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An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs. We observed the known interactions of MS with MSR and with MMACHC as well as MMADHC with MMACHC, but we also observed novel interactions for MSR with MMACHC and with MMADHC and MS with MMADHC. Furthermore, we show that the absence of MS or MMACHC expression disrupts the interactions between the other interactome members, in cblC and cblG fibroblasts and in HepG2 cells transfected with siRNAs. Our data show that the processing of Cbl in cytoplasm occurs in a multiprotein complex composed of at least MS, MSR, MMACHC and MMADHC, which could contribute to shuttle safely and efficiently Cbl towards MS. Our data suggest that defective protein-protein interactions among key players of this pathway could contribute to the molecular mechanisms of the cblC, cblG and cblE genetic defects and provide novel insights into our understanding of the pathophysiology of inherited disorders of Cbl metabolism.

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to acetaldehyde and ammonia. The radical intermediate observed during steady state turnover of substrate aminoethanol has been characterized by continuous wave electron paramagnetic resonance (EPR) spectroscopy [J. Am. Chem. Soc. 121 (1999) 10522]. This study presents simulations of EPR spectra of this radical intermediate. Quantitative fits to the EPR spectra are achieved with a model of isotropic exchange and magnetic dipolar interaction between the substrate-derived radical and the Co II in the corrin ring. The simulated parameters are compared with those of substrate analog 2-aminopropanol-derived radical in the same enzyme. The comparison confirms that the aminoethanol-derived product radical interacts more weakly with the Co II than the 2-aminopropanol-derived radical and suggests that the reduction of isotropic exchange between the aminoethanol-derived product radical and the Co II is probably due to orientational-dependent wave function overlap. Successful fits to the radical line shapes of different isotope substitutions unequivocally establish that the observed radical intermediate is an π-electron-based product radical. The derived principal hyperfine values for the 13 C α and 1 H α nucleus are consistent with previous electron nuclear double resonance (ENDOR) studies on similar radicals, thus providing reliable experimental hyperfine coupling constants for comparison with quantum mechanical-based calculations to gain further insight into the molecular structure of the observed radical.

Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P

The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B12 analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The Stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displaze human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

The uptake and metabolism of cobalamin (Cbl) has been studied in L-1210 murine leukemia cells propagating in vitro. Extracellular Cbl (protein bound and free) and intracellular Cbl (protein bound and free) were determined after culturing L-1210 cells in the presence of [57Co]cyanocobalamin (CN-Cbl) bound to transcobalamin II (transcobalamin, TC). The intracellular pool of free [57Co]Cbl increased during the first 24 h of culture and a substantial fraction of this free pool was effluxed from the cell to the medium. Upon depletion of extracellular TC-[57Co]CN-Cbl, the intracellular concentration of free Cbl decreased as did the efflux of Cbl to the medium. Internalized [57Co]CN-Cbl was converted to hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl) and 5'-deoxyadenosylcobalamin. These Cbl forms were found in both soluble (cytoplasmic) and insoluble (membrane) fractions. Intracellular protein-bound [57Co]Cbl fractionated with methionine synthase (MS) and methylmalonyl-CoA mutase (MU) activity. The major form of Cbl associated with the two enzymes was OH-Cbl. Cells propagated in medium containing N5-methyltetrahydrofolate and homocysteine showed a substantial increase in MS activity which paralleled the increase in the intracellular concentration of Me-Cbl and the Cbl bound to the enzyme.

