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At least 73 records · Page 4Linked to original sources
PubMed1979

Amino terminal sequence of hog intrinsic factor.

1. Hog intrinsic factor has been purified from gastric mucosa by labile ligand affinity chromatography. 2. The following N-terminal amino acid sequence has been determined: Thr-Arg-Ser-Ser-Cys-Ser-Val-Pro-Ser-Ala-Glu-Gln-Pro-Leu-Val-Asn-Gly-Ile- Gln-Val - Leu-Met. 3. However, approx 60% of the preparation lacked the first two residues. 4. A high degree of homology between the amino terminal sequence of human and hog intrinsic factor exists.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of a pancreatic intrinsic factor in the dog.

An intrinsic factor has been identified in the canine pancreas, and output and properties of this protein have been compared with those of gastric intrinsic factor in the dog. Mean concentrations of intrinsic factor and peak outputs per minute were approximately 5- to 10-fold higher in pure pancreatic juice after stimulation with secretin and cholecystokinin, respectively, than in pentagastrin-stimulated gastric juice. Purified gastric and pancreatic intrinsic factors had an identical molecular mass of 65 kDa, estimated by gel filtration on Sephacryl S-200, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated single bands corresponding to 53 kDa. Immunoblots showed that rabbit polyclonal antiserum to canine gastric intrinsic factor cross-reacted with canine pancreatic intrinsic factor. Gastric and pancreatic intrinsic factor-cyano[57Co]cobalamin complexes exhibited comparable association constants for ileal receptors in canine brush-border vesicles, while there was minimal binding to jejunal vesicles. These findings demonstrate that the canine pancreas is an important source of an intrinsic factor that closely resembles gastric intrinsic factor in the dog.

Animals↗
PubMed1992

In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.

Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.

Animals↗
PubMed1979

Solubilization and preliminary characterization of the human ileal vitamin B12-intrinsic factor receptor.

The human ileal intrinsic factor receptor was solubilized with Triton X-100 using an improved method originally devised for the porcine receptor. At pH 7.4 and in the presence of Ca2+ the receptor bound the vitamin B12 complexes of normal human and pig intrinsic factor but not that of an abnormal biologically inert human intrinsic factor. EGTA dissociated vitamin B12-intrinsic factor from the receptor complexes. The solubilized vitamin B12-intrinsic factor receptor complex consisted of three to four molecular species termed HC-L, HC-20 S, HC-12 S and HC-8.5 S (the three last-mentioned referring to sedimentation coefficients). Of these HC-20 S was the dominating component and had a Stokes radius of 18 nm. Radioactive calcium was shown to be bound to vitamin B12-intrinsic factor and to be contained in its complex with the receptor.

Animals↗
PubMed1984

The role of sialic acid in the binding of calcium ions to intrinsic factor and its intestinal receptor.

The binding of vitamin B-12-intrinsic factor (cobalamin-intrinsic factor) to its intestinal receptor requires Ca2+. Only low concentrations of Ca were shown to be needed; therefore apparently high-affinity Ca-binding sites, such as neuraminic acids, are involved. Accordingly, the Ca-binding properties of purified human intrinsic factor and purified hog intrinsic factor receptor were studied by gel filtration and by isoelectric focusing. Cobalamin-intrinsic factor binds 45Ca2+ as a stable complex and in electrofocusing the 45Ca activity resolves into several peaks corresponding to the acidic isoproteins of cobalamin-intrinsic factor. Digestion with neuraminidase abolishes the Ca-binding capacity of cobalamin-intrinsic factor. This intrinsic factor binds 45Ca2+ via sialic acids. The purified receptor also binds 45Ca2+ but the binding is not lost after neuraminidase treatment. Thus the receptor does not bind Ca2+ with sialic acid residues sterically accessible to neuraminidase. The desialylated receptor still binds both cobalamin-intrinsic factor and desialocobalamin-intrinsic factor.

Animals↗
PubMed1981

Topology of the hog intrinsic factor receptor in the intestine.

The intrinsic factor receptor was isolated from Triton X-100 extract of hog ileal mucosa using affinity chromatography on intrinsic factor bound to cobalamin-Sepharose. We verified that the receptor contains two subunits, alpha and beta. The purified receptor located in the detergent micelle was radioiodinated. The alpha subunit was labeled and dissociated from the receptor. When the receptor was immobilized on intrinsic factor cobalamin-Sepharose, the part of the receptor which binds intrinsic factor evidently faces the gel and the rest faces outward. When such gel-bound pure receptor was iodinated, the beta subunit was labeled. Iodination of the micellar cobalamin-intrinsic factor receptor complex also caused labeling of the beta subunit. This was interpreted as being due to a conformational change in the receptor affected by the binding of the substrate cobalamin-intrinsic factor, exposing groups accessible to iodination. The beta subunit was found to be hydrophobic, but the alpha subunit was soluble in phosphate buffer without detergent. The receptor was liberated from intestinal mucosa by papain treatment. The enzyme seems to solubilize an intrinsic factor-binding part of the receptor, apparently a part of the alpha subunit. The liberated papain-alpha was purified by affinity chromatography. In gel filtration, it seemed to occur in dimeric form, but its true Mr = 45,000, according to findings in sodium dodecyl sulfate-electrophoresis. In the light of the findings, the topology of the receptor is suggested to be as follows: the alpha subunit binding intrinsic factor faces out and the hydrophobic beta subunit faces in.

Animals↗
PubMed1988

Intrinsic factor in the human fetal stomach. An immunocytochemical study.

