Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Variant-specific differences in human unsaturated transcobalamin II

Electrophoresis and subsequent autoradiography of ⁵⁷Co-cobalamin (⁵⁷Co-Cbl)-labeled serum show intensity differences between the genetic variants of human transcobalamin II (TC2), suggesting differences in the unsaturated (apo-) TC2 concentration. In order to distinguish between variant-specific differences in the Cbl binding affinity and those in the total-TC2 concentration, techniques were developed to determine total, apo-, and holo-TC2. Prolonged incubation at 37° C with a 20-fold excess of ⁵⁷Co-Cbl resulted in an almost complete exchange of endogenously bound Cbl, which allowed determination of the total TC2. The holo-TC2 concentration of both gene products in TC2 heterozygotes could be estimated by comparison of the labeling levels of apo- and total TC2, using densitometric quantification of the autoradiographs. By means of ion-exchange chromatography, TC2 could be separated from other Cbl-binding proteins, permitting a simple quantitative assay of apo- and total TC2, the results of which correlate fairly well with those measured by an immunoadsorption assay. The results obtained in the present investigation indicate that the variant-specific variation in the apo-TC2 concentration is caused by differences in the total-TC2 concentration rather than in the Cbl binding affinity.     

Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.     

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

High affinity binding of the transcobalamin II–cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC–[⁵⁷Co]cyanocobalamin complex with a dissociation constant (K_d) of 4.9×10⁻¹¹ M. Uptake of the TC–[⁵⁷Co]cyanocobalamin complex at 37°C was saturable by 24 h. Binding of free [⁵⁷Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC–[⁵⁷Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC–cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC–cobalamin complex.     

Failure to detect beta-leucine in human blood or leucine 2,3-aminomutase in rat liver using capillary gas chromatography-mass spectrometry

It has been reported (Poston, J. (1976) J. Biol. Chem. 251, 1859-1863; (1982) 255, 10067-10072; (1984) 259, 2059-2061) that mammalian tissues contain an adenosylcobalamin-dependent enzyme, leucine 2,3-aminomutase, which catalyzes the interconversion of beta-leucine and leucine. It was also reported that beta-leucine is detectable in normal human serum (mean = 4.8 mumol/liter, n = 37) and is elevated in serum from patients with cobalamin deficiency (mean = 24.7 mumol/liter, n = 17). Serum levels of leucine were claimed to be decreased in the cobalamin deficient patients (mean = 52 mumol/liter) as compared with the normal subjects (mean = 81 mumol/liter). It was also reported that rat liver supernatant catalyzed the formation of beta-leucine, leucine, or both amino acids from iso-fatty acids, and that the generation of leucine from iso-fatty acids was stimulated by adenosylcobalamin and inhibited by unsaturated cobalamin-binding protein. We have synthesized t-butyldimethylsilyl derivatives of beta-leucine and leucine and have used capillary gas chromatography-mass spectrometry for their analysis. Using forms of beta-leucine and leucine that contain several deuterium atoms in place of several hydrogen atoms as internal standards, techniques have been developed which make it possible to detect and quantitate as little as 0.1 mumol/liter of beta-leucine or leucine in human serum and in incubations containing rat liver supernatant. beta-Leucine was not detectable, i.e. less than 0.1 mumol/liter, in any sera from 50 normal human subjects or in any sera from 50 cobalamin-deficient patients. The mean level of leucine in the 50 cobalamin-deficient sera was 219 mumol/liter, which was not decreased with respect to that in the 50 control sera (167 mumol/liter). Experiments in which beta-leucine, leucine, isostearic acid, or isocaproic acid were incubated with rat liver supernatant in the presence or absence of adenosylcobalamin or cobalamin-binding protein failed to demonstrate the formation of leucine or beta-leucine or their interconversion under any of the conditions studied. We conclude that beta-leucine is not present in human blood and that the existence of leucine 2,3-aminomutase in mammalian tissues remains to be established.     

High affinity binding of the transcobalamin II-cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC-[(57)Co]cyanocobalamin complex with a dissociation constant (K(d)) of 4.9 x 10(-11) M. Uptake of the TC-[(57)Co]cyanocobalamin complex at 37 degrees C was saturable by 24 h. Binding of free [(57)Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC-[(57)Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC-cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC-cobalamin complex.     

