Altered levels of S-adenosylmethionine and S-adenosylhomocysteine in the brains of L-isoaspartyl (D-Aspartyl) O-methyltransferase-deficient mice

L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein repair enzyme that initiates the conversion of abnormal D-aspartyl and L-isoaspartyl residues to the normal L-aspartyl form. In the course of this reaction, PCMT1 converts the methyl donor S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Due to the high level of activity of this enzyme, particularly in the brain, it seemed of interest to investigate whether the lack of PCMT1 activity might alter the concentrations of these small molecules. AdoMet and AdoHcy were measured in mice lacking PCMT1 (Pcmt1-/-), as well as in their heterozygous (Pcmt1+/-) and wild type (Pcmt1+/+) littermates. Higher levels of AdoMet and lower levels of AdoHcy were found in the brains of Pcmt1-/- mice, and to a lesser extent in Pcmt1+/- mice, when compared with Pcmt1+/+ mice. In addition, these levels appear to be most significantly altered in the hippocampus of the Pcmt1-/- mice. The changes in the AdoMet/AdoHcy ratio could not be attributed to increases in the activities of methionine adenosyltransferase II or S-adenosylhomocysteine hydrolase in the brain tissue of these mice. Because changes in the AdoMet/AdoHcy ratio could potentially alter the overall excitatory state of the brain, this effect may play a role in the progressive epilepsy seen in the Pcmt1-/- mice.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.     

Regulation of S-adenosylhomocysteine hydrolase in the halophilic cyanobacterium Aphanothece halophytica: a possible role in glycinebetaine biosynthesis

Aphanothece halophytica, a halophilic cyanobacterium capable of growing in saturated NaCl, accumulates high intracellular concentrations of glycinebetaine in response to increasing environmental NaCl. In this organism, intracellular levels of K⁺ rise dramatically with increasing external NaCl before an increase in glycinebetaine can be detected. Glycinebetaine synthesis requires three S-adenosylmethionine (AdoMet)-mediated transmethylations; each transmethylation reaction generates one molecule of the transmethylation inhibitor S-adenosylhomocysteine (AdoHcy). Thus, glycinebetaine synthesis should require continued removal of AdoHcy. In A. halophytica, catabolism of AdoHcy was shown to occur via the reversible reaction catalyzed by AdoHcy hydrolase (EC 3.3.1.1). Activity of AdoHcy hydrolase in the direction of synthesis of AdoHcy was inhibited by 0.4 M KCl in this organism. On the other hand, activity of AdoHcy hydrolase in the direction of AdoHcy hydrolysis was unaffected by 0.4 M KCl. Glycinebetaine increased synthesis of AdoHcy in the presence of 0.4 KCl, but had no effect on AdoHcy hydrolysis. Based upon these results, a mechanism is proposed for the regulation of glycinebetaine synthesis by K⁺ and glycinebetaine in A. halophytica. According to this mechanism, the regulatory response would be initiated by a K⁺-induced shift in the AdoMet/AdoHcy ratio.Abbreviations AdoMet S-adenosylmethionine - AdoHcy S-adenosyl homocysteine     

Relationship between tissue levels of S-adenosylmethionine, S-adenylhomocysteine, and transmethylation reactions.

The concentrations of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy), and various methyltransferases were determined in the cerebrum, cerebellum, and liver of rats during development and aging. The liver contained from 3 to 7 and from 10 to 15 nmol AdoHcy per gram in young and adult rats, respectively. The AdoMet concentration was 60 to 90 nmol/g liver from rats of the same age and sex. It did not vary significantly with age. In the brain the AdoMet concentration was 45 to 50 nmol/g at birth and decreased to 20 nmol/ g tissue with maturity of the organ. The level of AdoHcy in this organ was less than 1 nmol/g tissue throughout the life-span of the rat. Since the ratio of AdoMet to AdoHcy is relatively high, the rate of methylation of histones, DNA, or phosphatidylethanolamine in the liver or brain was not significantly influenced by AdoHcy. Under normal nutritional conditions, the tissue concentration of AdoMet is far above the Km values of histone and phosphatidylethanolamine methyltransferases. The levels of activity of these enzymes in liver and brain did not correlated with the cellular concentration of AdoHcy. Thi histone methyltransferase activity was elevated in rapidly proliferating tissues and declined markedly in the absence of histone biosynthesis. Phosphatidylethanolamine methyltransferase activity was elevated during development of the liver. The specific activity of the AdoHcy hydrolase remained relatively constant in the rat brain and liver. The activity of this enzyme was 10 times higher in liver than in brain, yet the concentration of AdoHcy was much lower in the latter organ. The tissue levels of this compound are evidently dependent on the rates of removal of homocysteine and adenosine. Adenosine deaminase was present in the liver and brain at relatively high concentrations, particularly during development.     

