Identification of a macromolecular factor in the ileum which binds intrinsic factor and immunologic identification of intrinsic factor in ileal extracts

Sheldon P. Rothenberg

doi:10.1172/JCI105783

Published April 1, 1968 - More info

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Abstract

The precipitate which resulted when ⁵⁷CoB₁₂ bound to normal human gastric juice was subjected to a 15% concentration of Na₂SO₄ contained virtually no radioactivity. However, after in vivo incubation of the gastric juice-⁵⁷CoB₁₂ mixture in the distal ileum of the guinea pig, the dialyzed extract of the washed mucosa contained a fraction of ⁵⁷CoB₁₂ which was precipitated at 15% Na₂SO₄. In addition, in vitro incubation of gastric juice-⁵⁷CoB₁₂ with an extract of the ileal mucosa or brush border membranes also resulted in the formation of a 15% Na₂SO₄-insoluble fraction which contained ⁵⁷CoB₁₂. The formation of this ⁵⁷CoB₁₂-containing insoluble fraction did not occur or was diminished by (a) addition of an excess of B₁₂-free normal human gastric juice. (b) reducing the incubation pH to 2, (c) incubating the mixture at 4°C, (d) pretreating the ileal extract at 56°C for 30 min, (e) incubating the reaction in sodium EDTA but not calcium EDTA, (f) incubating gastric juice-⁵⁷CoB₁₂ with an extract of jejunal mucosa. Sephadex gel filtration was used to demonstrate that the factor in the ileal extract which reacted with the gastric juice-⁵⁷CoB₁₂ filtered through G-100 and G-200 columns in the excluded volume.

When the ileal extract obtained after in vivo incubation with gastric juice-⁵⁷CoB₁₂ was subjected to starch gel electrophoresis one peak of radioactivity remained at the origin and another moved anodally. Eluates of each peak reacted with anti-intrinsic factor antibody indicating that at least the immunologically reacting portion of the intrinsic factor molecule was present in two fractions with different electrophoretic mobility.

These studies indicate that immunologically intact intrinsic factor can be extracted from the ileum after in vivo incubation with gastric juice-⁵⁷CoB₁₂, and that a macromolecular factor is present in the distal ileal mucosa which binds intrinsic factor both in vitro and in vivo, changing its solubility and electrophoretic properties. It is suggested that this ileal binding factor is the previously postulated intestinal receptor for intrinsic factor.