RIBOFLAVIN UPTAKE BY RAT SMALL-INTESTINAL BRUSH-BORDER MEMBRANE-VESICLES - A DUAL MECHANISM INVOLVING SPECIFIC MEMBRANE-BINDING

The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane v esicles. Vesicles were incubated at 25-degrees-C in the presence of [H -3]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [H-3]-riboflavin uptake was unaffec ted by Na+ or K+ gradients. The 5 sec uptake rate plotted as a functio n of the initial concentration of [H-3]-riboflavin in the medium (0.12 5 to 7.5 muM) revealed the presence of a dual mechanism, with a satura ble component (apparent kinetic constants: 0. 12 muM for K(m) and 0.36 pmol . mg-1 protein . 5 sec-1 for J(max)) prevailing at low concentra tions (<2 muM), and a nonsaturable component prevailing at higher conc entrations. The presence of a carrier-mediated system for riboflavin w as validated by countertransport experiments. At equilibrium, uptake w as almost completely accounted for by membrane binding, whereas at ear lier times the transport component accounted for about 30% of total up take. The plot of [H-3]-riboflavin binding at equilibrium as a functio n 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 constant s were 0.07 (K(d) in muM), 0.54 (B(max) in pmol . mg-1 protein), and 0 .11 (K(d)), 1.13 (B(max)), respectively, indicating the existence of s pecific riboflavin binding sites. The specificity of riboflavin bindin g to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a speci fic riboflavin entry mechanism in enterocytes.