The application of rapid kinetic techniques to the transport of thymidine and 3‐O‐methylglucose into mammalian cells in suspension culture

Abstract

Techniques are described by which the transport of nutrients into mammalian cells in suspension can be measured at intervals of 1.5 seconds. By application of these techniques, the existence of a saturable (Km = 85 muM), non-concentrative, transport system for thymidine was demonstrated in Novikoff rat hepatoma cells depleted of ATP. At concentrations of thymidine less than the Km, this system operated at velocities sufficient to nearly completely equilibrate intra- and extra-cellular thymidine pools within 8 seconds. In phosphorylating cells, the transport system operated with similar rapidity, so that intracellular phosphorylation was rate-limiting for the incorporation of thymidine into nucleotides. Uptake of 3-O-methylglucose occurred at comparable velocities, attaining 90% of equilibrium between internal and external pools within 25 seconds. Uptake of cytosine by simple diffusion was 100 times slower.

Mg 2+ as activator of uridine phosphorylation in coordination with other cellular responses to growth factors

“…As with uridine uptake, the uptake of thymidine is limited by its rate of intracellular phosphorylation (18). Unlike the uptake of uridine, however, the uptake of thymidine is not affected by serum stimulation of cells or by variations in Mg 2ϩ that produce large changes in uridine uptake (17).…”

Residual nitrobenzylthioinosine-resistant nucleoside transport in a transport mutant (AE1) of S49 murine T-lymphoma cells.

“…The Km and Vmax values for thymidine transport in ATP-depleted S49 cells at 25°C were 161 + 13 ,uM and 37 + 1 pmol/,ll of cell water per s, respectively (19). The cells were ATP depleted to prevent thymidine phosphorylation so that the transmembrane equilibration of unmodified substrate could be measured (17,25). In the context of the present study, we have directly compared the zero-trans influx of various nucleosides by S49 cells at a concentration (500 ,uM) approaching saturation of the transporter.…”

“…For experiments, cells were harvested from mid-to late-exponential-phase cultures and suspended to 1 x 107 to 5 x 107 cells per ml of basal medium 42B (BM42B [15] (17). The (25). Concentrations of intracellular substrate equivalents were expressed on the basis of intracellular water space determined by the use of 3H20 (25).…”

“…Concentrations of intracellular substrate equivalents were expressed on the basis of intracellular water space determined by the use of 3H20 (25). Where indicated, cells were depleted of ATP prior to transport measurements by incubation in glucose-free BM42B containing 5 mM KCN and 5 mM idoacetate at 37°C for 15 min as described previously (16,25).…”

“…For fractionating the acid-soluble pool of labeled cells, samples of cells were centrifuged through an oil layer directly into 100 of a solution composed of 0.5 N trichloroacetic acid and 10% (wt/vol) sucrose, which rapidly quenches all metabolism (18,25). The acid layer was further processed and analyzed by ascending paper chromatography with a solvent composed of 30 ml of 1 M ammonium acetate, pH 5, and 70 ml of 95% ethanol (solvent 28) as described previously (18).…”