Congenital disorders of vitamin B12 transport and their contributions to concepts. II.

Congenital deficiencies of Transcobalamin II (TC II) and R binders of vitamin B12 (B12, cobalamin, Cbl) have been described in several families. The deficiency of TC II exists as at least three variants. The deficiency of TC II is expressed by a profound megaloblastic pancytopenia during the first few weeks of life, but the serum Cbl is normal. In contrast, the deficiency of R binder is asymptomatic, tissues are replete in Cbl, but the serum Cbl is low. All of the R binder in the several body sources is under the same genetic control. Studies of the congenital deficiency TC II suggest the following: (1) The function of TC II is the promotion of cell uptake of physiologic amounts of Cbl, which can also be accomplished by very large amounts of Cbl, and not in any intracellular process. (2) TC II is essential for the absorption, postabsorptive distribution, and recycling of TC II. (3) The metabolic consequences of TC II deficiency are expressed primarily in rapidly dividing cells probably because they are dependent upon the constant need for new Cbl.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Specific pancreatic beta-cell surface antigens recognized by a xenogenic antiserum

An antiserum (R4) from a rabbit immunized with suspensions of C57BL/61 ob/ob mouse islet cells contains antibodies which in a 125I-protein A radioligand assay can be demonstrated to bind to single cell suspensions of normal Naval Medical Research Institute (NMRI) mouse islet cells. The binding of 125I-protein A to islet cells was about four times that of normal rabbit serum (NRS) after incubation at a 1/600 dilution of R4 antiserum quantitatively absorbed to mouse spleen lymphocytes (R4A antiserum) and hepatocytes. Subsequent absorption of the R4A antiserum to islet cells significantly reduced the binding of 125I-protein A to islet cells incubated with the doubly absorbed serum. Immunoprecipitation of radiolabeled islet cell lysates followed by SDS polyacrylamide gel electrophoresis and autoradiography suggested that the R4A antiserum recognized a Mr 40,000 glycoprotein. This glycoprotein was not detected in spleen lymphocytes. Electron microscope detection of gold-protein A complexes suggested that the binding of islet cell surface antibodies was cell specific. islet cell suspensions incubated with R4A antiserum and gold-protein A showed that 86 +/- 3 gold particles were bound per 100 beta-cells (mean +/- SE for six experiments). In contrast, the number of gold particles per 100 endocrine non-beta-cells was 8 +/- 1 which was similar to the number achieved with NRS (3 +/- 1) on all endocrine islet cells. Our observations suggest that the pancreatic islet cells, in particular the beta-cells, express a specific antigen.     

Immunoelectron microscopic study of somatostatin biosynthesis in dog pancreas

The biosynthesis of somatostatin has been studied at the ultrastructural level in pancreatic islets by using rabbit antiserum against synthetic somatostatin. To document that the antiserum specifically bound preprosomatostatin, we have tested the ability of the antiserum to precipitate the product synthesized in vitro. Poly(A) enriched RNA isolated from catfish islets was translated in both the wheat germ extract and nuclease-treated reticulocyte lysate systems. It was found that the in vitro translation product, preprosomatostatin, could be recognized by the antibody against synthetic somatostatin. The morphological study was then performed by immunoelectron microscopy by using the Fab-peroxidase conjugate technique. In dog pancreatic islets, somatostatin immunoreactive reaction product was seen only in the delta cells. In these cells, they were detected on bound ribosomes, in the cisternae of the rough endoplasmic reticulum (ER) and Golgi apparatus, in the Golgi associated vesicles, and in secretory vesicles. These findings suggest that somatostatin precursor molecules are synthesized on bound ribosomes and discharged into the cisternae of the rough ER. They are then transported to the Golgi apparatus and transferred to the secretory vesicles for secretion. The different staining intensities in the secretory vesicles would suggest that the processing of the precursor molecules of somatostatin probably takes place in the secretory vesicles.     

