (1990) Na(+)-dependent, active and Na(+)-independent, facilitated transport of formycin B in mouse spleen lymphocytes. Biochim Biophys Acta 1022:93-102
Plagemann, P G; Aran, J M (1990) Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells. Biochim Biophys Acta 1028:289-98
Plagemann, P G; Aran, J M; Wohlhueter, R M et al. (1990) Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides. Biochim Biophys Acta 1022:103-9
Plagemann, P G; Woffendin, C (1989) Dideoxycytidine permeation and salvage by mouse leukemia cells and human erythrocytes. Biochem Pharmacol 38:3469-75
Wohlhueter, R M; Plagemann, P G (1989) Measurement of transport versus metabolism in cultured cells. Methods Enzymol 173:714-32

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