Riboflavin uptake by rat small intestinal brush border membrane vesicles: a dual mechanism involving specific...

The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane vesicles. Vesicles were incubated at 25 degrees C in the presence of [3H]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [3H]-riboflavin uptake was unaffected by Na+ or K+ gradients. The 5 sec uptake rate plotted as a function of the initial concentration of [3H]-riboflavin in the medium (0.125 to 7.5 microM) revealed the presence of a dual mechanism, with a saturable component (apparent kinetic constants: 0.12 microM for Km and 0.36 pmol.mg-1 protein x 5 sec-1 for Jmax) prevailing at low concentrations (< 2 microM), and a nonsaturable component prevailing at higher concentrations. The presence of a carrier-mediated system for riboflavin was validated by countertransport experiments. At equilibrium, uptake was almost completely accounted for by membrane binding, whereas at earlier times the transport component accounted for about 30% of total uptake. The plot of [3H]-riboflavin binding at equilibrium as a function of its concentration in the medium was quite similar to that of the 5 sec uptake rate in both intact and osmotically shocked vesicles and demonstrated the occurrence of a saturable component: binding constants were 0.07 (Kd in microM), 0.54 (Bmax in pmol.mg-1 protein), and 0.11 (Kd), 1.13 (Bmax), respectively, indicating the existence of specific riboflavin binding sites. The specificity of riboflavin binding to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a specific riboflavin entry mechanism in enterocytes.