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.     

Imparied formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation

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.     

Detection and quantification of 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) in rat retina,optic nerve,and brain

Methanol poisoning is characterized by the accumulation of formic acid, a metabolite of methanol, which can lead to metabolic acidosis and ocular toxicity. Formate metabolism to CO₂ is governed by tissue H₄folate and 10-FTHFDH levels. Presumably, rats are not normally susceptible to formate toxicity because they possess high hepatic H₄folate and 10-FTHFDH levels. However, the ability of target tissues to metabolize formate is not known. Therefore, studies were performed to determine whether 10-FTHFDH was present in rat retina, optic nerve, and brain. 10-FTHFDH levels were determined using Western blot analysis of mitochondiral and postmitochondrial preparations from these tissues. Hepatic mitochandrial and postmitochondrial levels of 10-FTHFDH were 13 and 12 ng/μg protein, respectively. Postmitochondrial levels of 10-FTHFDH in rat retina, optic nerve and whole brain were 0.2, 1.3, and 2.1 ng/μg protein; mitochondrial values in retina and brain were 0.2 and 1.5 ng/μg protein, respectively. Postmitochondrial values obtained for rat brain regions were similar to those found for whole brain. These results suggest that, in rats, target tissues possess the capacity to metabolize formate to CO₂ and may be protected from formate toxicity through this folate-dependent system.     

Increased methionine synthetase activity in zinc-deficient rat liver

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.     

Oxidation of 5-methyltetrahydrofolate in cobalamin-inactivated rats.

Rats were exposed to nitrous oxide, which inactivates cob(I)alamin (Cbl). As in air-breathing rats methionine administration led to the conversion of hepatic 5-methyltetrahydrofolate (MeH4 folate) into formyltetrahydrofolate. The recovery of MeH4 folate levels in liver after its oxidation initiated by methionine was noted and the rate compared with that for air-breathing rats. Oxidation of MeH4 folate was less complete and occurred more slowly in Cbl-inactivated rats as compared with controls. However, recovery of MeH4 folate levels was more rapid in Cbl inactivation. S-Adenosylmethionine did not produce a significant change in MeH4 folate levels in Cbl-inactivated rats, whereas it did so in air-breathing animals.     

Oxidation of compounds metabolized through folate coenzyme pathways in vitamin B12-deficient rats

The effects of vitamin B₁₂ deficiency in rats and dietary supplementation with vitamin B₁₂ and/or l-methionine plus folate on the oxidation of compounds metabolized through folate coenzyme pathways were investigated. Rats fed a vitamin B₁₂-deficient diet oxidized significantly lower amounts in 60 min of l-histidine, glycine, sarcosine, formate, and l-serine to CO₂ than vitamin B₁₂-supplemented controls. Supplementation of the deficient diet with l-methionine plus folate restored the ability to oxidize the ring-2-carbon of l-histidine, the methyl group of sarcosine, and formate to the same level as that observed in animals receiving vitamin B₁₂. In contrast, oxidation of the 1-carbon of glycine and the 3-carbon of l-serine was not restored to control levels by addition of methionine plus folate to the vitamin B₁₂-deficient diet. Inhibition of the metabolism of the 2-carbon of glycine to CO₂ was partially overcome by additional dietary methionine and folate. Glycine synthase activity in homogenates paralleled the in vivo pattern of oxidation of the 1-carbon of glycine to CO₂, whereas sarcosine dehydrogenase activity appeared to increase 2-fold in vitamin B₁₂ deficiency.     

Impaired formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation

The effect of inactivation of cobalamin by N2O on 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 formyltetrahydrofolate 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.     

Detection by electron spin resonance of an exchange-coupled Cob(II)alamin...free radical pair species generated by anaerobic photolysis of polycrystalline adenosylcobalamin

Exposure to nitrous oxide (N₂O) $\text{in vivo}$ is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N₂O and this returns to normal on restoring the animals to an air environment.     

Effects of gibberellic acid and L-methionine on C1 metabolism in aerated carrot disk

Aeration of carrot storage tissue disks in water was accompanied by net folate synthesis and by changes in the specific activities of key folate-dependent enzymes. Disks aerated in 0.1 mM gibberellic acid (GA₃) for 48 hr contained higher concentrations of methyltetrahydrofolates but aeration in 5 mM L-methionine reduced net folate synthesis. Gibberellic acid also increased the specific activities of 5,10-methylenetetrahydrofolate reductase (E.C. 1.1.1.68), serine hydroxymethyltransferase (E.C. 2.1.2.1) and 5-methyltetrahydrofolate: homocysteine transmethylase. The levels of these enzymes in disks aerated in L-methionine (5 mM) were comparable or slightly higher than those of disks aerated in water. Activity of the reductase and 10-formyltetrahydrofolate synthetase (E.C. 6.3.4.3) was inhibited by L-methionine in vitro. Aeration increased ability to incorporate formate [¹⁴C] into serine, glycine and methionine. Disks aerated for 36 hr in 0.1 mM GA₃ incorporated greater amounts of ¹⁴C into free methionine but those aerated in L-methionine (5 mM) had less ability to metabolize formate and the specific radioactivities of free glycine, serine and methionine were low.     

Folate polyglutamate synthetase activity in the cobalamin-inactivated rat.

Exposure to N2O inactivates cob[I]alamin and interferes with the activity of methionine synthetase, of which cob[I]alamin is a coenzyme. Less directly, it stops the formation of folate polyglutamate from tetrahydrofolates. Studies on the activity of folate polyglutamate synthetase in rat liver in vivo were carried out. The synthetase activity increased after exposure to N2O for up to 48 h, but longer exposure was accompanied by a return of activity to baseline values. The rise in synthetase activity was prevented by supplying methionine, 5'-methylthioadenosine or 5-formyltetrahydrofolate. The fall in folate polyglutamate synthetase activity after 48 h was accompanied by a restoration of hepatic synthesis of folate polyglutamate despite continuation of N2O exposure.     

