S-adenosylmethionine Metabolism Research Articles

Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli.

We have developed a new research tool for the study of S-adenosylmethionine (AdoMet) metabolism by cloning the coliphage T3 AdoMet hydrolase (AdoMetase; EC 3.3.1.2) gene into the M13mp8 expression vector. The recombinant bacteriophage clones expressed an AdoMetase activity in Escherichia coli like that found in T3-infected cells. High levels of AdoMetase expression impaired AdoMet-mediated activities such as dam and dcm methylase-directed DNA modifications and the synthesis of spermidine from putrescine. Expression vectors containing the cloned AdoMetase gene thus provide an alternate approach to the use of chemical inhibitors or mutants defective in AdoMet biosynthesis to probe the effect of AdoMet limitation.

Effect of dietary methionine, arginine and ornithine on the metabolism and accumulation of polyamines, S-adenosylmethionine and macromolecules in rat liver and skeletal muscle.

The interrelationship and possible causality of polyamine synthesis and the transmethylation pathway in the growth-retarding effects of inadequate or excess dietary methionine was studied in young male rats. Feeding the rats for 2 weeks diets containing toxic concentrations of methionine had no effect on polyamine and S-adenosylmethionine metabolism in skeletal muscle, but resulted in markedly elevated concentrations of S-adenosylmethionine and S-adenosylhomocysteine and slightly decreased accumulation of spermine and RNA in the liver. These changes were accompanied by liver-specific stimulation of methionine adenosyltransferase and reduction of spermine synthase activities. Inadequate arginine feeding or supplementation of the diets with ornithine or excess arginine resulted in no apparent changes in tissue methionine or polyamine metabolism and did not alleviate the effects of varied dietary methionine supply. Inhibition of putrescine synthesis by supplementing the diets with 2-difluoromethylornithine did not modify the effects of toxic concentrations of dietary methionine. It is suggested that although hepatic spermine synthase is sensitive to excess methionine feeding, methionine toxicity is not mediated by defective polyamine metabolism.

Effects of alloxan on S-adenosylmethionine metabolism in the rat liver

Effects of alloxan on S-adenosylmethionine metabolism in the rat liver

S-adenosylmethionine metabolism as a target for adenosine toxicity.

Adenosine exerts marked cytostatic and cytotoxic actions to mammalian cells. The nucleoside has been reported to inhibit pyrimidine nucleotide synthesis, to foster cyclic AMP accumulation and to cause the accumulation of S-adenosylhomocysteine (AdoHcy).1,2 Adenosine kinase (EC 2.7.1.20) deficient mammalian cells do not phosphorylate adenosine, but adenosine still blocks their growth, and this Is not reversed by addition of uridine. 2,3 Thus, adenosine may exert toxicity at the nucleoside level. Also, some adenosine analogs are cytotoxic without being converted to nucleotides.4

Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine.

Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine.

METABOLISM OF 5′-METHYLTHIOADENOSINE IN METHIONINE DEPENDENT AND INDEPENDENT CELLS: 33

Methionine (Met) dependency was investigated in a Met-dependent (CCL 39) and in a Met-independent (Raji) cell line. Met dependency was similarly observed with two Met precursors, L-homocysteine and 5'-deoxy-5'-methylthioadenosine (MTA), suggesting that this phenomenon represents, in fact, the inability of a cell to grow on a metabolic precursor of Met. The study of MTA metabolism in CCL 39 and Raji cells indicates that Met dependency is not due to the inability to synthetize Met from MTA, nor to incorporate newly synthetized Met into proteins, nor into S-adenosylmethionine (SAM). However, there are differences in SAM metabolism and disposition between CCL 39 and Raji cells. The synthesis of SAM from labelled MTA is higher in CCL 39 than in Raji cells, but SAM is much more excreted by CCL 39 than by Raji cells, with ratios of extracellular to intracellular SAM of 20.3 and 5.1, respectively. However, when the cells are incubated with labelled Met, the synthesis of SAM is similar in both types of cells, and the ratios of extracellular to intracellular SAM are much lower than in the previous case (0.8 and 1.0 for CCL 39 and Raji cells, respectively). These results, and the fact that exogenous unlabelled Met moderately affects the incorporation of labelled MTA into intracellular SAM by CCL 39 cells, and profoundly depresses it in Raji cells, support the hypothesis of a metabolic compartmentation of exogenous and endogenous Met in Met-dependent cells.

