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Intrinsic factor-mediated absorption of cobalamin by guinea pig ileal cells
Abstract:
To investigate the fate of intrinsic factor and cobalamin during cobalamin absorption, we incubated enterocytes isolated from guinea pig ileum for periods of up to 30 min with (57)Co-labeled cyano-cobalamin bound either to human intrinsic factor or to rabbit intrinsic factor biosynthetically labeled with [(35)S]methionine. When the labeled complex was incubated for 30 min with isolated ileal cells under conditions that block cellular metabolism, virtually all cellular radioactivity could be removed by washing the cell surface with EDTA or acid. In contrast, washing removed only half the radioactivity from cells incubated at 37 degrees C in O(2). When residual cellular radioactivity was extracted and analyzed by gel filtration, 80-94% of both the (35)S and (57)Co radioactivity eluted in the same fractions as the original complex. The remaining 6-20% eluted as free [(57)Co]cobalamin or [(35)S]methionine. To examine events occurring after 30 min, we instilled into tied-off ileal loops of intact guinea pigs radiolabeled intrinsic factor-cobalamin complex and extracted nondissociable radioactivity 2-4.5 h later. The proportion of extracted (57)Co eluting as free cobalamin increased to 39-46%, that eluting as intrinsic factor-cobalamin complex declined to 22-45%, and 9-34% now eluted as a macromolecule that reacted with antitranscobalamin II antibody but not antiintrinsic factor antibody. Extracted (35)S radioactivity eluted in several peaks in addition to the intrinsic factor peak. These findings suggest that (a) after reversible attachment of intrinsic factor-cobalamin complex to its ileal surface receptor, an energy-dependent process prevents removal of the complex from the cell surface by EDTA or acid; (b) cobalamin dissociates from intrinsic factor and, as suggested by previous workers, binds to a molecule antigenically similar to transcobalamin II; and (c) intrinsic factor is slowly degraded and forms breakdown products that are detectable in ileal extracts.