The toxicity of methanol

Methanol toxicity in humans and monkeys is characterized by a latent period of many hours followed by a metabolic acidosis and ocular toxicity. This is not observed in most lower animals. The metabolic acidosis and blindness is apparently due to formic acid accumulation in humans and monkeys, a feature not seen in lower animals. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H4 folate). An excellent correlation between hepatic H4 folate and formate oxidation rates has been shown within and across species. Thus, humans and monkeys possess low hepatic H4 folate levels, low rates of formate oxidation and accumulation of formate after methanol. Formate, itself, produces blindness in monkeys in the absence of metabolic acidosis. In addition to low hepatic H4 folate concentrations, monkeys and humans also have low hepatic 10-formyl H4 folate dehydrogenase levels, the enzyme which is the ultimate catalyst for conversion of formate to carbon dioxide. This review presents the basis for the role of folic acid-dependent reactions in the regulation of methanol toxicity.     

TheSAM2 gene product catalyzes the formation of S-adenosyl-ethionine from ethionine inSaccharomyces cerevisiae

Ethionine is the toxic S-ethyl analog of the essential amino acid methionine. Whereas in prokaryotes the ethionine just competes with the methionine, in eukaryotes it can also be transformed into S-adenosyl-ethionine (Ado-Eth), competing with the S-adenosyl-methionine (Ado-Met). When the Ado-Met synthetase activity was studied in strains defective in either of the two isoenzymes, the one coded by theSAM1 gene was totally unable to convert ethionine into Ado-Eth and was inhibited by the analog, whereas the enzyme coded by theSAM2 gene was able to bind ethionine and was not inhibited by it. This has allowed the development of a procedure to measure Ado-Met synthetase and differentiate between the two isoenzymes present inSaccharomyces cerevisiae.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), on the subcellular distribution of hepatic folate coenzymes was determined. In controls, cytosolic folates were 5-methyltetrahydrofolate (45%), 5- and 10-formyltetrahydrofolate (9 and 19%, respectively), and tetrahydrofolate (27%). Exposure of rats to an atmosphere containing 80% nitrous oxide for 18 h resulted in a marked shift in this distribution pattern to 5-methyltetrahydrofolate, 84%; 5- and 10-formyltetrahydrofolate, 2.1 and 9.1%, respectively; and tetrahydrofolate, 4.7%. Activity of the cytosolic enzyme, methionine synthetase, was reduced by about 84% as compared to that of air breathing controls. In controls, mitochondrial folates were 5-methyltetrahydrofolate (7.3%), 5- and 10-formyltetrahydrofolate (11.5 and 33.1%, respectively), and tetrahydrofolate (48.1%). This distribution did not change after exposure to nitrous oxide. These results show that the effects of nitrous oxide inactivation of vitamin B12 are confined to the cytosol, at least in the short term, and suggest that there is little, if any, transport of free folates between the cytosolic and mitochondrial compartments.     

Methylthioadenosine serves as a single carbon source to the folate co-enzyme pool in rat bone marrow cells

[ribose-U-14C]Methylthioadenosine (MTA) was prepared by incubating methionine with [14C-U]ATP in the presence of methionine adenosyltransferase and the resulting S-adenosylmethionine was heated to release MTA. Labelled [14C]MTA, when incubated with rat bone marrow cells, yielded [14C]formate which was used in the synthesis of adenine and guanine. Unlike 14C from sodium, formate, serine and glycine, there was no decline in 14C utilization from MTA with bone marrow cells from rats in which cobalamin had been inactivated by exposure to nitrous oxide. It was concluded that methionine via MTA is a significant contributor of single-carbon units at the formate level of oxidation and that this pathway is maintained in cobalamin 'deficiency'.     

Regulation of 5-methyltetrahydrofolate synthesis.

After an intraperitoneal injection of 100 mumol of methionine to rats, there is rapid oxidation of the methyl group of hepatic 5-methyltetrahydrofolate to formate and CO2. Recovery of the methylfolate level starts 2.5 h after the methionine injection, when the hepatic methionine level and the S-adenosylmethionine/S-adenosylhomocysteine ratio have returned to baseline values. S-Adenosylmethionine concentration is still elevated at this time.     

Metabolism of 5-methylthioribose to methionine

During ethylene biosynthesis, the H₃CS- group of S-adenosylmethionine is released as 5′-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, [¹⁴C]MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of [ribose-U-¹⁴C]MTR with avocado extract resulted in the production of [¹⁴C]formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, l-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O₂ in the conversion of MTR to methionine is discussed.     

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4–8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [¹³C₁]methionine and [²H₃-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 μg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70–80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.     

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.     

Effect of nitrous oxide-induced inactivation of vitamin B12 on glycinamide ribonucleotide transformylase and 5-amino-4-imidazole carboxamide transformylase

Exposure to nitrous oxide (N2O) in vivo is accompanied by oxidation of cob[I]-alamin to the inactive cob[III]alamin [1]. There is loss of methionine synthetase activity [2] and evidence of depressed supply of single carbon units at the formate level of oxidation [3,4,5]. We measured the effect of inactivation of B12 on the folate-dependent transformylases concerned in purine synthesis. After 24 h exposure to N2O there was a significant fall in glycinamide ribonucleotide transformylase (EC 2.1.2.2) and a significant increase in 5-amino-4-imidazole carboxamide transformylase (EC 2.1.2.3).