Two enzymes involved in the intracellular metabolism of cobalamin have been identified and characterized: cyanocobalamin β-ligand transferase and microsomal cob(III)alamin reductase. The β-ligand transferase is a cytosolic enzyme utilizing FAD, NADPH and reduced glutathione. The product of the reaction has been identified as glutathionylcobalamin. NADH-linked cob(III)alamin reductase has been found in two subcellular fractions: microsomal and inner mitochondrial membrane. The product of the reduction catalyzed by the microsomal enzyme has been identified as cob(II)alamin. In cbl C mutant fibroblasts, the specific activities of cyanocobalamin β-ligand transferase and cob(III)alamin reductase were markedly decreased and have varied from 3%–30% and 36%–42% of normal, respectively. The specific activity of mitochondrial cob(III)alamin reductase was only 30% of normal in two cbl C mutants and normal in remaining mutant cell lines. In the cbl D cells, the specific activities were 33% and 55%. Mitochondrial cob(III)alamin reductase was not affected by cbl D mutation. Methionine synthase, L -methylmalonyl-CoA mutase and microsomal cytochrome c and b5 reductases are not affected by both mutations. The cbl E mutation affects only the activity of methionine synthase. These results support the hypothesis that the early enzymatic steps of intracellular metabolism of cobalamin are similar in the synthesis of both methylcobalamin and adenosylcobalamin and these steps are altered by the cbl C and cbl D mutations.

Intrinsic factor-cobalamin receptor (IFCR) activity in visceral yolk sac and placental membranes is regulated during pregnancy in rats. While the IFCR activity declined in the visceral yolk sac membranes by 15-fold, it rose nearly 20-fold in the placental membranes from fourteen to nineteen days of gestation. The visceral yolk sac membranes revealed a 230 kDa protein that co-migrated with pure rat renal IFCR. This 230 kDa band was also identified as IFCR in both the membranes by immunoblotting with anti-serum to rat renal IFCR. Immunoprecipitation of 35S labeled proteins obtained from in vitro translation using visceral yolk sac mRNA from 14-day pregnant rats, yielded on SDS-PAGE a single band of 220 kDa, while those obtained from 19-day pregnant rats did not. The binding of intrinsic factor-cyanol[57Co]cobalamin complex to the visceral yolk sac membranes was inhibited by princubation of these membranes with anti-serum to rat IFCR but not with anti-serum to rat asialoglycoprotein receptor or mannose or mannan or N-acetylglucosamine. Based on these results, we suggest that the IFCR activity, protein expression and mRNA levels in fetal membranes are regulated during pregnancy and may play an important role in the maternal-fetal transfer of cobalamin.

Two intermediate stages in cobalamin biosynthesis, amidation of carboxylic acid groups in the corrin ring and (R)-1-amino-2-propanol attachment at propionic acid position ƒ, have been studied using cell-free extracts from the obligate anaerobe Clostridium tetanomorphum. The preparation of an incomplete corrinoid, probably cobinic acid-a,c,d,e,g-pentaamide, as an in vitro amidation substrate was accomplished via mild acid hydrolysis of cobinamide. Weak, but reproducible activities for both amidation and (R)-1-amino-2-propanol attachment were found in crude, nucleic acid-free and DE-52 column-purified protein fractions. The amidation reaction was glutamine-dependent in crude fractions, but became ammonium ion-dependent in more purified fractions. Significant problems encountered were (a) the weak and unstable character of both enzyme activities, and (b) the irreversible changes in the visible spectra of the incomplete corrinoids employed as substrates caused by use of thiol-reducing agents in the buffers and assays.

Assay of the reactivity between the chicken anti-rabbit transcobalamin II antiserum and the sera of 19 vertebrate species was carried out by both immunodiffusion and Sephadex G-200 gel filtration column chromatography. Mammalian transcobalamin II cross-reacted with the antiserum whereas the serum vitamin B-12 binders of the bird, amphibian, reptile and fish did not. The biological activity of the purified rabbit transcobalamin II was assessed using reticulocytes or erythrocytes of human, rabbit, guinea pig and rat. The purified rabbit transcobalamin II promoted the uptake of vitamin B-12 by the cells but showed a great variation in its activity. It is suggested that the rabbit transcobalamin II is immunologically and biologically similar to the serum transcobalamin II of the mammalian species studied.

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[ 57 Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H + production as measured by amino[ 14 C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H 2 receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10 6 cells per h under basal conditions and 14.3 fmol in response to histamine.

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark. During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G 1 phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G 2 . In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.

