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Initial Rate Kinetics of the Transport of Adenosine and 4-Amino-7-(ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (Tubercidin) in cultured Cells

Cancer Research Unit (McEachern Laboratory) [E. R. H., C. E. C., A. R. P. P.] and Department of Biochemistry [C. E. C., A. R. P. P.], University of Alberta, Edmonton, Alberta, Canada T6G 2H7

A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing ³H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(ß-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with K_m values between 14 and 38 µM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.

² To whom requests for reprints should be addressed.

Received 10/27/81. Accepted 1/ 7/82.

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