Comparative studies on the methionine synthesis in sheep and rat tissues

The important features of the enzymes involved in methionine synthesis in sheep were found to be the low activity of betaine-homocysteine methyltransferase and the high activity of 5-methyltetrahydrofolate-homocysteine methyltransferase. The rate of the methionine synthesis in sheep liver was significantly lower than that in rats due to the low activity of hepatic betaine-homocysteine methyltransferase. The hepatic methionine recycling was stimulated by the addition of betaine in both species. These results indicate that in sheep 5-methyltetrahydrofolate-homocysteine methyltransferase plays a significant role in hepatic methionine synthesis along with betaine-homocysteine methyltransferase. In contrast, in the rat hepatic system methionine synthesis is virtually dependent on betaine-homocysteine methyltransferase.     

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-asenosylmethionine metabilism in the rat

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.     

Disturbance of methyl group metabolism in alloxan-diabetic sheep

Alloxan-induced diabetes results in changes in the activities of a number of enzymes related to methyl group metabolism in sheep. Decreases in the activities of phospholipid methyltransferase and betaine-homocysteine methyltransferase in diabetic sheep liver indicate a reduced rate of choline synthesis and oxidation. A 65-fold increase in the activity of glycine methyltransferase and a 4-fold rise in the activity of gamma-cystathionase in diabetic sheep liver with elevated urinary excretion of cyst(e)ine suggest that catabolism of the methyl group of methionine and homocysteine was enhanced in the diabetic state.     

Regulation of methyl group metabolism in lactating ewes

The effect of lactation on a number of enzymes involved in transmethylation reactions and the secretion of major methyl compounds into milk have been examined in sheep. The activities of hepatic phospholipid methyltransferase and 5-methyltetrahydrofolate-homocysteine methyltransferase were significantly higher in lactating ewes, compared with those in non-lactating ewes, while the activity of both hepatic and pancreatic glycine methyltransferase was significantly lower in the lactating state. No differences were observed in the activities of hepatic guanidoacetate methyltransferase, betaine-homocysteine methyltransferase and cystathionine beta-synthase on lactation. These results suggest that the extra demand for methyl groups for the secretion of methyl compounds in the milk is facilitated by enhancing the rate of de novo methyl group synthesis and lowering the rate of physiologically nonessential methylation.     

Nitrous oxide induced vitamin B12 deficiency: measurement of methylation reactions in the fruit bat (Rousettus aegyptiacus)

Nitrous oxide induced inhibition of methionine synthetase activity has been proposed as a suitable model for the myelopathy associated with vitamin B12 deficiency. This suggests a defect in methyl group metabolism. The fruit bat has been used previously as a model for dietary induced vitamin B12 deficiency. However in the nitrous oxide treated fruit bat with neurological symptoms: No changes in [14C]ethanolamine incorporation into liver and brain phospholipids could be detected. No changes in synaptosomal and myelin lipid methylation could be shown. No differences in the rate of synaptosomal and myelin protein methylation could be measured. Therefore the fruit bat myelopathy is not related to a methyl group transfer deficiency.     

Methionine synthesis by extracts of Salmonella typhimurium

1. Following the genetic studies by Smith (1961) and Smith & Childs (1963) with methionine auxotrophs of Salmonella typhimurium, methionine formation from homocysteine has been investigated with cell-free extracts of this organism. 2. As found with Escherichia coli (Woods, Foster & Guest, 1964), methyl groups are formed by an N(5)N(10)-methylenetetrahydrofolate reductase. They are then transferred to homocysteine by either a simple N(5)-methyltetrahydropteroyl-triglutamate-homocysteine methyltransferase or alternatively a cobalamin-dependent N(5)-methyltetrahydrofolate-homocysteine methyltransferase. 3. S. typhimurium differs from E. coli in being able to synthesize significant amounts of cobalamin.     

Effects of nitrous oxide inactivation of vitamin B12 and of methionine on folate coenzyme metabolism in rat liver, kidney, brain, small intestine and bone marrow

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), and of methionine on folate coenzyme metabolism were determined in rat liver, kidney, brain, small intestine and bone marrow cells. Nitrous oxide exposure led to an increase in the proportion of 5-methyltetrahydrofolate at the expense of other reduced folates in all tissues examined. Administration of methionine at levels up to 400 mg/kg resulted in the normalization of folate coenzyme patterns in liver as a result of the increased levels of S-adenosylmethionine. In other tissues examined, methionine had no effect on the levels of S-adenosylmethionine or S-adenosylhomocysteine, or on the distribution of folate coenzymes. These results are consistent with the methyl trap hypothesis as the explanation of the relationship between vitamin B12 and folate metabolism, and provide direct evidence that the sparing effect of methionine on folate metabolism is a phenomenon restricted to the liver.     

Inhibition of vitamin B12-dependent microbial growth by nitrous oxide

In methionine-free media, nitrous oxide inhibits the growth of an auxotrophic strain of Escherichia coli lacking a cobalamin-independent pathway for the de novo synthesis of methionine. Prototrophic E. coli is similarly inhibited by nitrous oxide if the cobalamin-independent pathway is selectively depressed by sulfanilamide. Nitrous oxide thus effectively inactivates cobalamin-dependent 5-methyltetrahydrofolate--homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) in intact bacteria.     

Cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine, producing tetrahydrofolate and methionine. Insufficient availability of cobalamin, or inhibition of methionine synthase by exposure to nitrous oxide, leads to diminished activity of this enzyme. In humans, severe inhibition of methionine synthase results in the development of megaloblastic anemia, and eventually in subacute combined degeneration of the spinal cord. It also results in diminished intracellular folate levels and a redistribution of folate derivatives. In this review, we summarize recent progress in understanding the catalysis and regulation of this important enzyme from both bacterial and mammalian sources. Because inhibition of mammalian methionine synthase can restrict the incorporation of methyltetrahydrofolate from the blood into cellular folate pools that can be used for nucleotide biosynthesis, it is a potential chemotherapeutic target. The review emphasizes the mechanistic information that will be needed in order to design rational inhibitors of the enzyme.     

Induction of rat liver glycine methyltransferase by high methionine diet

Dietary experiments were carried out to evaluate the physiological role of glycine methyltransferase. When rats received a 18% casein diet containing excess methionine, the activity of the enzyme in liver extracts increased with increasing methionine content in the diet. Adenosylmethionine synthetase and adenosylhomocysteinase activities were also elevated, while guanidoacetate methyltransferase activity showed no significant change. The glycine methyltransferase activity reached a maximal level after 4–6 days on the 3% methionine diet. Immunological titration showed that the increase in activity was associated with the increase in amount of the enzyme.     

Regulation of C1 metabolism by l-methionine in Saccharomyces cerevisiae

1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool.     

In vitro inactivation of methionine synthase by nitrous oxide

Nitrous oxide (N2O) is commonly used as an anesthetic agent. Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates. We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O. Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system. Reisolation of the inactivated E. coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation. However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin. We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical. Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity.     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Effect of interferon on phospholipid methylation by peripheral blood mononuclear cells

The effect of human interferon (IFN) preparations on the metabolic pathway leading to the synthesis of phosphatidylcholine (PC) by a stepwise addition of methyl groups to phosphatidylethanolamine (PE) was investigated in human peripheral blood mononuclear (PBMN) cells. An inhibition of the synthesis of PC via this pathway was regularly observed with both alpha- (recombinant or natural) and beta-IFN. This inhibition was apparent within the first 5 min of treatment, reached its maximum between 15 min and 1 hr, and persisted at the same level until 6 hr, the last time point examined. Each of the transmethylated products of PE underwent a similar inhibition, as measured by the turnover rate of individual products. The intracellular pool of the methyl donors, methionine and S-adenosyl-methionine (SAM), was shown to be unaffected. The methyltransferase activity of IFN-pretreated cell extracts was unchanged. These findings support the hypothesis that IFN induces a functional change in phospholipid methylation at the level of organized membrane-bound phospholipid methyltransferase enzymes in intact cells.     

Tissue metabolism of methionine in sheep

The rate of oxidation of the carboxyl and methyl carbons of [14C]methionine to CO2 by homogenates of liver, kidney cortex, pancreas, muscle and small intestinal mucosa was studied in two breeds of sheep (Merino and Poll Dorset Horn) at three ages (2 weeks, 3 months, 4 years). Sodium alpha-keto-gamma- methiolbutyrate (0 X 4 mM) stimulated production of CO2 from the carboxyl carbon of methionine, but not from the methyl carbon. Sodium pyruvate did not affect the recovery of CO2 from either carboxyl or methyl of methionine. Sodium formate (15 mM) suppressed the conversion of the methyl carbon of methionine to CO2 by liver and kidney homogenates to 4 and 50%, respectively, of control values, but did not affect the percentage of carboxyl carbon of methionine recovered in CO2 with either tissue. With addition of S-methyl-L-cysteine (40 mM) and 3- methylthiopropionate (10 mM) the percentage of methyl and carboxyl carbons recovered in CO2 was reduced to about 20% of control values in homogenates of both tissues. Activity per gram of tissue was higher in liver and kidney cortex than in pancreas, intestinal mucosa, or muscle, with no significant differences due to breed (Merino or Poll Dorset Horn) or sex (ewe, ram or wether) of sheep. Conversion of both the carboxyl and methyl carbons to CO2 by liver was significantly lower in 2-week-old lambs than in older animals (P less than 0.01). The activity of other tissues was not markedly affected by age. Results are discussed in relation to evidence of alternative pathways of methionine catabolism, and capacities of the tissues of the sheep to catabolize methionine by alternative pathways.     

Developmental changes in the activities of enzymes related to methyl group metabolism in sheep tissues

The activities of choline oxidase and betaine-homocysteine methyltransferase increased markedly in pre-ruminant lamb liver after birth and subsequently decreased when the lambs reached the ruminant state, while the developmental changes in hepatic 5-methyl-H4folate-homocysteine methyltransferase were negatively correlated with those of betaine-homocysteine methyltransferase. Hepatic phospholipid methyltransferase was elevated almost four-fold by the 10th postnatal day, but declined thereafter. Hepatic glycine methyltransferase in one-day-old lambs increased 55-fold, compared with that of fetuses, and thereafter decreased dramatically with age. Guanidoacetate methyltransferase, glycine methyltransferase and betaine-homocysteine methyltransferase in sheep pancreas increased markedly with age and were many times higher than the hepatic enzymes in adult sheep. Choline oxidase, betaine-homocysteine methyltransferase, cystathionine beta-synthase and glycine methyltransferase in adult sheep liver were much lower than those in rat. These results illustrate the conservative features of methyl group metabolism in postruminant sheep.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.