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

Abstract

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°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 K_m of 19–22 nm and a V_max 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 × 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.