The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-¹⁴C]glycine to ¹⁴CO₂ was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-¹⁴C]serine to ¹⁴CO₂ both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-¹⁴C]histidine to ¹⁴CO₂ is more sensitive indicator of folate deficiency than the rate of oxidation of [3-¹⁴C] serine to ¹⁴CO₂ although both are presumably tetrahydrofolate dependent.     

The regulation of one-carbon oxidation in the rat by nitrous oxide and methionine

Formate is oxidized to CO₂ in the rat by folate-dependent reactions. Nitrous oxide treatment inhibited hepatic methionine synthetase activity, reduced hepatic S-adenosyl-l-methionine (Ado-Met) and tetrahydrofolate (H₄ folate) concentrations and decreased the rate of formate oxidation in the rat. The administration of methionine to nitrous oxide-treated rats increased hepatic Ado-Met concentrations and restored hepatic H₄folate levels and formate oxidation to control values but did not reverse the inhibition of methionine synthetase. Positive correlations were observed between hepatic Ado-Met levels and H₄folate concentrations and between hepatic H₄folate concentrations and formate oxidation. These results suggest that alterations in hepatic H₄folate concentrations may profoundly influence the oxidation of one-carbon compounds. They confirm the importance of the methionine synthetase reaction as a major source of regeneration of H₄folate. These findings also indicate that methionine acts at a site other than the methionine synthetase reaction to restore hepatic H₄folate concentrations and formate oxidation to control values in nitrous oxide-treated rats.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats.

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-14C]glycine to 14CO2 was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-14C]serine to 14CO2 both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-14C]histidine to 14CO2 is a more sensitive indicator of folate deficiency than the rate of oxidation of [3-14C]serine to 14CO2 although both are presumably tetrahydrofolate dependent.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.     

Dietary folate affects the response of rats to nickel deprivation

Because vitamin B₁₂ and Ni are known to interact and because of the similar metabolic roles of vitamin B₁₂ and folate, an experiment was performed to determine the effect of dietary folate on Ni deprivation in rats. A 2×2 factorially arranged experiment used groups of nine weanling Sprague-Dawley rats. Dietary variables were Ni, as NiCl₂·6H₂O, 0 or 1 μg/g; and folic acid, 0 or 2 mg/kg. The basal diet, based on skim milk, contained less than 20 ng Ni/g. After 54 d, an interaction between dietary Ni and folate affected several variables including erythrocyte folate, plasma amino acids, and femur trace elements. For example, folate deprivation decreased erythrocyte folate; folate supplementation to the Ni-supplemented rats caused a larger increase in erythrocyte folate concentration than did folate supplementation to the Ni-deprived rats. Also, dietary Ni affected several plasma amino acids important in one-carbon metabolism (e.g., Ni deprivation increased the plasma concentrations of glycine and serine). This study shows that dietary Ni, folate, and their interaction can affect variables associated with one-carbon metabolism. This study does not show a specific site of action of Ni but it indicates that Ni may be important in processes related to the vitamin B₁₂-dependent pathway in methionine metabolism, possibly one-carbon metabolism. US Department of Agriculture, Agricultural Research Service, Northern Plans Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO₂ or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [⁷⁵Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.Abbreviations DTE dithioerythritol - TES N-Tris (hydroxymethyl) methyl-2-amino-ethane sulfonic acid     

The role of methionine in regulating folate-dependent reactions in isolated rat hepatocytes

In isolated rat hepatocytes, histidine and formate, are oxidized to CO₂ by folate-dependent reactions. These reactions are stimulated two- to fourfold by the addition of l-methionine, dl-homocysteine, S-adenosyl-l-methionine (Ado-Met), or S-adenosyl-l-homocysteine (Ado-Hcy). These compounds all increase the hepatocyte concentration of Ado-Met and Ado-Hcy. Substrates of hepatic catechol O-methyltransferase, such as l-Dopa methyl ester and 3,4-dihydroxyphenylacetic acid, decrease the hepatocyte concentration of Ado-Met in the presence or absence of added l-methionine or dl-homocysteine. The catechols do not affect the concentration of Ado-Hcy, but they inhibit the oxidation of formate and histidine. Thus, there is an excellent positive correlation between the rate of histidine and formate oxidation and the concentration of Ado-Met. There is no correlation between the rate of these reactions and either the Ado-Hcy concentration or the concentration ratio of Ado-Met:Ado-Hcy. Ado-Met inhibition of rat hepatic 5,10-methylene tetrahydrofolate reductase activity is reversed by Ado-Hcy, but the dependency of rat hepatic 5-methyltetrahydrofolate-homocysteine transmethylase activity (methionine synthetase) on Ado-Met is not altered by Ado-Hcy. These results indicate that methionine, through its conversion to Ado-Met, regulates folate-dependent reactions in isolated hepatocytes by increasing activity of methionine synthetase which leads to an increased concentration of tetrahydrofolate. That methionine and Ado-Met increase the hepatocyte concentration of nonmethyltetrahydrofolate compounds and decrease the hepatocyte concentration of 5-methyltetrahydrofolate supports this hypothesis.     

