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Intrinsic factor (IF) has been expressed previously in Baculovirus with a yield (0.1-1 mg/l) that was inadequate for structural and metabolic studies. IF cDNAs were cloned into the shuttle vector pPIC9 of P. pastoris, and the proteins were induced and purified by cobalamin (Cbl) affinity chromatography. Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF. Expression of human IF was achieved at 1040 mg/l, but of rat IF at only 1-2 mg/l. Reaction of human IF with a photo-activatable derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequencing revealed at least three regions (residues 129-151, 234-254, and +294) linked to Cbl. Both recombinant human and rat [125I]IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding capacity of human IF for the rat receptor was only 10% compared with rat IF. All six amino acids within the previously identified N-terminal binding region of human IF were mutated to be identical to rat IF, but the resulting chimeric IF still bound poorly to rat membranes. Mutations of residues 26/27 (Glu26 to Asp and Asn27 to Gln) and 32/34 (Ser32 to Thr and Tyr34 to Arg) showed changes in both Ka and Vmax, with great effects on Vmax. In conclusion, P. pastoris is an expression system that produces functional human IF at a higher yield than in the baculovirus system. Cbl binding was directly demonstrated at multiple sites along the linear sequence of human IF. The receptor binding function of the amino terminal sequence 25 62 has been confirmed, but it is insufficient to reproduce all the features of IF-Cbl binding.
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A 11 1/2-year-old girl of Turkish origin showed symptoms and signs of pernicious anaemia with low B12 level in blood but normal gastric acid secretion. There were no antibodies to mucosal cells or intrinsic factor. Quantitative determination of intrinsic factor in gastric juice, done by a RIA method, proved absence of the factor. Both parents had normal values for haemoglobin, normal B12-absorption tests and slightly decreased B12 blood levels. The gastric acid secretion was normal in both, but intrinsic factor in the gastric juice was decreased markedly. These findings support the hypothesis that congenital pernicious anaemia, e.g. congenital absence of intrinsic factor, is transmitted by an autosomal recessive trait.
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A method employing non-radioactive vitamin B(12) and microbiological assay is described for estimating intrinsic factor in gastric juice and for detecting antibody to intrinsic factor in serum. Satisfactory agreement was obtained between the results by this method and by a modification of the method of Ardeman and Chanarin (1963). During the first hour after gastric stimulation 11 patients with pernicious anaemia secreted between 0 and 240 units of intrinsic factor compared with between 1,600 and 39,000 units in 21 patients with other conditions. The results in three out of four patients with gastric atrophy were higher than those in pernicious anaemia but lower than in other conditions.
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A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic factor antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel at pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for a least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor.
The precipitate which resulted when (57)CoB(12) bound to normal human gastric juice was subjected to a 15% concentration of Na(2)SO(4) contained virtually no radioactivity. However, after in vivo incubation of the gastric juice-(57)CoB(12) mixture in the distal ileum of the guinea pig, the dialyzed extract of the washed mucosa contained a fraction of (57)CoB(12) which was precipitated at 15% Na(2)SO(4). In addition, in vitro incubation of gastric juice-(57)CoB(12) with an extract of the ileal mucosa or brush border membranes also resulted in the formation of a 15% Na(2)SO(4)-insoluble fraction which contained (57)CoB(12). The formation of this (57)CoB(12)-containing insoluble fraction did not occur or was diminished by (a) addition of an excess of B(12)-free normal human gastric juice. (b) reducing the incubation pH to 2, (c) incubating the mixture at 4 degrees C, (d) pretreating the ileal extract at 56 degrees C for 30 min, (e) incubating the reaction in sodium EDTA but not calcium EDTA, (f) incubating gastric juice-(57)CoB(12) 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-(57)CoB(12) filtered through G-100 and G-200 columns in the excluded volume. When the ileal extract obtained after in vivo incubation with gastric juice-(57)CoB(12) 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-(57)CoB(12), 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.