Homocysteine Methyltransferase Activity Research Articles

Betaine alleviates hepatic lipid accumulation via enhancing hepatic lipid export and fatty acid oxidation in rats fed with a high-fat diet.

To assess the effects of betaine on hepatic lipid accumulation and investigate the underlying mechanism, thirty-two male Sprague-Dawley rats weighing 100 (sd 2·50) g were divided into four groups, and started on one of four treatments: basal diet, basal diet with betaine administration, high-fat diet and high-fat diet with betaine administration. The results showed that no significant difference of body weight was found among experimental groups. Compared with high-fat diet-fed rats, a betaine supplementation decreased (P< 0·05) hepatic TAG accumulation induced by high-fat diet, which was also supported by hepatic histology results. Additionally, hepatic betaine-homocysteine methyltransferase concentration [corrected] as well as its mRNA abundance and lecithin level were found increased (P< 0·05) by betaine supplementation in both basal diet-fed rats and high-fat diet-fed rats. Betaine administration in high-fat diet-fed rats exhibited a higher (P< 0·05) concentration [corrected] of hepatic carnitine palmitoyltransferase 1 (CPT1) compared with high-fat diet-fed rats. High-fat diet inhibited (P< 0·05) the gene expression of hepatic PPARα and CPT1. However, betaine administration in high-fat diet-fed rats elevated (P< 0·05) the gene expression of PPARα and CPT1. Moreover, concentration, gene and protein expressions of hepatic fibroblast growth factor 21 (FGF21) were increased (P< 0·05) in response to betaine administration in high-fat diet group; meanwhile the gene expression of hepatic AMP-activated protein kinase was increased (P< 0·05) as well. The results suggest that betaine administration enhanced hepatic lipid export and fatty acid oxidation in high-fat diet-fed rats, thus effectively alleviating fat accumulation in the liver.

Homocysteine Methyltransferases Mht1 and Sam4 Prevent the Accumulation of Age-damaged (R,S)-AdoMet in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosyl-l-methionine (AdoMet) is spontaneously degraded by inversion of its sulfonium center to form the R,S diastereomer. Unlike its precursor, (S,S)-AdoMet, (R,S)-AdoMet has no known cellular function and may have some toxicity. Although the rate of (R,S)-AdoMet formation under physiological conditions is significant, it has not been detected at substantial levels in vivo in a wide range of organisms. These observations imply that there are mechanisms that either dispose of (R,S)-AdoMet or convert it back to (S,S)-AdoMet. Previously, we identified two homocysteine methyltransferases (Mht1 and Sam4) in yeast capable of recognizing and metabolizing (R,S)-AdoMet. We found similar activities in worms, plants, and flies. However, it was not established whether these activities could prevent R,S accumulation. In this work, we show that both the Mht1 and Sam4 enzymes are capable of preventing R,S accumulation in Saccharomyces cerevisiae grown to stationary phase; deletion of both genes results in significant (R,S)-AdoMet accumulation. To our knowledge, this is the first time that such an accumulation of (R,S)-AdoMet has been reported in any organism. We show that yeast cells can take up (R,S)-AdoMet from the medium using the same transporter (Sam3) used to import (S,S)-AdoMet. Our results suggest that yeast cells have evolved efficient mechanisms not only for dealing with the spontaneous intracellular generation of the (R,S)-AdoMet degradation product but for utilizing environmental sources as a nutrient.

Recognition of Age-damaged (R,S)-Adenosyl-L-methionine by Two Methyltransferases in the Yeast Saccharomyces cerevisiae

The biological methyl donor S-adenosylmethionine (AdoMet) can exist in two diastereoisomeric states with respect to its sulfonium ion. The S configuration, (S,S)-AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the R form, producing (R,S)-AdoMet. As of yet, (R,S)-AdoMet has no known physiological function and may inhibit cellular reactions. In this study, we found two Saccharomyces cerevisiae enzymes that are capable of recognizing (R,S)-AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, identified previously as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine, respectively. We found here that Sam4 recognizes both (S,S)- and (R,S)-AdoMet, but that its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet, whereas no activity is seen with the S,S form. R,S-Specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)-AdoMet in these organisms.

A nuclear-magnetic-resonance-based assay for betaine–homocysteine methyltransferase activity

Betaine–homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by 1H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1 h. Using 2 mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N, N-dimethylglycine, the CV is 6.3% ( n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N, N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n=6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K m for glycine betaine is 0.19 ± 0.03 mM with a V max of 17 ± 0.7 nMol min −1 mg −1.

