Specificity of Riboflavin Molecular Groups for Riboflavin Binding to Rat Small Intestinal Brush Border Membrane

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The binding of riboflavin to rat small intestinal brush border membrane at equilibrium was formerly shown to have a saturable, specific component, prevailing at the intraluminal physiological concentrations of the vitamin. In this study, the specificity of riboflavin binding to rat small intestinal brush border vesicles was further investigated using structural analogues of riboflavin. The vesicles, prepared by Ca²⁺-precipitation, were incubated at 25°C, for 20min, in the presence of [³H]-riboflavin at physiological intraluminal concentrations for rat, and each analogue, at appropriate concentrations. Three groups of analogues were used, that were derived from the riboflavin molecule by modifying one of the following positions: the ribityl side chain, position 3, and position 8 of the isoalloxazine moiety. Group specificity was assessed by determining the inhibition potency of each analogue on the saturable component of riboflavin binding to the vesicles. Inhibition constants were calculated, according to Dixon, for lumiflavin, lumichrome, and for analogues substituted at position 8. Specific riboflavin binding was inhibited competitively by most of the analogues used. Substitutions at the ribityl side chain or at position 3 of the isoalloxazine moiety reduced the inhibition power. Substitutions at position 8 enhanced the inhibition power in direct proportion to the bulk of the substituents. We conclude that the ribityl side-chain and the NH group at position 3 are essential for recognition by the specific binding sites, whereas the methyl group at position 8 is important but not essential. The analogues that bind to specific membrane sites for riboflavin share specificity requirements with many riboflavin binding proteins, and are also good substrates for the intracellular phosphorylating enzyme flavokinase. Thus, the riboflavin-binding component in the membrane is likely to be a protein with high specificity. Cellular internalization of the membrane bound vitamin is probably achieved by phosphorylation of the vitamin bound to the inner side of the membrane.