Abstract
We have studied Fe(III)-citrate and Fe(II)-ascorbate uptake by purified intestinal brush-border membrane vesicles from normal (iron-replete) and iron-deficient mice. In iron-replete mice using a final Fe(III) concentration of 1.43 microM, 25-30 pmol of Fe(III)/mg of protein were bound to the membranes versus 65-70 pmol in iron-deficient mice. Fe(II) uptake in normal mice using a final Fe(II) concentration of 1.79 microM was 1600-1800 pmol/mg of protein versus 3600-4000 pmol in iron-deficient mice. Evidence that Fe(II) was transported into the vesicles by a membrane carrier-mediated process was obtained by observing saturation kinetics under conditions of isotope exchange at equilibrium in mice rendered iron-deficient, but not in iron-replete mice. Eighty per cent of the transported Fe(II) could be removed by strong chelating agents. The remainder was exchangeable with Fe(II) in the medium when measured under equilibrium conditions. We can explain these results by the following model; iron uptake appears to be a 2-fold process. The first step is the transport of Fe(II) across the membrane by a carrier-mediated process which is biologically regulated. The second step is the subsequent binding of iron on the inside of the membrane. The number of binding sites is also regulated by the iron status of the mouse. The membrane binding affinity for Fe(II) appears to be weaker than that for dithiothreitol but stronger than for ascorbate.
Highlights
We have studied Fe(II1)-citrate and Fe(I1)-ascorbate uptake by purified intestinal brush-border membrane vesicles from normal and iron-deficient mice
Assuming that the intravesicular glucose concentration at equilibrium is identical to the medium concentration, the intravesicular space can be calculated from the included glucose uptake at equilibrium
Experimental observations have provided evidence for two major mechanisms which may be involved in the initiation of iron absorption.One mechanism involves Fe-macromolecular complexes in the gut lumen [25], principally transferrin, which may bind to mucosal cell receptors and become internalized in endocytotic vesicles similar to the events thought to occur intransferrin-reticulocyteinteractions [26]
Summary
Brush-Border Membrane Preparation-For each experiment, the brush-border membrane vesicles were prepared from the upper half of the small intestine of two DBA/SJ male mice (Jackson Laboratories, Bar Harbor, ME) and were used within 5 h after preparation. The purified membrane vesicles werecollected by centrifuging the supernatant a4t 8,000 X g for 15 min and suspending in Solution A plus 0.1 mM MgSO, to the desired concentration priorto theiron uptake experiments. Uptake was terminated by removal of a sample and dilution with a 50-fold excess of an ice-cold stop solution consisting of 0.9% NaCl, 10 mM Tris/HEPES, and0.2mM phlorizin. Results were corrected for nonspecific binding (incubation without membranes) of labeled iron to the filters This correction was always less than 1%of the total counts. + Percoll Gradient Centrifugation of Membranes-Membranes were incubated in Solution B D with 10.75p~ Fe(I1) for 15 min at 37 "C for maximal iron uptake and thendiluted into a 5-fold volume excess of ice-cold stop solution. Sigma supplied HEPES,PIPES, cholamine, Dmannitol, and FeS04.Dithiothreitol was obtained from ICN Nutritional Biochemicals (Cleveland, OH), and Percoll was from Pharmacia (Uppsala, Sweden)
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