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.     

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.     

Methionine metabolism in mammals. Adaptation to methionine excess

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.     

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.     

Perturbation of methionine metabolism in sheep with nitrous-oxide-induced inactivation of cobalamin

Exposure of sheep to 36% nitrous oxide for 8 days (2-hr per day) led to 90%, 82% and 74% inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase in the liver, heart and brain, respectively, while there was no significant decrease in the activity of methylmalonyl-CoA mutase. There was also no change of betaine-homocysteine methyltransferase activity. The level of plasma methionine in nitrous-oxide-exposed sheep fell to 30% of its initial value. S-Adenosylmethionine level was reduced to 50% of the control value in the liver, and was also significantly decreased in the heart, but not in the brain. Excretion of formiminoglutamic acid and homocystine was also observed in the urine of sheep exposed to nitrous oxide. These results demonstrate that inhibition of 5-methyltetrahydrofolate-homocysteine methyltransferase causes a pronounced perturbation of methionine metabolism in sheep, suggesting that dietary methionine plus methionine synthesized from the methyl groups of betaine are not sufficient to meet the methyl needs for biological methylation reactions in this species and, in turn, emphasizing the role of 5-methyltetrahydrofolate-homocysteine methyltransferase in methionine synthesis in the sheep.     

The effect of diet and of methionine loading on activity of enzymes in the transulfuration pathway in sheep

In three experiments, activity of hepatic enzymes associated with metabolism of methionine through the transulfuration pathway were studied with respect to possible effects of diet and methionine infusion per abomasum. In experiment 1 no differences in methionine adenosyltransferase (MAT) or cystathionine lambda-lyase (CGL) were detected between lucerne and wheaten straw diets, or between effects of fasting for 48 h and 96 h after feeding lucrene chaff as opposed to fasting after feeding wheaten straw. Fasting for 96 h resulted in a trend toward increasing CGL and MAT specific activities on both diets. In experiment 2 MAT was depressed significantly by infusion of methionine at 1.4 g/day and to a greater extent by infusion at 4.2 g/day, whilst CGL was not significantly affected. In experiment 3 MAT specific activity decreased significantly in response to both levels of methionine supplementation. Betaine-homocysteine methyltransferase activity was increased by methionine infusion. CGL decreased in all treatments but there was a larger decrease in those animals receiving methionine infusion. No significant changes were observed in relation to other enzymes examined which included cystathionine beta-synthase and threonine dehydratase. These observations are consistent with the hypothesis that in sheep the increase in methionine in blood plasma which occurs when methionine is absorbed in increased amounts may be due to reduced entry into the transulfuration pathway because of a repression of MAT activity.     

Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine

The hepatic activity of betaine-homocysteine methyltransferase is a complex function of the content of methionine in the diet. Enzyme levels are lower in the livers of rats fed a 0.3% methionine diet than in livers of animals maintained on either methionine-free or excessivemethionine (1.0%) rations. The finding that activities are increased at both extremes of the spectrum of dietary methionine intake suggests the possibility that the betaine-homocysteine methyltransferase reaction may function both to maintain tissue concentrations of methionine when intake of this amino acid is limited and to remove homocysteine when methionine intake is excessive.     

Cystathionine as a precursor of methionine in Escherichia coli and Aerobacter aerogenes

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and Stanley K. Shapiro. Cystathionine as a precursor of methionine in Escherichai coli and Aerobacter aerogenes. J. Bacteriol. 92:1331-1336. 1966.-Cystathionine has been shown to be a precursor of methionine biosynthesis in Escherichia coli and Aerobacter aerogenes. A double enzyme assay was developed to show the formation of homocysteine from cystathionine. The results obtained support the concept that cystathionine serves as a precursor of methionine via the intermediate formation of homocysteine. The latter compound is methylated by the homocysteine methyltransferase of these microorganisms. Sulfhydryl and keto acid assays were used to demonstrate cystathionase activity. Methionine represses both homocysteine methyltransferase formation and cystathionase formation. However, the presence of methionine in reaction mixtures resulted in product inhibition of homocysteine methyltransferase activity, but not of cystathionase activity.     

A mutation in the Mod subunit of EcoP15I restriction enzyme converts the DNA methyltransferase to a site-specific endonuclease

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.     

Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous area 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl-3H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells.     

Reduced activity of Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed methionine content

In the S- methylmethionine cycle of plants, homocysteine methyltransferase (HMT) catalyzes the formation of two molecules of methionine from homocysteine and S- methylmethionine, and methionine methyltransferase (MMT) catalyzes the formation of methionine from S- methylmethionine using S- adenosylmethionine as a methyl group donor. Somewhat surprisingly, two independently isolated knockdown mutations of HMT2 (At3g63250), one of three Arabidopsis thaliana genes encoding homocysteine methyltransferase, increased free methionine abundance in seeds. Crosses and flower stalk grafting experiments demonstrate that the maternal genotype at the top of the flower stalk determines the seed S- methylmethionine and methionine phenotype of hmt2 mutants. Uptake, transport and inter-conversion of [¹³C] S- methylmethionine and [¹³C]methionine in hmt2 , mmt and wild-type plants show that S- methylmethionine is a non-essential intermediate in the movement of methionine from vegetative tissue to the seeds. Together, these results support a model whereby elevated S- methylmethionine in hmt2 vegetative tissue is transported to seeds and either directly or indirectly results in the biosynthesis of additional methionine. Manipulation of the S- methylmethionine cycle may provide a new approach for improving the nutritional value of major grain crops such as rice, as methionine is a limiting essential amino acid for mammalian diets.     