Betaine-Homocysteine Methyltransferase-2: cDNA Cloning, Gene Sequence, Physical Mapping, and Expression of the Human and Mouse Genes

Anomalies in folate and homocysteine metabolism can result in homocysteinemia and are implicated in disorders ranging from vascular disease to neural tube defects. Two enzymes are known to methylate homocysteine, vitamin B12-dependent methionine synthase (MTR) and betaine-homocysteine methyltransferase (BHMT). BHMT uses betaine, an intermediate of choline oxidation, as a methyl donor and is expressed primarily in the liver and kidney. We report the discovery of a novel betaine-homocysteine methyltransferase gene in humans and mice. The human BHMT2 gene is predicted to encode a 363-amino-acid protein (40.3 kDa) that shows 73% amino acid identity to BHMT. The BHMT2 transcript in humans is most abundant in adult liver and kidney and is found at reduced levels in the brain, heart, and skeletal muscle. The mouse Bhmt2 gene shows 69% amino acid identity and 79% similarity to the mouse Bhmt gene and 82% amino acid identity and 87% similarity to the human BHMT2 gene. Bhmt2 is expressed in fetal heart, lung, liver, kidney and eye. The discovery of a third gene with putative homocysteine methyltransferase activity is important for understanding the biochemical balance in using methyltetrahydrofolate and betaine as methyl donors as well as the metabolic flux between folate and choline metabolism in health and disease.

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.

Homocysteine-Dependent Demethylation of Trimethylselenonium Ion and Selenobetaine with Methionine Formation

In the presence of rat liver cytosol and homocysteine, trimethylselenonium ion (TMSe+) underwent time-dependent demethylation to dimethylselenide with the concurrent formation of methionine. Convenient methods were developed for assay of this activity using either radioactive methods based on the gamma emitting isotope 75Se or nonradioactive HPLC assay of methionine. The rate of demethylation was linear with protein concentration and dependent on homocysteine, which could not be replaced by cysteine, glutathione, or dithiothreitol. The TMSe+ demethylation rate was inhibited by the addition of betaine, sulfobetaine (dimethylthetin), or dimethylglycine. The Km for TMSe+ was 8 mM compared to 0.04 mM for betaine, but the rate of TMSe+ demethylation was approximately 50-fold that of betaine when both were assayed at 25 mM. Methionine was also produced from selenobetaine, selenobetaine methylester, and sulfobetaine. The selenium analogues of betaine inhibited the demethylation of TMSe+ with only minor decreases in methionine production, indicating substrate competition. In preliminary studies aimed at the partial purification of the TMSe+:homocysteine methyltransferase activity, the enzyme was found to have chromatographic and heat stability characteristics similar to betaine:homocysteine methyltransferase. The data indicate that betaine:homocysteine methyltransferase, or a very similar enzyme, is involved in the demethylation of TMSe+ and show that TMSe, an in vivo urinary selenium metabolite of many selenium compounds, is not biologically inert.

Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease

Elevated plasma homocysteine enhances the risk of thrombosis and premature arteriosclerosis. We have assessed the activity of the 3 prime enzymes of homocysteine metabolism in cultured human venous endothelial cells, in a study of their possible protective roles. In cells from 4 individuals, cultured in Dulbecco's modified Eagle medium, the mean activity ± S.D. of cystathionine β-synthase (nmol of product/h per mg of cell protein, at 37°C) was 3.58 ± 3.11 at pH 8.6. The assay used was our newly developed amino acid analyser-based procedure. The activity of 5-methyltetrahydrofolate:homocysteine methyltransferase at pH 7.4 was 4.12 ± 1.25 and betaine:homocysteine methyltransferase (BHMT) was undetectable (< 1.4 nmol/h per mg protein). Cells were also cultured in a medium aimed at stimulating methionine biosynthesis, containing methionine-deficient Dulbecco's modified Eagle medium to which L-homocystine (100 μmol/1) and methylcobalamin (1 μmol/1) had been added. In these cells 5-methyltetrahydrofolate:homocysteine methyltransferase activity increased to 7.95 ± 1.45, P < 0.001, there was a non-significant decrease in cystathionine β-synthase activity to 2.16 ± 1.52 and BHMT activity was still undetectable. These cells were more resistant to in vitro homocysteine-induced detachment than were cells from the same line cultured in Dulbecco's modified Eagle medium alone. Our findings establish that human endothelial cells express 2 of the 3 primary enzymes of homocysteine catabolism. They suggest that persons who are deficient in cystathionine β-synthase or 5-methyltetrahydrofolate:homocysteine methyltransferase activity may not only develop homocysteinemia, but also have vascular endothelium which is more susceptible to damage by homocysteine than persons with normal enzyme levels.