The stomachs from 15 human fetuses and 5 neonates were examined by immunocytochemical methods for the presence of intrinsic factor. Intrinsic factor was localised in cells within the gastric mucosa of all fetuses from 11 weeks of gestation onwards. The cells, which were immunoreactive for intrinsic factor, were located mainly at the base of the developing gastric glands in both the pylorus and corpus of the stomach. Occasional cells located at the isthmus of the gastric glands and amongst the surface columnar cells were strongly immunoreactive for intrinsic factor. With conventional staining these cells had the morphological and histochemical characteristics of parietal cells.

Gastric Mucosa↗
PubMed1999

An enzyme immunoassay for intrinsic factor in urine.

An enzyme immunoassay for intrinsic factor has been used on urine. The assay can measure intrinsic factor in native urine from healthy people and from patients with pernicious anaemia with no antibodies. The urinary intrinsic factor concentration in healthy individuals ranged from 40 to 54 pmol/l. Intrinsic factor antibodies, demonstrated by testing the recovery of added intrinsic factor, interfered with the assay. Cobalamin at high concentrations also affected the assay result. A low intrinsic factor concentration or the presence of antibodies to intrinsic factor was found in the urine of individuals with pernicious anaemia.

Antibody Specificity↗
PubMed1980

Commercially-supplied binders for plasma cobalamin (vitamin B12), analysis--"purified" intrinsic factor, "cobinamide"-blocked R-protein binder, and non-purified intrinsic factor-R-protein binder--compared to microbiological assay.

Recent evidence (Kolhouse et al., N. Engl. J. Med. 299: 785-792, 1978) demonstrates that commercial cobalamin (Vitamin B12) radioassay kits contain nonspecific R-protein binding agents that can give falsely normal results in patients who are actually cobalamin deficient. We tested three kits: with "purified" intrinsic factor as the binder, with intrinsic factor and the nonspecific R-protein sites blocked with "cobinamide," and non-purified intrinsic factor-R-protein binder. Results with use of the first two compared well with those by a microbiological assay (Lactobacillus leichmannii) and are in harmony with clinical impressions.

Biological Assay↗
PubMed1975

Cellular immunity to intrinsic factor in pernicious anemia.

Cellular immunity to hog intrinsic factor was detected by a modified agarose-leukocyte migration test in 18 patients with pernicious amemia. Lymphocytes from 17 out of 18 patients with pernicious anemia gave positive responses to a concentrate of hog intrinsic factor; the intrinsic factor present in 1 mg. of this concentrate bound 128 ng. of vatamin B12. Six patients with atrophic gastritis, 7 with regional enteritis, and 9 out of 10 healthy adults did not respond to this preparation. No correlation existed between the presence of serum autoantibodies to intrinsic factor and in vitro lymphocyte responsiveness to intrinsic factor. The results demonstrate that cellular immunity to intrinsic factor concentrates is present in the majority of patients with pernicious anemia.

Adult↗
PubMed1990

Monoclonal antibodies to human intrinsic factor.

Mice were immunized with human intrinsic factor, and their lymph node cells were fused with a myeloma cell line by standard hybridoma techniques. Eleven of the resulting 227 hybridomas secreted immunoglobulin G capable of binding to intrinsic factor-cobalamin complex. Cloning by limiting dilution gave 6 clones secreting anti-intrinsic factor antibodies that bound human intrinsic factor-cobalamin complex with affinities of 13-116 nM; 3 antibodies also bound rabbit intrinsic factor-cobalamin complex. Five antibodies inhibited to some degree the binding of cobalamin by intrinsic factor, and 2 also prevented attachment of intrinsic factor-cobalamin complex to guinea pig ileal receptors. Anti-rabbit intrinsic factor antibodies specifically precipitated a peptide of molecular weight 53,000, corresponding to the molecular weight of rabbit intrinsic factor from homogenates of rabbit gastric mucosal explants biosynthetically labeled with [35S]methionine and from culture medium in which the explants were incubated. Indirect fluorescence immunocytochemistry with the antibodies in human and rabbit gastric mucosal sections showed intense selective staining of parietal cells. These results (a) document species differences between human and rabbit intrinsic factors not previously demonstrable with polyclonal anti-intrinsic factor sera; (b) confirm earlier evidence that cobalamin binding and receptor functions occur at separate sites in intrinsic factor; and (c) provide a useful approach to studying structure-function relations of the intrinsic function molecule.

Animals↗
PubMed1980

Immunocytochemical localization of human intrinsic factor: the nonstimulated stomach.

Human intrinsic factor (IF) is found on the surface of parietal cells at the light microscopic level by immunofluorescence, but there is no direct evidence that IF is synthesized in and secreted from this cell. In order to provide this evidence, IF was localized at the ultrastructural level in human stomach, using an immunochemically, monospecific antibody against purified human IF. We defined optimal immunocytochemical conditions and identified the subcellular location of IF in fundic biopsies from 4 fasting subjects. Intrinsic factor was found on the perinuclear membrane, on rough endoplasmic reticulum, Golgi apparatus, tubulovesicles, and membranes of the multivesicular body in parietal cells. Intrinsic factor was absent from nuclei, mitochondria, basolateral surface membranes, blunt microvilli, and the cytosol. Thus, we have defined the parietal cell as the site of IF synthesis. The observed distribution of intracellular IF suggested that an interrelationship existed between the stained membranous structures, and that IF secretion may depend upon membrane translocation. Further immunocytochemical and physiologic studies should increase our understanding of the cellular physiology of IF secretion as well as the parietal cell.

Adult↗
PubMed1994

von Willebrand factor as a regulator of intrinsic factor X activation.

Factor VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