Effects of maternal vitamin B12 deficiency from end of gestation to weaning on the growth and haematological and immunological parameters in mouse dams and offspring

Vitamin B₁₂-deficiency may induce specific symptoms as neurological alterations and unspecific symptoms such as anaemia and growth retardation. In this study, maternal vitamin B₁₂ deficiency from end of gestation to weaning was evaluated in mouse dams, which was provoked by feeding a vitamin B₁₂-deficient diet. The animals were divided into two groups (control and deficient). The control group received the vitamin B₁₂-deficient diet supplemented with commercial vitamin B₁₂. Compared to the control, the vitamin B₁₂-deficient dams and their offspring showed a significant decrease of body weight (by 20 and 39%, respectively), serum vitamin B₁₂ concentration (by 61 and 67%, respectively), haematological values as haematocrit (25 and 26%, respectively), and IgA producer cells (by 36 and 54%, respectively). In both, vitamin B₁₂-deficient mouse dams and their offspring, histological alterations of small intestine were observed, whereas growth retardation occurred in the offspring only. This experimental murine model allows assessing the incidence of maternal cobalamin deficiency in offspring and would be useful for evaluating novel adjuncts such as functional foods to prevent vitamin B₁₂ deficiency.     

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H₂-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO₂. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO₂, H₂ and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H₂, formate or e.g. valine were added as H-donors. In pure culture on alanine under N₂, strain al-2 grew very poorly and produced H₂ up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H₂-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H₂S, CH₄ or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (0.1 M); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO₂. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO₂ and H₂. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.     

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B(12) or methionine. 3. Concentrations of ATP, NAD(+), NADH and NADPH were lower in freeze-clamped liver from vitamin B(12)-deficient sheep than in liver from pair-fed, vitamin B(12)-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K(+)/Na(+) ratios found in deficient livers. 4. The livers of vitamin B(12)-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.     

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism

1. A study was made of the effects of injected l-methionine on the activity of several enzymes of folate metabolism, and on the transport of methotrexate in liver preparations from vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). 2. The activities of dihydrofolate reductase (EC 1.5.1.3) and 5-methyltetrahydrofolate-homocysteine transmethylase were significantly decreased in the liver of vitamin B(12)-deficient animals, but were unaffected by l-methionine. 3. The concentration of S-adenosyl-l-methionine in the liver of deficient animals was about one-half of that in normal animals, and was restored to normal by either vitamin B(12) or l-methionine. 4. Methylenetetrahydrofolate reductase (EC 1.1.1.68) from sheep liver was inhibited by S-adenosyl-l-methionine in vitro, but not by concentrations of S-adenosyl-l-methionine found in the liver of vitamin B(12)-deficient animals after injection of physiological amounts of l-methionine. 5. Pteroylpolyglutamate synthetase activity was significantly increased in the liver of vitamin B(12)-deficient animals, and was decreased by intravenous injections of l-methionine. 6. l-Methionine injections increased the initial rate of uptake of methotrexate in liver slices from deficient animals and acted synergistically with vitamin B(12) to increase the quantity taken up in 40min. The failure of folate metabolism in vitamin B(12) deficiency can be satisfactorily explained if l-methionine similarly affects the membrane transport of naturally occurring folates. 7. Further details of the results have been deposited as Supplementary Publication SUP 50028 (4 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Effect of thiouracil in modifying folate function in severe vitamin B12 deficiency

