Concerning a published paper on cobalamin-binding proteins.
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Animals↗
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Transfer of cobalamin from the cobalamin-binding protein of egg yolk to R binder of human saliva and gastric juice.
Patients may fail to absorb cobalamin (vitamin B12) bound to food even when they have adequate intrinsic factor to absorb free cobalamin normally. We studied cobalamin transfer from egg yolk cobalamin-binding protein to human saliva and gastric juice as a model of this important first step in cobalamin assimilation. The cobalamin-binding protein of egg yolk eluted with human R binder on Sephadex gel chromatography and bound cobalamin with a comparable affinity, but it did not cross-react with R binder immunologically. Transfer of cobalamin from egg yolk to saliva or gastric juice R binder did not occur at neutral pH. Slight transfer (8%-12% of the 57Co-cobalamin bound to egg yolk) occurred when the saliva was acidified to pH 1.5. This minor transfer by acid was not inhibited by pepstatin A, a pepsin inhibitor. Acidification caused variable transfer to gastric juice R binder (12%-40%) that appeared to be partially due to residual gastric pepsin activity. Adding 1200 U of pepsin per milliliter enhanced cobalamin transfer to saliva or gastric juice R binders (39%-58% transfer). At no time was cobalamin transferred directly to intrinsic factor; R binder-deficient gastric juice failed to accept cobalamin from egg yolk. The transfer of cobalamin from egg yolk to human R binder requires both an acid pH and pepsin activity. While as little as 30 U of pepsin added per milliliter of saliva promoted transfer of cobalamin, the requirement for an acid pH was very strict. Virtually no transfer occurred when pH exceeded 2.0, regardless of the amount of pepsin present. Acid provided an optimal pH for pepsin activity and, to a lesser extent, affected transfer by a mechanism unrelated to pepsin. Our data suggest that compromised pepsin secretion and, probably even more importantly, compromised acid secretion interfere with transfer of food cobalamin to R binder.
Anemia, Pernicious↗
Portal and biliary phases of enterohepatic circulation of corrinoids in humans.
The assimilation of labeled cobalamin and the transport of corrinoids in portal blood, peripheral venous blood, and bile were studied in eight cholecystectomized patients, after ingestion of a dose of cyano[57Co]cobalamin (0.5 microCi). The radioactivity appeared in the portal vein after a delay of 1.5-2 hours and in the peripheral vein 1 hour later. In bile, it reached a maximum at 24-72 hours; the excreted cobalamin corresponded to 1.42% +/- 0.92% of the dose ingested. The output of total corrinoids was 1.85 nmol/day. The high-performance liquid chromatography analysis of bile showed the presence of methylcobalamin, 5'-deoxyadenosylcobalamin, hydroxocobalamin, and an unknown corrinoid. This corrinoid bound to R binder but not to the intrinsic factor, and it had the same retention time as cobinamide. The R binder was the single cobalamin-binding protein found in bile. It was completely saturated in some periods of bile secretion. The corrinoids corresponding to such a period were eluted in Sephacryl S 300 gel filtration (Pharmacia Fine Chemicals, Uppsala, Sweden) in two peaks corresponding to saturated R binder and to free cobalamin. The mean level of total corrinoid was significantly higher in the portal vein (593 +/- 238 pmol/L) than in the peripheral vein (376 +/- 114 pmol/L) (P less than 0.01). This "cobalamin analogue" fraction was hypothetical because it was calculated from the difference between total corrinoid concentration and the so called "true cobalamin" concentration. This difference corresponded to the cobalamin analogue fraction. These data show that bile removes not only cobalamin but also cobalamin analogues and that R binder is the single carrier protein involved in their excretion.
Aged↗
The minor components of the rat asialoglycoprotein receptor are apically located in neonatal enterocytes.
Because specific uptake of the asialoglycoprotein haptocorrin has been reported in suckling distal intestine, evidence of the asialoglycoprotein receptor in rat ileum was sought. On Western blot, two different polyclonal antisera against purified rat holoreceptor recognized only proteins of the size of the minor receptor components (51 and 55 kilodaltons) in both suckling and adult rat intestine. Messenger RNA encoding the minor component (RHL-2/3) was detected in total RNA extracted from rat ileum. Calcium-specific binding of porcine or rat haptocorrin-[57Co]cobalamin complexes was detected in the brush border membranes of distal suckling rat and porcine small intestine. This binding was almost completely blocked by another asialoglycoprotein, asialofetuin. The pH optimum for binding was 6-9, with a Ka of 0.25 nmol/L and a capacity of 4.6 fmol/mg protein. These properties, with the exception of the low capacity, are all consistent with those of the intact receptor on hepatocytes. The intestinal receptor was localized not to the basolateral membrane, as in the liver, but to the apical brush border, as suggested by the binding data. Furthermore, immunoperoxidase stains of paraffin-embedded tissue detected strong binding to the brush border and apical phagolysosome of mid and distal suckling rat intestine. These data show that, contrary to expectations, the minor components of the asialoglycoprotein receptor are functional and expressed in the apical membrane of the suckling intestine. The abundance of the protein by Western blot suggests possible roles for it in the neonatal gut other than haptocorrin binding.
