Properties of the Active Thiamine Transport System in Ehrlich Ascites Tumor Cells

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Abstract
Thiamine transport in Ehrlich ascites tumor cells was investigated. The rate of [ 35 S]thiamine transport in the presence of an Na + gradient was dependent on temperature but not on the presence of glucose, although the steady-state level of the uptake was reduced in the absence of glucose. Approximately 65% of thiamine taken up by cells for 5 min at 37°C in the presence of glucose was present in unaltered form, and the distribution ratio of intra-/extracellular thiamine concentration was 1.8. This ratio increased to 2.3 either in the absence of glucose or in the presence of 2,4-dinitrophenol and KCN without glucose, which indicated a concentrative uptake of thiamine after incubation for 5 min under any conditions employed. When the incubation was prolonged to 60 min in the presence of glucose, thiamine taken up was metabolized to thiamine pyrophosphate; 10.7% of the total uptake was thiamine and its distribution ratio was 1.0. The initial rate of transport as a function of thiamine concentrations showed saturation kinetics with a Km value of 43.5 nM and a Vmax of 0.71 pmol/mg of protein/min. Pyrithiamine and chloroethylthiamine, antimetabolites of thiamine, competitively inhibited thiamine uptake with K1 values of 21.0 and 10.1 nM, respectively, but oxythiamine was not inhibitory. These results indicate the presence of a specific thiamine carrier of high affinity to the substrate. When the effect of Na + concentration on the kinetics of thiamine transport was determined, it was found that a decrease in Na + concentration increased the Km value for thiamine without changing the Vmax value. Treatment of the cells with gramicidin reduced the rate of thiamine uptake to a half of that in untreated cells. The uptake rate in the presence of an Na + gradient (out > in) was also independent of cellular ATP level in the range from 0.1 to 2.4 mm. These results indicate that thiamine is actively transported by coupling to the Na + gradient as a direct driving force.

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