The effects of thiouracil in correcting defects in folic acid function produced by B12 deficiency were studied. Addition of the thyroid inhibitor, thiouracil, to a low methionine diet containing B12, increased the oxidation of [2-14C]histidine to carbon dioxide, and increased liver folate levels. Addition of 10% pectin to the diet accentuated B12 deficiency as evidenced by a greatly decreased rate of histidine oxidation (0.19%) and an increased excretion of methylmalonic acid. Addition of thiouracil to the diet restored folate function as measured by increased histidine oxidation and increased liver folate levels similar to that produced by addition of methionine to a B12-deficient diet. Thiouracil decreased methylmalonate excretion, and increased hepatic levels of B12 in animals on both B12-deficient and -supplemented diets. Hepatic methionine synthase was increased by thiouracil, which may be the result of the elevated B12 levels. S-Adenosylmethionine and the enzyme methionine adenosyltransferase were also increased by thiouracil. Thus it is possible that the effect of thiouracil in increasing folate function consists both in the effect of thiouracil in decreasing levels of methylenetetrahydrofolate reductase, and also in its action in increasing S-adenosylmethionine which exerts a feedback inhibition of this enzyme.     

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. Am J Physiol Endocrinol Metab 301: E000-E000, 2011. First published September 20, 2011; 10.1152/ajpendo.00345.2011.-We carried out a (1)H-NMR metabolomic analysis of sera from vitamin B(12)-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 μM in control rats to 402 μM in vitamin B(12)-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B(12)- and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B(6)-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine β-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways.     

In Vivo Kinetics of Formate Metabolism in Folate-deficient and Folate-replete Rats

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [¹³C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h⁻¹·100 g of body weight⁻¹ in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate.     

Effects of increasing amounts of extruded linseed in the diet on apparent ruminal synthesis of some B vitamins in dairy cows

Many studies have shown that metabolic efficiency of ruminants can be significantly decreased when B-vitamin supply is insufficient. Under the present state of knowledge, the amounts of B vitamins available for intestinal absorption cannot be predicted based on diet composition. Therefore, in an attempt to increase our understanding of the effects of dietary factors, on B-vitamin supply for dairy cows, the effects of increasing amounts of extruded linseed in diets based on hay (permanent grassland hay, H; Experiment 1) or corn silage (CS; Experiment 2) on apparent ruminal synthesis (ARS) of thiamin, riboflavin, niacin, vitamin B₆, folates and vitamin B₁₂ were evaluated. In each experiment, four lactating Holstein cows fitted with cannulas in the rumen and the proximal duodenum were used in a 4 × 4 Latin square design. In both experiments, the dietary treatments consisted of an increasing supply of extruded linseed representing 0%, 5%, 10% or 15% of diet DM. The forage : concentrate ratios were 50 : 50 and 60 : 40 for Experiments 1 and 2, respectively. Duodenal flow was determined using YbCl₃ as a marker. The ARS of each B vitamin was calculated as duodenal flow – daily intake. In both experiments, treatments did not affect thiamin, riboflavin, niacin and vitamin B₁₂ duodenal flow or ARS. Increasing the dietary concentration of extruded linseed decreased folate intake in Experiment 1 and vitamin B₆ intake in Experiment 2 but resulted in a greater duodenal flow of vitamin B₆ and folates regardless of the forage used in basal diet. Greater dietary linseed concentrations decreased vitamin B₆ apparent degradation in the rumen in CS-based diet only and increased folate ARS in both H- and CS-based diets. Increasing linseed concentration of isonitrogenous and isoenergetic diets increased vitamin B₆ and folate supply to dairy cows, both with H- and CS-based diets.     

Effects of Vitamin B12 Analogues with Alternations in the Side Chains of the Corrin Ring on Urinary Methylmalonate Excretion in Vitamin B12-Deficient Rats

To study how much the side chains of the corrin ring of vitamin B₁₂ are involved in the physiological roles of the vitamin, five vitamin B₁₂ analogues (cyanocobalamin-b-monocarboxylate, cyanocobalamin-d-monocarboxylate, cyanocobalamin-e-monocarboxylate, cyano-13-epicobalamin, and cyanocobalamin(c-lactam)) with alternations in the side chains were synthesized chemically and then administered orally and intravenously to vitamin B₁₂-deficient rats. Male rats fed a vitamin B₁₂-deficient diet for 11 wk developed a severe vitamin B₁₂ deficiency with a high urinary methylmalonate excretion (223.8 ± 136.2 μmol/d) and ~97% (1.2±0.7ng/g tissue) lower hepatic vitamin B₁₂ content. Oral and intravenous administration of cyanocobalamin-b-,-d-, and -e-monocarboxylates and cyano-13-epicobalamin could not improve the severe vitamin B₁₂-deficient status of the rats, indicating that the b-, d-, and e-propionamide side chains of the corrin ring of vitamin B₁₂ are important in the absorption, transport, and function of the vitamin in rats. Urinary methylmalonate excretion of the rats that were intravenously administered cyanocobalamin(c-lactam) increased twice as much as those of the other analogue-supplemented rats, suggesting that cyanocobalamin(c-lactam) act as a powerful Cbl-antagonist. The results also indicate that mammalian cells do not contain a system for synthesizing complete vitamin B₁₂ from these analogues.     

