Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein.
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Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells. (+info)
Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney.
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High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities. (+info)
Intrinsic factor in the human fetal stomach. An immunocytochemical study.
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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. (+info)
Radioautographic localisation of iodinated human intrinsic factor in the guinea pig ileum using electron microscopy.
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The uptake of iodinated human intrinsic factor by guinea pig ileum was studied in vivo using electron microscopy radioautography. Iodination of intrinsic factor cobalamin complex with N-chlorobenzene-sulphonamide beads did not modify its physicochemical properties in gel filtration, nor in iso-electrofocusing. It didn't affect its biological activity either in vitro in presence of solubilised receptor, or in vivo in the Schilling test. The 125I-intrinsic factor cobalamin complex was instilled in vivo in ileal blind loops in presence of either CaCl2, EDTA, bile or intestinal juice. The labelled complex was predominantly located in the intermicrovillous pits of the apical membrane and in the apical cytoplasm. The uptake was about five-fold lower in presence of EDTA. On the apical membrane, the number of grains (per 80 micron length of epithelium) increased from 2.7 (0.2) up to 9.4 (1.7) respectively for 15 minutes and two hours of delay. This suggests a recycling of the intrinsic factor receptor complex. In the apical compartment, the silver grains were often detected over non-coated vesicles and over the infoldings of the lateral membrane. These results show that intrinsic factor is internalised into enterocytes during cobalamin absorption, and that a part of intrinsic factor enters the blood circulation through transcytosis. (+info)
Lymphocyte subpopulations in patients with hydroxocobalamin responsive megaloblastic anaemia.
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Lymphocyte subpopulations and intrinsic factor and gastric parietal cell antibodies have been measured in 23 patients with megaloblastic anaemia who responded to treatment with hydroxocobalamin. The ratio of helper (OKT4) to suppressor (OKT8) lymphocytes was significantly increased in patients with intrinsic factor antibody compared with those who lacked the antibody. No such correlation was found for gastric parietal cell antibody. Alterations in the lymphocyte helper to suppressor (OKT4:OKT8) ratio may be associated with pernicious anaemia. (+info)
Combined congenital deficiencies of intrinsic factor and R binder.
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Coexisting deficiencies of both intrinsic factor (IF) and R binder were identified in an Algerian boy who presented with severe megaloblastic anemia, growth retardation, and neurologic dysfunction with typical features of subacute combined degeneration of the spinal cord. The anemia responded completely to cyanocobalamin and folic acid. IF was absent from gastric juice, but acid secretion and gastric mucosa were normal. R binders were absent from gastric juices as well as from serum, saliva, and polymorphonuclear leukocytes. The patient's father exhibited absence of R binder in his serum with a low serum vitamin B12 level and was asymptomatic. This unique case of simultaneous IF and R binder deficiencies suggests a genetic association between these two functionally and immunologically dissimilar, but structurally close vitamin B12-binding proteins. (+info)
Rapid protein A assay for intrinsic factor and its binding antibody.
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A simple and rapid method for the measurement of cobalamin bound intrinsic factor (Cbl-IF) complex and intrinsic factor binding antibody is described. The method is based on the principle of affinity chromatography and adapted to a batch separation technique. A specific ligand staphylococcal protein A was coupled to Sepharose to form a convenient solid phase matrix. The intrinsic factor binding antibody in patients with pernicious anaemia was used to form an immune complex with Cbl-IF. This complex was adsorbed on to staphylococcal protein A. Gastric juice from control subjects and patients with pernicious anaemia was assayed for intrinsic factor activity and the results correlated very closely with two other established methods. Sera from 30 control subjects were assayed for binding intrinsic factor antibody and all were found to be negative; of 15 patients with pernicious anaemia, six were positive. These patients were selected with blocking antibody. The method does not require technologically advanced protein separation techniques and could therefore be applied in any clinical laboratory using radioisotopes. It could also be adapted to assay cobalamin in body fluids or in food. (+info)
Isolation and structural characterization of a cDNA clone encoding rat gastric intrinsic factor.
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Rat intrinsic factor (IF) has been purified and proteolytic fragments were sequenced. A cDNA library was constructed from size-enriched gastric poly(A)+ RNA and screened for IF-positive clones by antibody and synthetic oligodeoxynucleotide probe hybridization. An IF clone was isolated and sequenced, revealing a predicted primary amino acid sequence in the coding region of 421 amino acids and a putative signal sequence of 22 amino acids. The primary translation product of IF produced in a cell-free translation system displayed cobalamin (Cbl)-binding activity without proteolytic processing or glycosylation. The amino-terminal region of IF showed significant secondary structural and hydropathic homologies with the nucleotide-binding domain in NAD-dependent oxidoreductases. Alignment of the first 80 residues of IF, following the signal peptide, demonstrated homology with the nucleotide-binding domain of cytoplasmic malate dehydrogenase. Based on these data, we propose a model of IF tertiary structure in which the Cbl-binding domain resides in the NH2-terminal half of the protein. (+info)