Specific antisera suitable for solid-phase radioimmunoassay of 11beta-hydroxyandrost-4-ene-3,17-dione

Specific antiserum has been developed for use in measuring 11β-hydroxyandrost-4-ene-3, 17-dione by radioimmunoassay (RIA). Rabbit antiserum was generated by employing the conjugate prepared by coupling 6β,11β-dihydroxyandrost-4-ene-3,17-dione 6-hemisuccinate with bovine serum albumin. The antiserum bound 68% of 50 picograms of 11β-hydroxyandrost-4-ene-3,17-dione-[1,2,6,7-³H] during characterization at a dilution of 1:12,500. Among the numerous steroids tested for cross-reactivity, 5α-androstane-3,17-dione, androst-4-ene-3,17-dione, and 11β-hydroxy-5α-androstane-3, 17-dione showed 2%, 5%, and 30% cross-reactivity respectively. The Rivanol-treated antiserum was coupled to Enzacryl AA, in order to study the feasibility of a solid-phase RIA, and this complex showed 50% binding with the labeled antigen at a dilution of 1:3000. The complex retained high specificity and should prove useful in a simple solid-phase RIA.     

III. Serum and sputum immunoglobulin E levels in respiratory diseases in adults

Using a solid-phase radioimmunoassay, serum IgE level was determined in 46 normal subjects, 53 patients with bronchial asthma, 44 patients with chronic bronchitis and / or emphysema, and 19 patients with restrictive lung disease. Sputum IgE was measured simultaneously in 51 of the subjects. The range of serum IgE concentration in the normal subjects was wide. It varied between 15 and 750 ng/ml with a mean of 135 ng. Asthmatic patients had significantly higher levels of serum IgE with a mean of 579 ng/ml, but only 30% fell outside the normal 95% confidence limits. Patients with chronic bronchitis, emphysema and restrictive lung diseases had normal IgE levels. There was a significant correlation between serum and sputum IgE levels.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone

A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simultaneously incubating zearalenone with zearalenone antiserum over zearalenone-6'-carboxymethyloxime poly-L-lysine solid phase and then determining the bound rabbit immunoglobulin with goat anti-rabbit peroxidase conjugate. Response range for zearalenone in the resulting competition curve was between 1 and 50 ng/ml. Reactivities of this antiserum for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were, respectively, 50, 12, 6, and 3% of that found for zearalenone. By using the competitive indirect ELISA, zearalenone was detectable in methanol-water extracts of corn, wheat, and pig feed samples.     

Immunoassay of 5-methyltetrahydrofolate: Use of 125I-labeled Protein A as the tracer molecule for specific antibody

A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. ¹²⁵I-Labeled staphylococcal Protein A (¹²⁵I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of ¹²⁵I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific: 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15–20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits.     

Conformational changes of transcobalamin induced by aquocobalamin binding. Mechanism of substitution of the cobalt-coordinated group in the bound ligand

Binding of aquo-, cyano-, or azidocobalamin (Cbl.OH(2), Cbl.CN, and Cbl.N(3), respectively) to the recombinant human transcobalamin (TC) and haptocorrin from human plasma was investigated via stopped-flow spectroscopy. Association of cobalamins with haptocorrin always proceeded in one step. TC, however, displayed a certain selectivity for the ligands: Cbl.CN or Cbl.N(3) bound in one step with k(+1) = 1 x 10(8) M(-1) s(-1) (20 degrees C), whereas binding of Cbl.OH(2) under the same conditions occurred in two steps with k(+1) = 3 x 10( 7) M(-1) s(-1) (E(a) = 30 kJ/mol) and k(+2) = 0.02 s(-1) (E(a) = 120 kJ/mol). The second step of Cbl.OH(2) binding was interpreted as a transformation of the initial "open" intermediate TC.Cbl.OH(2) to the "closed" conformation TC(Cbl) with displaced water. The backward transition from the closed to the open conformation was the reason for the identical rate-limiting steps during substitution of H(2)O in TC.Cbl.OH(2) for cyanide or azide according to the reaction TC(Cbl) --> TC.Cbl.OH(2) + CN(-)/N(3)(-). The cyano and azido forms of holo-TC which were produced behaved as the open proteins. Different conformations of holo-TC, determined by the nature of the active group in the bound Cbl, may direct transportation of cobalamins in the organism.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.