A FERRIC REDUCTASE-ACTIVITY IS FOUND IN BRUSH-BORDER MEMBRANE-VESICLES ISOLATED FROM CACO-2 CELLS

Brush border membrane Vesicles isolated from Caco-2 cells were used to examine whether there is an apical membrane-associated ferric reducta se activity in small intestinal enterocytes. A ferric reductase activi ty which was dependent on NADH or NADPH as reductants was shown. Reduc tion of Fe(III) was quantified by the formation of a stable Fe(II)/fer rozine complex. The ferric reductase revealed saturation kinetics with a K-m of 4.12 +/- 0.65 mu mol/L and a V-max of 3.11 +/- 0.043 nmol/(m in mg protein) for NADH. About 25% of the electrons for the NADH-depen dent ferric iron reduction were transferred indirectly from the supero xide anion as verified by the superoxide dismutase inhibitable ferric iron reduction rate. However, the main part of Fe(III) reduction occur s directly by catalyzed electron transfer from NADH to ferric iron thr ough (an) enzyme(s) located in the brush border membrane. The ferric r eductase activity was inhibited by Pt(II) and especially p-chloromercu ribenzoate. Ferricyanide, which is also reduced by the enzyme, is a co mpetitive inhibitor of the Fe(III)/nitrilotriacetate (NTA) complex wit h a K-i of 43 mu mol/L. These results suggest that brush border membra nes of enterocytes possess a ferric reductase that reduces ferric to f errous iron before the iron is transported through the microvillous me mbrane.