Increase in S-adenosylhomocysteine concentration in interferon-treated HeLa cells and inhibition of methylation of vesicular stomatitis virus mRNA

A fraction of the viral mRNA synthesized in interferon-treated HeLa cells infected with vesicular stomatitis virus (VSV) lacks the 7-methyl group in the 5'-terminal guanosine of the cap; this mRNA is not associated with polyribosomes and does not bind to ribosomes in an assay for initiation of protein synthesis (de Ferra, F., and Baglioni, C. (1981) Virology 112, 426-435). To establish whether this defect in methylation is due to changes in the level of the methyl donor S-adenosylmethionine (AdoMet) and of its competitive inhibitor S-adenosylhomocysteine (AdoHcy), we measured the concentration of these compounds in HeLa cells treated with interferon. An increase in both AdoMet and AdoHcy was detected 3 to 6 h after addition of interferon. The level of these compounds increased gradually and in proportion to the interferon concentration used. With 125 reference units/ml of beta interferon, for example, the AdoHcy concentration increased more than 3-fold and that of AdoMet about 1.5-fold with a consequent change in the AdoHcy/AdoMet ratio. An increased AdoHcy/AdoMet ratio was also found in HeLa cells treated with pure alpha 2 interferon produced in Escherichia coli by recombinant DNA techniques. When the methylation of VSV mRNA was measured in assays carried out with permeabilized virions at the AdoHcy and AdoMet concentrations found in interferon-treated cells, a preferential inhibition of the viral (guanine-7-)methyltransferase activity was observed. Such an inhibition may account for the synthesis of VSV mRNA lacking the 7-methyl group of guanosine in the cap.     

Diet-dependent survival of protein repair-deficient mice

Protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein-repair enzyme, and mice lacking this enzyme accumulate damaged proteins in multiple tissues, die at an early age from progressive epilepsy and have an increased S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy) ratio in brain tissue. It has been proposed that the alteration of AdoMet and AdoHcy levels might contribute to the seizure phenotype, particularly as AdoHcy has anticonvulsant properties. To investigate whether altered AdoMet and AdoHcy levels might contribute to the seizures and thus the survivability of the repair-deficient mice, a folate-deficient amino acid-based diet was administered to the mice in place of a standard chow diet. We found that the low-folate diet significantly decreases the AdoMet/AdoHcy ratio in brain tissue and results in an almost threefold extension of mean life span in the protein repair-deficient mice. These results indicate that the increased AdoMet/AdoHcy ratio may contribute to the lowered seizure threshold in young PCMT1-deficient mice. However, mean survival was also extended almost twofold for mice on a control folate-replete amino acid-based diet compared to mice on the standard chow diet. Survival after 40 days was similar in the mice on the low- and high-folate amino acid-based diets, suggesting that the survival of older PCMT1-deficient mice is not affected by the higher brain AdoMet/AdoHcy ratio. Additionally, the surviving older repair-deficient mice have a significant increase in body weight when compared to age-matched normal mice, independent of the type of diet. This weight increase was not accompanied by an increase in consumption levels, indicating that the repair-deficient mice may also have an altered metabolic state.     

Adenosine dialdehyde and neplanocin A: Potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N2a cells

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase, E.C. 3.3.1.1) catalyzes the metabolism of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy) in mouse neuroblastoma N2a cells. AdoHcy hydrolase in N2a cells can be inhibited completely by adenosine dialdehyde (Ado dialdehyde) or neplanocin A. The inhibitory effects of Ado dialdehyde (2.5 μM) and neplanocin A (1 μM) on cellular AdoHcy hydrolase were time-dependent, with total enzyme inhibition occurring after 30 min and 15 min of incubation, respectively. The inhibition of AdoHcy hydrolase produced by Ado dialdehyde and neplanocin A persisted for up to 72 h of incubation, and was paralleled by a time-dependent increase in endogenous AdoHcy levels reaching a maximum 4-fold elevation after 8 h of incubation with Ado dialdehyde and an 11-fold increase in the neplanocin A-treated cells. This increase in AdoHcy levels produced a subsequent inhibition of S-adenosylmethionine (AdoMet)-dependent cellular methylations (e.g. protein carboxylmethylation (PCM), lipid methylation). In addition, neplanocin A was metabolically converted to the corresponding AdoMet analog, S-neplanocylmethionine (NepMet), in neuroblastoma N2a cells. NepMet reached maximum levels after 8 h of incubation of the cells with neplanocin A.     