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.     

Methionine metabolism in mammals. The methionine-sparing effect of cystine

Cystine can replace approximately 70% of the dietary requirement for methionine. We used standard enzyme assays, determinations of the hepatic concentrations of metabolites and an in vitro system which simulates the regulatory site formed by the enzymes which utilize homocysteine in this study of the mechanism for this adaptation. A significant alteration in the pattern of hepatic homocysteine metabolism occurs following the substitution of cystine for methionine. The major change is a marked reduction in the synthesis of cystathionine. Decreases in both the level of cystathionine synthase and in the concentration of adenosyl-methionine, a positive effector of the enzyme, explain this finding. Despite significant increases in the hepatic levels of betaine-homocysteine methyltransferase and methyltetrahydrofolate-homocysteine methyltransferase, flow through these reactions remains relatively constant. The betaine enzyme may be essential for efficient methionine conservation. In the absence of choline, cystine cannot replace methionine in an adequate diet limited in the latter amino acid.     

Effects of prolonged ethanol feeding on methionine metabolism in rat liver

Pairs of rats were fed control and alcohol liquid diets for periods of 1, 2, 3, and 4 months. The animals were then killed, and their livers analyzed for betaine, S-adenosylmethionine (SAM), methionine synthetase activity, and betaine--homocysteine methyltransferase (BHMT) activity. The results of this time-course study showed that chronic ethanol feeding inhibited the activity of the methionine synthetase throughout the study, but increased the activity of BHMT and lowered betaine levels. These data suggest that the rat, because of its ability to produce betaine from choline, has the capacity to compensate for the ethanol-induced impairment of methionine synthetase and maintain vital tissue levels of SAM over prolonged periods of time via an adaptive increase in BHMT activity.     

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.     

Methionine and cysteine metabolism in Fasciola hepatica

Radiolabel from the methyl groups of serine and methyltetrahydrofolate was readily incorporated into methionine in adult Fasciola hepatica, and a substantial proportion of the label from [³⁵S]methionine appeared in cysteine. The data suggest that methionine synthesis is via methyltetrahydrofolate-homocysteine methyltransferase and that there is cysteine synthesis from methionine. Cystathionine-β-synthase and γ-cystathionase activities were demonstrated in homogenates.     

Induction and Repression in the S-Adenosylmethionine and Methionine Biosynthetic Systems of Saccharomyces cerevisiae

Two methionine biosynthetic enzymes and the methionine adenosyltransferase are repressed in Saccharomyces cerevisiae when grown under conditions where the intracellular levels of S-adenosylmethionine are high. The nature of the co-repressor molecule of this repression was investigated by following the intracellular levels of methionine, S-adenosylmethionine, and S-adenosylhomocysteine, as well as enzyme activities, after growth under various conditions. Under all of the conditions found to repress these enzymes, there is an accompanying induction of the S-adenosylmethionine-homocysteine methyltransferase which suggests that this enzyme may play a key role in the regulation of S-adenosylmethionine and methionine balance and synthesis. S-methylmethionine also induces the methyltransferase, but unlike S-adenosylmethionine, it does not repress the methionine adenosyltransferase or other methionine biosynthetic enzymes tested.     

Reverse methionine biosynthesis from S-adenosylmethionine in eukaryotic cells

The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.     

Changes in DNA methyltransferase induced by treatment with N-2-acetylaminofluorene

We have compared the levels of DNA methyltransferases from rat liver and spleen in both sexes following a single injection of N-2-acetylaminofluorene (AAF). Enzyme extracts from treated animals were obtained at different intervals (2-34 days) after treatment. The extracts were assayed in the presence of chicken erythrocyte DNA and S-adenosyl-L-[Me-3H]methionine. A 55% increase in male rat-liver methyltransferase activity measured by Me-3H incorporation into DNA occurred on day 14. By contrast, female methyltransferase after a similar period revealed a 33% decrease in activity. Between days 21 and 34, there is a progressive return to normal methyltransferase levels. Spleen-derived enzyme studied between days 7 and 14, showed a decrease in methylating activity in both sexes. After replacing corn seed oil by ethanol as the vehicle for AAF injection, we observed a change in liver methyltransferase 48 h after injection. Quantification of radioactive eluates in m5C fractions together with the increase in the integrated area identified as m5C in HPLC chromatograms allowed positive identification of methylated products.     

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.