Indirect measurement of betaine - homocysteine methyltransferase activity using 1H nmr spectroscopy

The (re)methylation of homocysteine to methionine is an essential process for continuation of the cycle whereby Sadenosylmethionine acts as the major methyl donor for biosynthesis. There are two enzymes capable of mediating the homocysteine to m e t h i o n i n e c o n v e r s i o n : N 5 methyltetrahydrofolate-homocysteine methyltransferase with the methyl group provided by N5-methyltetrahydrofolate o r betaine-homocysteine methyl transferase (BHMT), the methyl group being donated by betaine leaving N-dimethylglycine (DMG) as product. Attempts have been made to assess the relative contributions of these reactions e.g. by estimating the production of sarcosine in patients with sarcosinaemia [l]. However, the accumulation of DMG (the immediate precursor of sarcosine) was not measured. We have investigated patients with the disorder multiple acyl CoA dehydrogenase deficiency (glutaric aciduria type 11; [2]) . The disorder is caused by a deficiency in either electron transfer flavoprotein (ETF) o r ETFubiquinone reductase (ETF-QO) and has been characterised by the excretion of several short-chain dicarboxylic acids as a result of the block in metabolism of precursor CoA analogues and subsequent 61 and 61-1 oxidation [ 3 1 . The enzymes DMG dehydrogenase and sarcosine dehydrogenase are also ETF(-QO) dependent and whereas sarcosine excretion has been reported in such patients no investigation of DMG metabolism has been carried out. Urine from a neonate with multiple acyl-CoA dehydrogenase deficiency was immediately frozen at -20 prior to nmr analysis. Spectra were recorded at 400 and 500 MHz with a sweep of 12 ppm, a 90 pulse and recycling time of 5 s . 120 scans were generally sufficient to give good signal to noise. The spectrum from the patient shows several abnormal resonances. Signals from adipic, suberic , sebacic, glutaric and isovaleric acids are present. At 2.93 and 3.73 ppm are large signals from DMG and at 2.74 ppm a small signal from sarcosine. The excretion rate of DMG expressed as creatinine equivalents was 1.5. mol/mol. Excretion of DMG in healthy neonates is usually less than 0.3 mol/mol creatinine and sarcosine < 0.1 (Davies and Iles, unpublished). Perhaps surprisingly betaine excretion was within the normal range for a child of this age [41. As the excretion of DMG was so large in comparison to sarcosine it is likely that the inhibition of DMG

Betaine metabolism in human neonates and developing rats

Proton nuclear magnetic resonance spectroscopy has been used to demonstrate the presence of high concentrations of betaine (up to 0.75 mol/mol creatinine) in the urine of normal healthy human neonates. Betaine is not normally excreted in adults. Excretion of betaine from birth to 7 days old was monitored. The excretion of betaine in rats from 21 days after birth to 40–45 days old was also monitored. A peak in excretion in the rats of 1.5-3 mol/mol creatinine occurred between days 30–35. The presence of a high concentration of betaine in the urine is unlikely to be caused by a relative lack of betaine homocysteine methyl transferase activity compared with adults but may relate to the disposal of dietary choline during development.

Effect of chronic primidone treatment on folate-dependent one-carbon metabolism in the rat

Rats were treated chronically with primidone (100 mg/kg/12 hr, p.o.) for up to 8 weeks. The effects of this treatment on one-carbon metabolism were determined in brain and liver. Serine hydroxymethyltransferase activity increased in both brain (44%) and liver (50%). Methylene-tetrahydrofolate reductase activity increased in liver (26%) with a significant correlation to the length of treatment, but in brain it was unchanged. Methyltetrahydrofolate:homocysteine methyltransferase activity increased in brain (43%) with a significant correlation to length of treatment, but in liver no effect was observed. Methionine adenosyltransferase activity in brain was significantly lower than control at only one point after 8 weeks of chronic treatment. S-Adenosylmethionine concentration in liver increased gradually (23%) during treatment. S-Adenosylhomocysteine concentrations decreased in brain (33%) and increased in liver (23%) with chronic primidone treatment. These data support the hypothesis that chronic primidone treatment leads to folate depletion through interference with folate metabolism.

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.

Effect of chronic valproate treatment on folate-dependent methyl biosynthesis in the rat

Folate deficiency has been associated with chronic anticonvulsant therapy. Characterization of the effects of individual anticonvulsants has been undertaken. Chronic treatment of rats with sodium valproate caused a decrease in liver folate concentration with concomitant increases in brain and plasma folate concentrations. After several weeks, these trends were reversed and folate concentrations tended to normalize. Chronic valproate treatment affected the activities of folate-dependent one-carbon enzymes: Serine hydroxymethyltransferase activity in liver was increased; methylenetetrahydrofolate reductase activity in both brain and liver was decreased; and methyltetrahydrofolate:homocysteine methyltransferase activity in both brain and liver decreased initially but returned toward normal with continued treatment. Methionine adenosyltransferase activity in brain declined after several weeks of treatment but the concentration of S-adenosylmethionine in liver increased with chronic valproate treatment. These data are consistent with the hypothesis that the effects of anticonvulsants on folates are a consequence of the mechanism of action of the anticonvulsant.

