A Thiamine/H + Antiport Mechanism for Thiamine Entry into Brush Border Membrane Vesicles from Rat Small Intestine

Abstract

Outwardly oriented H+ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pHin5:pHout7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10-13% of intravesicular transport. At the same gradient, the Km and Jmax values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 +/- 1.4 microM and 14.9 +/- 3 pmol.mg-1 protein.6 sec-1 respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H+ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H+ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H+ antiport, and imipramine. Conversely, the guanidine/H+ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H+ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9.