Regulation of mitochondrial glycine decarboxylase from pea mitochondria

Reactions of glycine cleavage were assayed in mitochondria isolated from cotyledons of germinating pea seeds. These reactions, which included the exchange of bicarbonate with C-1 of glycine and an NAD-stimulated decarboxylation of glycine, were maximal under aerobic conditions at pH 7·8. The apparent Michaelis-Menten constants for glycine and bicarbonate in the exchange reaction were 1·8 and 12·5 mM respectively. The K_m for NAD in the decarboxylation reaction was 47 μM. Maximal enzyme activity was observed when mitochon-drial integrity was maintained. Up to 40% inhibition of the decarboxylation reaction was observed when NADH, NADPH or l-methionine were added to the reaction system. When glycine-[2-¹⁴C] was incubated with the isolated mitochondria, labelled CO₂ was evolved in nanomolar quantities. It is concluded that glycine decarboxylase may be of importance in supplying C-1 units for the de novo synthesis of methionine in pea mitochondria.     

Kinetics of neutral amino-acid transport by isolated gill tissue of the bivalve Mya arenaria (L.)

An in vitro technique was used to examine the absorption by the gill of Mya arenaria (L.) of six neutral l-amino acids chosen for differences in their side chains, viz., short chain — glycine and alanine, long chain — leucine, sulphur containing — methionine, aromatic — phenylalanine, hydroxylic — serine. The uptake of all these substrates was active and carrier-mediated, and was analysed by Michaelis-Menten kinetics. Values of K_t, the transport constant, decreased with increasing length of the side chain for glycine, l-serine, l-alanine, l-methionine, l-phenylalanine, and l-leucine, while values for V_max, the maximum velocity of uptake, decreased as chain length increased, except in the case of l-serine. Inhibition experiments suggested that at least one transport locus was common to all the neutral amino acids examined, but homogeneity of transport was only demonstrated in the case of methionine and leucine. The transport of the basic amino acid l-lysine overlapped with several of the-neutral amino acids. These results emphasize the need to consider the mutual inhibitory effects between amino acids absorbed from sea water, when calculations are made of the value of this source of nutrition to marine invertebrates.     

Changes in the tissue concentration of folate and vitamin B12 during maturation and ageing

Age-dependent changes in folate and vitamin B₁₂ metabolism of mice have been investigated. The concentration of folate in liver plasma and blood showed a postnatal increase to a maximum at approx. 25 weeks. Total folate concentrations then remained constant whereas free folate decreased slowly up to week 98. Conversely both total and free folate of the brain were reduced extensively during the first 10 weeks of life after which time total folate concentration stabilised whilst that of free folate continued to decline slowly. The concentration of vitamin B₁₂ in brain, liver and plasma showed an initial rapid increase. The vitamin continued to accumulate more slowly in the brain and liver from weeks 10 to 98. The concentration of vitamin B₁₂ in the plasma appeared to achieve equilibrium after a period of accumulation lasting 25 weeks. These results suggest that during maturation the characteristics of folate metabolism of the brain are distinct from those of peripheral tissues, and that folate, unlike vitamin B₁₂ metabolism, undergoes continuing change with advancing age.     

Microbial production of vitamin B12 antimetabolites: II. 2-Amino-4-keto-3-methylpentanoic acids from bacillus cereus 439

2-Amino-4-keto-3-methylpentanoic acids were isolated as a diastereomeric mixture from Bacillus cereus 439 fermentations and found to be vitamin B₁₂ antimetabolites in a bioassay system based on the vitamin B₁₂-requiring Escherichia coli (Davis 113-3). A similar diastereomeric mixture with bioactivity was synthesized by condensation of 2-bromo-3-butanone with sodio diethyl acetamidomalonate followed by hydrolysis with 6 N HCl and purification by ion-exchange chromatography. The growth inhibitory effects of the antimetabolite were reversed by vitamin B₁₂, l-methionine, l-isoleucine, l-leucine, l-valine, and d-alanine.