Reduction of hepatic tetrahydrofolate and inhibition of exhalation of 14CO2 formed from [dimethylamino-14C]aminopyrine in nitrous oxide-treated rats.

The exhalation of 14CO2 after the administration of [dimethylamino-14C]aminopyrine to an organism is assumed to reflect the demethylation of aminopyrine by hepatic mixed-function oxidase activity. The formaldehyde formed as a result of the demethylation of aminopyrine is then sequentially oxidized to formic acid and CO2. The last step in the pathway, i.e., formate oxidation, is dependent upon tetrahydrofolate; thus, factors which alter hepatic tetrahydrofolate potentially may modify 14C-aminopyrine metabolism to 14CO2 in vivo. Exposure of rats to nitrous oxide (N2O) produces a significant reduction in hepatic tetrahydrofolate as a result of the inhibition of 5-methyltetrahydrofolate:homocysteine methyltransferase activity (E.C. 2.1.1.13). In the present study, exposure of rats to N2O/O2 (1:1) for 4 hr prior to the administration of 14C-aminopyrine (40 or 400 mumoles per kg) produced a 60% reduction in the peak rate of 14CO2 exhalation and a 45% decrease in the total 14CO2 exhaled within 2 hr. In control experiments, exposure of rats to nitrogen/O2 (1:1) produced no effect on 14C-aminopyrine metabolism to 14CO2. Administration of methionine (1.3 mmoles per kg) 30 min prior to 14C-aminopyrine administration reversed the inhibition of 14CO2 exhalation and reduction in hepatic tetrahydrofolate observed in N2O-exposed animals. Aminopyrine (400 mumoles per kg) administration to air-breathing rats did not affect the level of urinary formate, but exposure to N2O produced a 40-fold increase. Aminopyrine administration to N2O-exposed rats produced a 75% increase in urinary formate as compared to rats treated with N2O alone.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Methyltetrahydrofolate-related enzymes and DNA polymerase α in normal and malignant hematopoietic cells

The activities of 5-methyltetrahydrofolate (5- CH 3-THF)-related enzymes [5- CH 3-THF homocysteine methyltransferase and 5,10-methylenetetrahydrofolate (5,10- CH 2-THF) reductase] and DNA polymerase α were measured in normal and malignant hematopoietic cells. The 5- CH 3-THF homocysteine methyltransferase activity was significantly correlated with 5,10- CH 2-THF reductase activity, indicating that the hematopoietic cells with active biosynthesis of tetrahydrofolate from 5- CH 3-THF also actively synthesize 5- CH 3-THF from 5,10- CH 2-THF. The activities of 5- CH 3-THF-related enzymes had a tendency to be high in lymphoid cells and low in myeloid cells, and were not correlated with the percentage of blasts and immature cells in the samples examined. Fairly good correlations were observed among these three enzymes in non-malignant bone marrow cells. However, the activities of two of the enzymes correlated only weakly overall with DNA polymerase α activity in normal and malignant hematopoietic cells. Generally speaking, DNA polymerase α activity correlated well with the percentage of blasts and immature cells in the samples examined.

5-Methyltetrahydrofolate Related Enzymes and DNA Polymerase α Activities in Bone Marrow Cells from Patients with Vitamin B12 Deficient Megaloblastic Anemia

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10-methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10-methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

5-Methyltetrahydrofolate related enzymes and DNA polymerase alpha activities in bone marrow cells from patients with vitamin B12 deficient megaloblastic anemia

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10- methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10- methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

Vitamin B12-dependent methyltetrahydrofolate: Homocysteine methyltransferase activity in normal and leukemic human hematopoietic cells

The cyanogen bromide method was applied to the assay of vitamin B12-dependent methyltetrahydrofolate:homocysteine methyltransferase activity in normal and leukemic human hematopoietic cells.Normal peripheral lymphocytes and leukemia cells of lymphoid origin such as CLL and ALL, contained higher levels of enzyme activity than did normal human bone marrow cells. Normal granulocytes and leukemia cells of myeloid origin, such as CML in the chronic phase and AML, contained lower enzyme activity. Leukemia cells of CML in blast crisis showed higher mean activity than in the chronic phase of the disease.

Betaine-homocysteine methyltransferase in the fungus [formula omitted

A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus Aspergillus nidulans . Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis in vivo . Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.

S-adenosylmethionine:homocysteine methyltransferase during embryogenesis in Musca domestica

S-Adenosylmethionine:homocysteine methyltransferase activity was monitored during embryogenesis of the housefly, Musca domestica. A rapid decrease in the activity of S-adenosylmethionine:homocysteine methyltransferase was observed during the first 3 hr of embryogenesis. Activity continued to decline less rapidly until hatching at 12 hr. An inverse relationship was found to exist between the activities of S-adenosylmethionine:homocysteine methyltransferase and the tRNA methyltransferases during Musca embryogenesis.