Animals↗
Reversal of protein-bound vitamin B12 malabsorption with antibiotics in atrophic gastritis.
The role of bacteria in the bioavailability of protein-bound vitamin B12 was examined in eight elderly subjects who had atrophic gastritis and in eight normal controls. On separate days and in random order, vitamin B12 absorption tests were performed using either radiolabeled crystalline or protein-bound vitamin B12. At the same time, bacterial samples were collected from the upper gastrointestinal tract. The tests and gastrointestinal aspirates were performed before and during tetracycline therapy. Crystalline vitamin B12 was absorbed to the same extent in the two study groups. Atrophic gastritis subjects absorbed significantly less protein-bound vitamin B12 than normal controls (mean +/- SEM, 0.7% +/- 0.2% vs. 1.9% +/- 0.5%, respectively). However, protein-bound vitamin B12 absorption in these subjects normalized after antibiotic therapy. These results suggest that the small amounts of vitamin B12 released from the protein binders is readily absorbed (as shown in vitro) and/or metabolized by bacteria.
Achlorhydria↗
Vitamin B12 and folic acid studies by saturation analysis techniques. A review.
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Antibodies↗
B12 -- dependent methionine synthetase as a potential target for cancer chemotherapy.
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5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Serial measurements of beta-2-microglobulin in plasma correlates with white blood cell counts and haptocorrin in chronic myeloid leukemia.
The fate of beta-2-microglobulin was investigated in 17 patients with chronic myeloid leukemia by correlation analysis of the plasma concentration and various clinical measurements. The plasma concentration of beta-2-microglobulin correlated individually with white blood cell count, differential counts of band and segmented neutrophils, metamyelocytes and myelocytes, and haptocorrin concentration during the clinical course (p less than 0.002). The neutrophil granulocytes from the differential stage of myelocytes appears to be a major source of beta-2-microglobulin in chronic myeloid leukemia, and our results suggest that beta-microglobulin is is contained within the specific granules or is liberated from the plasma membranes synchronously with degranulation of neutrophils.
Adolescent↗
Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor.
The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.
Chemical Phenomena↗
Purification and partial characterization of a cobalamin-binding protein from chicken egg yolk.
Cobalamin-binding protein has been purified from chicken egg yolk by using DEAE-cellulose with a NaCl gradient. The resultant protein fraction was subjected to bioaffinity chromatography. The Mr was 38,000 by SDS-PAGE and 39,000 by gel filtration, and indicated that it was a glycoprotein. The Stokes radius was 4.3 nm and the pI 4.1. The protein bound 57CO.B12 with a molar ratio of 1:1 and a Kd of 0.41 microM. The CBP composed 296 amino acids residues. The protein-ligand interaction was inhibited by Cbl analogues.
Amino Acids↗
Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium.
We have recently reported that human neutrophils can be permeabilized with the cholesterol complexing agent saponin and that these cells can be induced to secrete the granule enzyme lysozyme in response to micromolar levels of free calcium. We now report that digitonin can be used in place of saponin and that it has several advantages. Permeabilization of human neutrophils was accomplished with 10 micrograms/ml digitonin in a high potassium medium. Normally impermeant solutes such as [14C]sucrose and inulin [14C]carboxylic acid gained access to one half of the intracellular water space marked with [3H]H2O. Between 30 and 100% of the cytoplasmic enzyme, lactate dehydrogenase, leaked from the intracellular space. The permeabilization process and calcium-triggered granule secretion were critically dependent upon temperature, time and digitonin concentration. Permeabilized neutrophils secreted beta-glucuronidase, lysozyme and vitamin B-12 binding-protein, constituents of both azurophil and specific granules, when exposed to micromolar levels of free calcium. Release of specific granule constituents appeared to be more sensitive to free calcium than release from azurophil granules. Although the amount of permeabilization varied considerably with each batch of cells, release of these granule markers was a consistent finding. Release of granule markers was accompanied by resealing of the cells to high-molecular-weight (Mr greater than 5000) solutes. Electron microscopic evidence also suggested that granule and plasma membranes were intact following digitonin treatment and that fusion of these membranes occurred in response to calcium. These results suggest that elevation of intracellular free-calcium levels is a sufficient condition for lysosomal enzyme release.
Calcium Chloride↗
The effects of heavy metal cations and sulfhydryl reagents on degranulation from digitonin-permeabilized neutrophils.
Digitonin-permeabilized neutrophils were exposed to micromolar levels of a variety of heavy metal cations and sulfhydryl oxidants to gain insight into the potential biochemical mechanisms underlying neutrophil degranulation. The results from this study suggest that the oxidation of intracellular sulfhydryl groups may play a role in neutrophil signal transduction. Evidence to support this conclusion is based on the observation that cupric phenanthroline and Cu2+/cysteine, agents reported to induce disulfide bond formation, evoke significant granule enzyme release when presented to permeabilized neutrophils. The stimulatory actions of these compounds occur in the absence of Ca2+ and are blocked by the sulfhydryl reducing agent, dithiothreitol. In addition, we observed marked potentiation of Ca2+-induced secretion by potentially physiological levels of Ni2+. Although we are unaware of any Ni2+-requiring enzymes in eukaryotic cells that are likely to be pertinent to degranulation, the ability of this divalent metal cation to lower the Ca2+ requirements for granule secretion suggests that it may play an important regulatory role in Ca2+-dependent processes. Finally, we observed significant granule release when permeabilized neutrophils were exposed to the heavy metal cations, Hg2+ and Ag+. The apparent stimulatory actions of these metals were the result of lysis rather than degranulation. Thus, the ability of these metals to lyse intracellular organelles such as lysosomal granules may contribute to their toxicological properties.
