INITIAL RATE KINETICS OF THE TRANSPORT OF ADENOSINE AND 4-AMINO-7-(BETA-D-RIBOFURANOSYL)PYRROLO[2,3-D]PYRIMIDINE (TUBERCIDIN) IN CULTURED-CELLS

Abstract

A procedure is described for determining early time courses of nucleoside uptake by culture cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (s) 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 1st few s of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(.beta.-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 [human cervical carcinoma] cells and in 2 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 2 L5178Y cell types and were saturable in the 3 cell lines with Km values between 14-38 .mu.M. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.