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Iron uptake by rat duodenal microvillous membrane vesicles: evidence for a carrier mediated transport system
Author(s) -
STREMMEL W.,
LOTZ G.,
NIEDERAU C.,
TESCHKE R.,
STROHMEYER G.
Publication year - 1987
Publication title -
european journal of clinical investigation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.164
H-Index - 107
eISSN - 1365-2362
pISSN - 0014-2972
DOI - 10.1111/j.1365-2362.1987.tb02393.x
Subject(s) - chemistry , membrane , vesicle , chromatography , size exclusion chromatography , biochemistry , biophysics , enzyme , biology
. The mechanism of iron translocation from intestinal lumen to portal plasma is poorly understood. To examine these processes, uptake of Fe 2+ and Fe 3+ by rat duodenal microvillous membrane vesicles prepared by a Ca 2+ precipitation procedure was studied. Membrane aliquots were incubated with increasing concentrations of 59 FeCl 3 in the presence of a one‐thousand‐fold molar excess of citrate or 59 FeSO 4 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 FeCl 3 (4°C), and uptake of 59 Fe was determined by a vacuum filtration assay. Initial uptake velocity of 59 FeCl 3 and 59 FeSO 4 was determined from the slope of the cumulative uptake curves, which was linear for the first 60 s. Initial uptake rates of both, 59 Fe 3+ and 59 Fe 2+ revealed an identical saturable uptake component with a K m of 19–22 n m and a V max of 8 pmol min −1 mg protein −1 . In addition, transport of Fe 2+ revealed a linear unspecific uptake phase, which was predominant at high substrate concentrations. Saturable uptake of Fe 2+ and Fe 3+ 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 m m EDTA yielded a single 52 000 dalton protein. The protein co‐chromatographed over an Ultro‐Pac TSK G 3000SW HPLC column together with 59 FeCl 3 and 59 FeSO 4 . 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.