Inhalation of nitrous oxide oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamn-dependeent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N2O but was restored to pre-N2O levels after 6 days despite continuing N2O exposure. Brain methionine fell within 12 h of N2O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homoysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N2O inhalation was continued. There was an initial rise in liver S-adenosyl-methionine levels followed by a steady fall to 40% of its initial level after 11 days of N2O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.

Rat intrinsic factor was bound to vitamin B-12-Sepharose to produce intrinsic factor-vitamin B-12-Sepharose. Intestinal receptor for intrinsic factor-vitamin B-12 complex was purified from rat ileal extract by affinity chromatography using the intrinsic factor-vitamin B-12-Sepharose as an affinity adsorbent with recovery of 48.5% and specific activity increased 335 fold of original sample.

An investigation has been made of the effect of varying pH at constant ionic strength on vitamin B 12 binding by human serum and by two transcbalamin fractions separated from serum by gel filtration. It was found that the methodology used had a considerable influence on the results obtained. Genuine effects of pH were largely confined to reduced vitamin B 12 binding at very acid and very alkaline pH. However, due to an adsorption artefact involving transcobalamin II, certain methods appeared to demonstrate a marked decrease in vitamin B 12 binding between pH 4.5 and pH 10.3, especially in the range of pH 5.3–7.5.

A novel procedure for the purification of methylmalonyl-CA mutase from Propionibacterium shermanii has been described which employs affinity chromatography on a column of immobilized vitamin B-12 linked covalently to Sepharose. The method has the advantage of being simple and rapid, thus enabling the purification of the enzyme to near homogeneity with good yields.

The one-electron reduction of vitamin B1 2 and its coenzyme has been studied by γ-irradiating the frozen methanolic solutions of these compounds. A new optical absorption spectrum was obtained by γ-irradiation of aqueous methanol solution of vitamin B1 2 at 77°K; this spectrum changed by slight warming to that of B1 2 r. ESR spectra were also recorded. The ESR signal at g ≈ 2.3 for the irradiated solution at 77°K was ascribed to the vitamin B1 2 anion, in which an unpaired electron is localized in the σ★ orbital of the cobalt-ligand bond. Upon slight warming the ESR signal changed to that of B1 2 r. In contrast to the vitamin, the ESR spectrum of irradiated coenzyme B1 2 solution in C2 H3 O2 H3 + 2H2 O at 77°K showed no signal due to a paramagnetic cobalt complex but an asymmetric signal of g ≈ 2.0, which is attributable to an organic radical presumably derived from the adenosyl group. The optical spectrum indicates the formation of B1 2 s (diamagnetic) by the CoC bond cleavage. Upon slight warming the optical and ESR spectra changed to those ascribed to B1 2 r. The present study thus revealed that the one-electron reduction of vitamin B1 2 and its coenzyme B1 2 gives rise to the heterolytic cleavage of the CoC bonds by the selective attack of electrons, and the former produces B1 2 r and the latter, B1 2 s.

Purification of ethanolamine ammonia-lyase (EC 4.3.1.7) from a Clostridium sp. grown at the University of Sussex, U.K. and the National Institutes of Health, U.S.A., has been compared and an improved isotopic assay for the enzyme has been developed. Successful purification of this enzyme from Sussex-grown cells requires modification of the published procedure (Kaplan and Stadtman (1968) J. Biol, Chem. 243, 1787-1793) principally a 70% decrease in volume during precipitation with 0.4 M NaCl. This modification also increases the yield from N.I.H.-grown cells. Purified enzyme, resolved of inactive cobalamins, has the same high specific activity from both sources and behaves in the same way on disc gel electrophoresis. Sussex enzyme, before resolution, has less than 20% of the specific activity of unresolved N.I.H. enzyme and contains over 50% more inactive cobalamin. The bound cobalamin from both preparations has been identified as a "base-on" Co11 psi-cobalamin.

The uptake of intrinsic factor-cobalamin complex by guinea pig ileal brush borders is inhibited by particle-free sonicates of distal but not proximal brush border preparations. This finding suggests that sonication releases intrinsic factor receptor from ileal brush borders in a form which is not sedimented by centrifugation for 1 h at 105 000× g .

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.

Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.

