Abstract
Thiamine transport in Ehrlich ascites tumor cells was investigated. The rate of [35S] 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 5min 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 5min under any conditions employed. When the incubation was prolonged to 60min 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 10.1nM, 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 2.4mM. These results indicate that thiamine is actively transported by coupling to the Na+ gradient as a direct driving force.