Structure and mechanism of mRNA cap (guanine-N7) methyltransferase

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.     

Inhibitory effect of Epimedium extract on S-adenosyl-L-homocysteine hydrolase and biomethylation

In the present paper, the inhibitory effect of Epimedium extract on the activity of S-adenosyl-L-homocysteine (AdoHcy) Hydrolase was studied. The results showed that Epimedium extract inhibited the activity of recombinant human AdoHcy hydrolase in a dose-dependent manner. This inhibitory effect was also observed in hepatic cell line 7701 and hepatoma HepG2, however, the effect in 7701 cells was more potent than in HepG2 cells. The extract could significantly reduce AdoMet/AdoHcy ratio in 7701 cells in a dose-dependent manner, suggesting reduced biomethylation level in 7701 cells. In contrast, it resulted in elevated AdoMet/AdoHcy ratio in the HepG2 cells. The result of MALDI-MS assay indicated that epimedin A and ikarisoside F from the extract could bind to AdoHcy hydrolase. The present data suggested that Epimedium extract could inhibit the activity of AdoHcy hydrolase, thus regulating the cellular biomethylation as well as reducing cellular Hcy level. These results will provide new clues to the mechanisms of Epimedium in curing of cardiovascular disease and regulating tumor cell growth.     

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.     

The Methylation Cycle and its Possible Functions in Barley Endosperm Development

Barley endosperm development can be subdivided into the pre-storage, intermediate, storage and desiccation phase. Nothing is known about DNA methylation events involved in different endosperm-specific developmental programmes. A complete set of methylation cycle enzyme genes was identified and investigated by mRNA expression analysis. During the pre-storage phase, methionine synthase and S-adenosylmethionine (AdoMet) synthase genes are expressed at high levels, mainly to produce AdoMet, which might be used for methylation processes as indicated by high expression of methyltransferases HvMET1, HvCMT1 and HvDnmt3-1 as well as AdoHcy hydrolase genes. The methyltransferases, core histones and DNA-unwinding ATPases are co-expressed at the mRNA level. On the contrary, storage protein (prolamin) gene expression is repressed due to CpG methylation. Expression of genes responsible for starch biosynthesis is also developmentally regulated but not methylation-dependent. Thus, during pre-storage phase, activity of HvMET1 and HvCMT1 possibly maintains DNA replication and suppresses specific pathways of maturation. Besides, HvDnmt3-1 might be responsible for differentiation-specific de novo methylation. Expression of methyltransferases HvDnmt3-2 and HvCMT2 peaks during the onset of massive starch accumulation. The enzymes are likely responsible for DNA methylation involved in determining plastid division and amyloplast differentiation as concluded from the patterns of co-expressed genes. Levels of AdoMet decarboxylase mRNA, but not methyltransferase- and AdoHcy mRNA, increase at the beginning of desiccation together with methionine synthase and AdoMet synthase levels. This increase may be indicative for utilization of AdoMet in polyamine production protecting aleuron and embryo cell membranes during desiccation.     

Tissue-specific effects of testosterone on S-adenosylmethionine formation and utilization in the mouse.

Exogenous administration of testosterone produced several metabolic tissue-specific changes in female mouse kidneys, but not in the liver. The hormone induced ornithine decarboxylase (ODC) activity, and also profoundly influenced metabolism of S-adenosylmethionine (AdoMet). Therefore, the activity of the AdoMet-synthesizing enzyme (AdoMet synthetase) and of cystathionine synthase, which commits homocysteine irreversibly to the transsulfuration pathway, were significantly increased. In contrast to the level of AdoMet in the liver the renal level of this metabolite was augmented, whereas the level of S-adenosylhomocysteine (AdoHcy) did not change. This resulted in an increase of the AdoMet/AdoHcy ratio. In testosterone-treated mice, pulse-labelled with [methyl-14C]methionine, the radioactivity recovered in the kidneys doubled, but in the liver remained the same. The rise in radioactivity recovered occurred mainly in TCA-soluble compounds and lipids, and to a smaller extent, in proteins and nucleic acids.     

The role of hyperhomocysteinemia in nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation.