The effect of inactivation of cobalamin by N2O in the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-14C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl[14C]tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N2O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N2O but fell significantly after 7 days exposure. There was a significant fall in the amount of formlytetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.

We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B12-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B12r. By varying the magnitude of the exchange of coupling we have quite accurately simulated the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammon-ia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.

Initial rates of 59Fe3+ uptake by mouse duodenal fragments (in vitro) and tied-off duodenal segments (in vivo) have been characterised for control and hypoxic animals. 59Fe3+ uptake by duodenal fragments was rapid, selective and dependent on medium Fe3+-nitrilotriacetate concentration. Most of the 59Fe3+ uptake (70–75%) occurred via the mucosal route and was dependent on the metabolic state of the tissue. Mucosal uptake showed an adaptive increase following exposure of animals to 3 days hypoxia; the enhancement was due to a 2–3-fold increase in Vmaxapp, without any significant changes in the Kmapp. Studies of upper small intestine transit times showed a mean residence time of 4–5 min for 59Fe-labelled mouse chow, emphasising the importance of initial uptake measurements. Time courses for in vivo total mucosal uptake exhibited linearity over a wide variety of absorption rates after correction for the permeation by intact metal-chelate complex. The corrected uptake showed a hyperbolic dependence on medium Fe3+-nitrilotriacetate concentration. Kinetic studies revealed a 2–3-fold increase in total mucosal uptake in hypoxia. Mucosa-to-carcass transfer of 59Fe was also markedly increased by chronic hypoxia. The in vitro system exhibits similar qualitative and quantitative kinetics for Fe3+ transport via the mucosal membrane to those obtained in vivo. The results observed in vitro are thus valid and provide a convenient method for further studies on Fe3+ transport in animals and in man.

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 with 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 the half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.

1. 1. Methylcobalamin, adenosylcobalamin, hydroxocobalamin and cyanocobalamin have been estimated by a chromato-bioautographic technique in 16 tissues from healthy rats and in five guinea pig tissues. 2. 2. Plasma and erythrocyte cobalamins have been estimated in rats, cats and guinea pigs and the results compared with those in man. 3. 3. Unidentified corrins were detected in 8 of the 16 rat tissues and in 3 of the 5 guinea pig tissues analysed, but were not present in tissues from specific pathogen-free rats nor in the standard laboratory diet. 4. 4. Adenosylcobalamin was the major corrin in 8 of the 16 rat tissues. In the remainder hydroxocobalamin predominated or was present in equal proportions with adenosylcobalamin. Methylcobalamin was detected in the majority of rat tissues but at levels much lower than those in human tissues. Small amounts of cyanocobalamin were detected also and levels were higher than those of methylcobalamin in 8 of the 16 tissues. 5. 5. In the rat, cat and guinea pig, levels of methylcobalamin and hydroxocobalamin were higher in erythrocytes than in plasma, a pattern almost the complete reverse of that in man.

To avoid using protein-denaturing agents for desorption, when purifying cobalamin-binding protein by biospecific affinity chromatography, an affinity column has been prepared where cobalamin is attached through a temperature-labile linkage to insolubilized 3, 3'-diaminodipropylamine. On desorption the protein is obtained in solution saturated with cobalamin. The method has been used for purification of intrinsic factor and transcobalamin I.

1. 1. 72 h uptake of cyano [57Co]cobalamin and formation of 57Co-labelled methylcobalamin, adenosylcobalamin and hydroxocobalamin has been estimated with and without the addition of methylcobalamin analogues in phytohaemagglutinin-stimulated lymphocytes from healthy human subjects. 2. 2. Difluorochloromethylcobalamin reduced cell uptake of cyanocobalamin and caused a disproportionated reduction in synthesis of adenosylcobalamin. 3. 3. Methylcobalamin-palladium trichloride reduced cell uptake of cyanobalamin more effectively than did difluorochloromethylcobalamin and reduced the formation of methylcobalamin, adenosylcobalamin and hydroxocobalamin in proportion. 4. 4. The results that in addition to inhibiting uptake of cyanocobalamin, one or both compounds may have interfered directly with the mechanism of synthesis of the cobalamin coenzymes.

