Iron uptake by rat duodenal microvillous membrane vesicles: evidence for a carrier mediated transport system

The mechanism of iron translocation from intestinal lumen to portal plasma is poorly understood. To examine these processes, uptake of Fe2+ and Fe3+ by rat duodenal microvillous membrane vesicles prepared by a Ca2+ precipitation procedure was studied. Membrane aliquots were incubated with increasing concentrations of 59FeCl3 in the presence of a one-thousand-fold molar excess of citrate or 59FeSO4 with a twenty-fold molar excess of L-ascorbic acid. After various time intervals the incubation reaction was stopped by addition of 0.1 mM FeCl3 (4 degrees C), and uptake of 59Fe was determined by a vacuum filtration assay. Initial uptake velocity of 59FeCl3 and 59FeSO4 was determined from the slope of the cumulative uptake curves, which was linear for the first 60 s. Initial uptake rates of both, 59Fe3+ and 59Fe2+ revealed an identical saturable uptake component with a Km of 19-22 nM and Vmax of 8 pmol min-1 mg protein-1. In addition, transport of Fe2+ revealed a linear unspecific uptake phase, which was predominant at high substrate concentrations. Saturable uptake of Fe2+ and Fe3+ was temperature dependent, and significantly reduced by trypsin pretreatment of the microvillous membrane vesicles, indicating the involvement of a protein in the uptake process. This suggestion was pursued by isolation of an iron binding protein from duodenal brushborder membranes. After solubilization of microvillous plasma membranes with 1% Triton X 100, affinity chromatography of the membrane protein mixture over an iron chelate gel derived from epoxy activated Sepharose and elution with 50 mM EDTA yielded a single 52,000 dalton protein. The protein co-chromatographed over an Ultro-Pac TSK G 3000SW HPLC column together with 59FeCl3 and 59FeSO4. It showed no immunologic activity to rabbit antibodies against whole rat serum or rat transferrin. Furthermore, by photoaffinity labelling technique a single iron binding protein with a molecular weight of about 52,000 dalton was identified in microvillous membranes of the rat duodenum. These data are compatible with the hypothesis that intestinal iron absorption is mediated by a specific carrier-dependent transport system.