Calcium↗
The influence of net surface charge on the interaction of uropathogenic Escherichia coli with human neutrophils.
Escherichia coli strains, grown to suppress fimbrial expression, synthesised enhanced quantities of polysaccharide capsule, which significantly lessened their binding to heparin sepharose columns. In the presence of poly-L-lysine, these strains were strongly retained on the columns confirming their highly anionic nature. Uropathogenic strains of E. coli expressing type 1 fimbrial adhesins activated the respiratory burst, the degranulation response and the release of leukotrienes from human neutrophils (PMN) to a significantly greater extent than the same strains grown in a medium to suppress this fimbrial expression. The addition of the poly-cation poly-L-lysine, however, selectively increased neutrophil activation in response to these non-fimbriate strains. This dose-dependent effect was reversed by the addition of heparin suggesting a mechanism dependent on surface charge. The results of this study suggest that non-specific mechanisms involving the neutralisation of surface charge, in addition to specific receptor and adhesin mediated events could affect neutrophil activation at sites of infection.
Cell Membrane↗
Ca2(+)-induced secretion by electropermeabilized human neutrophils. The roles of Ca2+, nucleotides and protein kinase C.
Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2(+)-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP beta S ATP gamma S, and App[NH]p were effective. GTP gamma S and GDP beta S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 degrees C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Effect of glycemic control on vitamin B12 metabolism in diabetes mellitus.
To determine the effect of glycemic control on vitamin B12 (B12) metabolism in diabetes mellitus, we studied B12 metabolism in 19 diabetic patients with poor glycemic control and 15 normal individuals. The diabetic patients had significantly higher total B12 binding capacity (3303 +/- 963 pg/ml), higher serum B12 levels (1173 +/- 503 pg/ml) and unsaturated B12 binding capacity (2131 +/- 902 pg/ml) when compared with the normal controls, but there was no difference in R-binder levels and the B12 binding ratio between the two groups. During a 2-week admission to establish glycemic control, the fructosamine levels in the diabetic patients decreased from 556 to 428 mumol/l and the total B12 binding capacity as well as unsaturated B12 binding capacity were significantly improved to the normal range (P < 0.01), but serum B12 levels, R-binder levels and the B12 binding ratio were not changed. There was a significant association between serum fructosamine levels and the total B12 binding capacity in poorly controlled diabetic patients and the decrease of fructosamine was correlated significantly with the change of total B12 binding capacity and serum B12 levels in diabetic patients. These results indicate the effects of glycemic control on B12 metabolism in diabetes mellitus.
Adult↗
Purification and properties of chicken egg-white cobalamin-binding protein.
A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities.
Amino Acid Sequence↗
A derivative of R-type cyanocobalamin binding proteins in the human intestine. A candidate antibacterial molecule.
Human jejunal fluid contains a protein which has a molecular radius of 3.37 nm, an Mr or 60600 and a mean pI of 3.35 and binds cobalamin with a K of 0.1 . 10(9) l/mol. This protein also couples cobalamin analogues lacking the nucleotide moiety, cross-reacts with R-type protein and is resistant to proteolysis in the intestine. These findings refute the hypothesis that cobalamin-analogue binders are present and effectively inhibit the bacterial uptake of analogues in the intestine.
Anti-Bacterial Agents↗
In vivo evidence that intrinsic factor-cobalamin complex traverses the human intestine intact.
Ingested cyano[57Co]cobalamin was recovered as coupled to intrinsic factor from the jejunum of healthy humans. This vitamin-protein complex and the analogous complex from patients having exocrine pancreatic insufficiency were indistinguishable from each other in terms of molecular radius (3.30 nm), ionic nature (eight well-defined isoproteins isoelectric at pH 4.71, 4.84, 4.90, 5.13, 5.23, 5.31, 5.40 and 5.69), and antigenic structure. These findings indicate that the pancreatic proteases do not alter the intrinsic factor cobalamin complex in vivo. Purified R type protein-cyano [57Co]cobalamin complex recovered from patients with exocrine pancreatic insufficiency was similar to the analogous gastric protein in terms of molecular radius (alpha = 4.78 nm) and types of isoproteins (seven well-defined isoproteins isoelectric at pH 2.70, 3.03, 3.38, 3.74, 3.87, 4.05 and 4.20). However, this R protein complex from patients and the intrinsic factor complex from both control subjects and from patients was comprised of more of the acidic type of isoproteins, thereby supporting the notion that carbohydrate-rich isoproteins are more resistant to digestion in the intestine.
Biological Transport↗