A 340 000-dalton component “C-III” was found when Triton X-100-containing extracts of ileal mucosa were incubated with human or porcine intrinsic factor vitamin B-12 preparations. It was not formed when abnormal human intrinsic factor, unable to attach to the intrinsic factor receptor, was used. Prolonged storage promoted the transfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor receptor species C-I and C-II. The component was also present in ileal extracts prepared with or without detergent and it bound vitamin B-12 directly. Immunologically and by electrofocusing it could be classified as a cobalophilin but its molecular dimensions were larger than described for cobalophilin. It thus represents a novel vitamin B-12 binding protein, possibly a macromolecular acceptor of vitamin B-12 which accepts vitamin B-12 bound via intrinsic factor to the ileal intrinsic factor receptor. In the presence of EDTA or at low pH, vitamin B-12-intrinsic factor did not bind to any of the receptor species and under the same conditions it could all be dissociated from the receptor complexes but not from C-III. The dissociated receptor was able to recombine with vitamin B-12-intrinsic factor and it appeared to bind free and vitamin B-12-bound intrinsic factor in vivo.

The vitamin B-12-intrinsic factor receptor was shown to be present in pig ileum and localized on the brush borders of the enterocytes, and to be solubilisable with Triton X-100. At neutral pH and in the presence of Ca2+ it bound the vitamin B-12-intrinsic factor but not the vitamin B-12-cobalophilin complex. The solubilised vitamin B-12-intrinsic factor-receptor complex consisted of two molecular species with clear Stokes radii (13.11 and 33.53 nm), sedimentation coefficients (15.1 and 45.1 S) and molecular weights (1 600 000 and 12 000 000). A third smaller macromolecule possibly also represented the receptor. Some receptor activity was present in extracts prepared with buffers lacking detergent. There was evidence that the receptor is a membrane lipoprotein from which the lipids reversibly dissociate. Free intrinsic factor also bound to the solubilized receptor and its vitamin B-12-binding site seemed not to be involved in the attachment to the receptor. A small portion of the vitamin B-12-intrinsic factor spontaneously dissociated from the receptor and nearly all dissociated in the presence of Na2-EDTA. The Stokes radius of the dissociated vitamin B-12-intrinsic factor complex was 0.17 nm smaller than before binding to the receptor. Intrinsic factor and cobalophilin were present in ileal extracts and observations were made on their molecular characteristics. These proteins, polymers of the vitamin B-12-intrinsic factor complex and binding of vitamin B-12 and its protein complexes to detergent micelles may give spurious receptor-like effects which must be properly controlled.

Rabbit haptocorrin (R-binder) has been purified from serum by affinity chromatography on cobalamin-Sepharose and Blue Sepharose. It proved to be a protein with a relative molecular mass of 60 000 and an amino acid content very similar to that of other haptocorrins (Nexo, E. and Olesen, H. (1981) in B12 (Dolphin, D., ed.), Wiley Interscience, New York/London, in the press). The pH optimum (pH 6–9) for binding of cyanocobalamin and the affinity to dicyanocobinamide were like those of human and hog haptocorrins. In spectral studies, the extinction coefficient of cyanocobalamin at 363 nm (γ1-band) increased by about 16% on binding to rabbit haptocorrin. Binding of azidocobalamin gave spectra changes similar to those for binding to rabbit transcobalamin.

