Thiamin transport by human erythrocytes and ghosts

Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20 degrees C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 microM and at 37 degrees C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes, Km = 0.12, 0.11 and 0.10 microM and Jmax = 0.01, 0.02 and 0.03 pmol.microliter-1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts, Km = 0.16 and 0.51 microM and Jmax = 0.01 and 0.04 pmol.microliter-1 intracellular water after 15 and 30 min incubation times, respectively. At 20 degrees C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence of D-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein- (probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN- showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.