Hyperhomocysteinemia (HHcy) is associated with impaired endothelial-dependent vasodilatation and increased risk of atherosclerosis and thrombosis. Here, we summarize some of our previous work on the effect of HHcy on pathways involved in endothelium-dependent vasodilatation, and present new data concerning the endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation. We showed that the 894 G>T single-nucleotide polymorphism in the human endothelial nitric oxide synthase gene (eNOS) increased the risk of recurrent venous thrombosis in individuals with elevated homocysteine levels, indicating that the pathophysiological mechanism in HHcy involves impaired NO-mediated vasodilatation. In addition, the EDHF-mediated vasodilatation of the renal artery was disturbed in diet-induced hyperhomocysteinemic rats. Interestingly, we demonstrated that pretreatment of rats with periodate-oxidized adenosine (Adox), which is an inhibitor of S-adenosylhomocysteine hydrolase, prevented the methionine-induced rise in plasma total Hcy (tHcy) levels but not the inhibition of the EDHF pathway. Furthermore, we demonstrated that S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) levels were increased in the kidneys of diet-induced HHcy rats, resulting in a decreased AdoMet:AdoHcy ratio. In addition, we demonstrated that mRNA expression of Connexin 40, which is one of the structural subunits of gap-junctions, was down-regulated in endothelial cells of HHcy rats, and correlated with elevated AdoHcy levels in kidney of these rats. These finding suggest a key role for AdoHcy in relation to decreased Cx40 mRNA expression and impaired EDHF-mediated vasodilatation of HHcy rats.     

Decreased Transmethylation of Biogenic Amines After In Vivo Elevation of Brain S-Adenosyl-l-Homocysteine

Abstract: The ability of S -adenosyl- l -homocysteine (AdoHcy) to inhibit biologic transmethylation reactions in vitro has led us to explore the possibility of pharmacologically manipulating AdoHcy levels in vivo and examining the consequences of these alterations on the transmethylation of some biogenic amines. Swiss-Webster mice were injected intraperitoneally with different doses of adenosine (Ado) and d,l -homocysteine thiolactone (Hcy) and were killed at various times thereafter. S -Adenosyl- l -methionine (AdoMet) and AdoHcy concentrations were determined by using a modified isotope dilution-ion exchange chromatography-high pressure liquid chromatography technique sensitive to less than 10 pmol. Increasing doses of Ado + Hcy (50-1000 mg/kg of each) produced a dose-related increase in blood, liver, and brain AdoHcy levels. At a dose level of 200 mg/kg Ado + Hcy, AdoHcy levels were markedly elevated, with minimal concomitant perturbations of AdoMet. This elevation was maximal 40 min after giving Ado + Hcy, returning to control values within 6 h. Ado + Hcy treatment resulted in decreased activities of catechol- O -methyltransferase, histamine- N -methyltransferase, and AdoHcy hydrolase in vitro. The cerebral catabolism of intraventricularly administered [³H]histamine (HA) was decreased in a dose-related manner by Ado + Hcy treatment as evidenced by higher amounts of nonutilized [³H]HA in brain, concurrent decreases in [³H]methylhistamine formation, and decreases in the transmethylation conversion index. Steady state levels of HA also showed dose-related increases after Ado + Hcy treatment. It is concluded that injections of Ado + Hcy can markedly elevate AdoHcy levels in vivo , which can, in turn, decrease the rate of transmethylation reactions.     

Protein Arginine Methylation Is More Prone to Inhibition by S-Adenosylhomocysteine than DNA Methylation in Vascular Endothelial Cells

Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

E1 mice epilepsy shows genetic polymorphism for S-Adenosyl-L-homocysteine hydrolase

E1 mice are an animal model of human epilepsy (idiopathic complex partial seizures). We have previously demonstrated abrupt poly(A)(+) RNA expression in liver from 1-day-old E1 mouse [Mita et al., 1991. Devl. Brain Res. 64, 27-35]. In the present study, we constructed a cDNA library of the poly(A)(+) RNA. By analyzing cDNA clones and nucleotide sequences, we found a clone that was homologous to a rat gene of S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1.) (SAHH) (a key enzyme in the active methyl transfer pathway) and showed the gene polymorphism/RFLP(PstI) between the epileptic strain, E1, and the non-epileptic mother strain, ddY, as indicated in a gel electrophoresis by cleaving 2.6 kb with PstI into 1.9 kb and 0.7 kb fragment bands. F1(E1xddY) showed the heterozygosity. An attempt to determine the mutation on the genomic SAHH gene in the E1 disclosed a single nucleotide polymorphism indicated by a C-->T transition in the 8th intron, by which the PstI site was created. SAHH enzymatic activity in the liver in 1-day-old E1 mice was slight (approximately 10%), and in fact was significantly lower than that of the control ddY. Results suggested that the abrupt primary mRNA transcribed on the SAHH gene in the liver of 1-day-old E1 mice was processed partially or incompletely because of the presence of the point mutation in the intron. Accordingly, poor energy supply by the insufficient SAHH enzymatic activity in the brain postnatally may be responsible for epileptogenesis in this animal model. It is concluded that a single nucleotide SAHH gene polymorphism may be associated with epilepsy in E1 mice.