Vitamin B-12 is released from the purified gastric intrinsic factor-[ 57 Co]vitamin B-12 (intrinsic factor- [ 57 Co]vitamin B-12) complex, when incubated with rat intestinal mucosa. Maximum specific activity for splitting the complex is localized in ileal brush border. Release of [ 57 Co]vitamin B-12 is not due to its mere exchange during incubation with endogenous non-radioactive vitamin B-12. The splitting process has specific requirement for Ca 2+ and ATP and it is thermolabile, time- as well as temperature-dependent. It is also inactivated by the presence of p -chloromercuribenzoate. Further, the vitamin B-12-releasing factor has been isolated from solubilized brush border and is purified 70-fold by (NH 4 ) 2 SO 4 precipitation, gel filtration and Con. A-Sepharose 4B affinity chromatography. In SDS-polyacrylamide gel electrophoresis, it is resolved into a single band of about 25 kDa, indicating its purity. The releasing factor exhibits maximum activity at pH 7.4; isoelectric focusing reveals only one major form with p I 7.52. With intrinsic factor-[ 57 Co]vitamin B-12-complex as the substrate, apparent K m and V max values obtained are 128.2·10 −12 M/1 and 117.6 pg·h −1 100 μg protein, respectively. Amino acid and carbohydrate analyses reveal the glycoprotein nature of the factor. Intrinsic factor-[ 57 Co]vitamin B-12 complex is not susceptible to unspecific proteolytic digestion/ Similarly, the releasing factor does not hydrolyse other proteins. Thus, the observed substrate-specificity of the releasing factor differentiates it from other known proteolytic enzymes of ileal brush borders.

The vitamin B- 12 binding proteins in Rhesus and African Green monkey serum and red cells were isolated by ion-exchange and molecular sieve chromatography. Vitamin B 12 added in vitro to monkey serum and lysed red cells was bound by monkey Transcobalamin II and a red cell binder, respectively, and these vitamin B 12 binding substances cross-reacted with antisera to their human homologues. Most of the native vitamin B 12 in monkey serum was also attached to monkey Transcobalamin II rather than to Transcobalamin I which is the predominant binder of native vitamin B 12 in human serum.

The subcellular distribution of cobalamin during absorption in the dog ileum has been studied using analytical subcellular fractionation. Animals dosed orally with cyano[57Co]cobalamin were killed 2 h later, and postnuclear supernatant fractions prepared from homogenates of the ileal mucosa were subjected to isopycnic centrifugation on reorientating sucrose density gradients. Marker enzymes for the principal subcellular organelles and cyano[57Co]cobalamin were assayed in the gradient fractions. At 2 h, the distribution of cyano[57Co]cobalamin exhibited a major lysosomal localisation with only 30% of the counts being recovered in the soluble fractions. This observation was confirmed by preparing postnuclear supernatant fractions in digitonin, which selectively disrupted lysosomes and released their contents into the soluble fractions. Lysosomal localisation during passage through the ileal enterocyte strongly supports absorption of cobalamin by a process of receptor-mediated endocytosis in the dog.

By a series of chromatographic and gel-filtration steps, the 2 components in human gastric juice carrying intrinsic-factor activity, binders S and I, were prepared as their cyanocobalamin complexes. The former was found to be homogeneous and the latter almos pure. Complex S has a mol. wt. of about 115 000 and contains 2 moles of cobalamin per mole and about 13% carbohydrate. Complex I is somewhat smaller and is apparently a degradation product of S, the original intrinsic factor. Both complexes are probably polymers, most likely dimers.

The purine requirement for growth of L. leichmannii in the absence of cobalamin has been examined and the results of this study do not substantiate an involvement of cobalamin in purine synthesis. Excessive adenine accumulation, excretion, and toxicity, resulting in part from a rapid conversion of guanine (or derivative) to adenine appears to be responsible for the nutritional purine requirement. Growth of this organism may be obtained in the absence of purines and cobalamin if organisms pregrown in the presence of cobalamin are supplemented with high concentrations of 5-amino-4-imidazole carboxamide. The purine requirement is thought to be initiated by events which become manifested by a retarded rate of DNA synthesis.

Gradients for isoelectric focusing in the pH range 2–5 were developed by adding acids to commercially available carrier ampholytes (Ampholine, L.K.B.). The effects of current direction, voltage, focussing time, stabilising medium and pH gradient composition were studied to find the optimum focussing conditions. In addition, the electric potential was measured in loco during the run in order to determine the field strength in different parts of the column. Details for the production of suitable acid gradients are given. Using these gradients it was possible to fractionate the R-type vitamin B 12 -binding protein of human amniotic fluid into six fractions with isoelectric points between pH 2.3 and 4.2.