Mechanism of Transport of Riboflavin in Rabbit Intestinal Brush Border Membrane Vesicles

Abstract

Abstract

Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (<76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All ³H radioactivity that appeared in the intravesicular space after incubation with [³H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na⁺ or a K⁺ gradient (out >in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent K_m of 7.24 ± 1.06 and 8.88 ± 0.90 μM and V_max of 24.31 ± 1.48 and 34.24 ± 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [³H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and d-ribose all failed to inhibit [³H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [³H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K⁺-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na⁺ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.