Differential regulation of polyamine synthesis and transmethylation reactions in methylthioadenosine phosphorylase deficient mammalian cells

The consumption of S-adenosylmethionine during polyamine synthesis and transmethylation reactions yields stoichiometric amounts of 5′-deoxy-5′-methylthioadenosine and S-adenosylhomocysteine, respectively. Information concerning the regulation of the two routes of S-adenosylmethionine metabolism in viable cells under changing growth conditions is limited. The present experiments have measured the time-dependent accumulation of 5′-deoxy-5′-methylthioadenosine and l-homocysteine in the medium of four malignant human and murine cell lines deficient in 5′-deoxy-5′-methylthioadenosine phosphorylase (5′-methylthioadenosine: orthophosphate methylthioribosyltransferase). Included in this group were anchorage-independent and anchorage-dependent cells. The enzyme-deficient cells did not detectably cleave 5′-deoxy-5′-methylthioadenosine, and did not appreciably metabolize homocysteine. A comparison of 5′-deoxy-5′-methylthioadenosine and homocysteine excretion therefore provided a noninvasive method for estimating the relative rates of polyamine synthesis and transmethylation. Early after the release of human CEM lymphoblasts from density dependent growth arrest, 5′-deoxy-5′-methylthioadenosine production increased, and exceeded homocysteine synthesis. 5′-Deoxy-5′-methylthioadenosine formation reached a maximum of 0.9 nmol/12 h per 10 6 cells prior to the onset of exponential growth. The kinetics of homocysteine synthesis were different. Homocysteine accumulation was proportional to the specific growth rate, and achieved a peak of 3.1 nmol/12 h per 10 6 cells during mid-exponential phase, at which time 5′-deoxy-5′-methylthioadenosine production was falling. Similar patterns of 5′-deoxy-5′-methylthioadenosine and homocysteine excretion were observed in other 5′-deoxy-5′-methylthioadenosine phosphorylase deficient cell lines. These data show that polyamine synthesis and transmethylation are differentially regulated during the growth cycle of mammalian cells.

Effects of 5′deoxy-5′-methylthioadenosine on the metabolism of S-adenosyl methionine

The treatment of transformed rat cells with micromolar amounts of 5′deoxy 5′methyl thioadenosine induces rapid effects on the rate of methylation of DNA concomitantly with alterations of intracellular pools of S-adenosyl methionine and S-adenosyl homocysteine. Pulse chase labelling experiments indicate that 5′deoxy 5′methylthioadenosine does not inhibit the degradation of S-adenosyl homocysteine but inhibits the comsumption of S-adenosyl methionine. In vitro transmethylation assays performed with heterologous DNA show that low doses of the thioethernucleoside do not significantly affect the DNA methyltransferase activity of cellular extracts. The biological role of 5′deoxy 5′methylthioadenosine, a natural molecule formed during the synthesis of polyamines is discussed.

Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.

Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.

In vivo effects of α- dl-Difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor α- dl-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72–80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (< 1 nmol mg −1 protein) after 36 h treatment. α- dl-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [ 3H]ornithine but were able to convert [ 3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50–100%, while protein synthesis increased up to 4-fold in 36-h treated cells. α- dl-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender → short stumpy transformation.

S-Adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.

Studies of the metabolism of 5′-isobutylthioadenosine (SIBA): Phosphorolytic cleavage by methylthioadenosine phosphorylase

5’.Isobutylthioadenosine, a synthetic analogue of S-adenosylhomocysteine [ 11, is a powerful antiproliferative drug. It has been reported to inhibit cell transformation induced by oncogenic RNA or DNA viruses [2,3J, the growth of transformed mouse mammary cells [4], the mitogen-stimulated blastogenesis of lymphocytes [S] and the capping of herpes virus mRNA 161. It has also been demonstrated that an antimalarial activity is exerted by SIBA against P~~s~d~rn fizb5pmwm in cultures [7]. Evidences have been recently reported indicating a rapid degradation of SIBA in chick embryo fibroblasts, which has been attributed to S-adenosylhomocysteine hydroIase (EC 3.3.1 .l) [8]. Despite the variety of effects exerted by SIBA at cehular level, the mechanism of action of the molecule is still largely obscure. It has been postulated that the antiproliferative action could be related to the inhibitory effect exerted by SIBA on tRNA methylases 191, on protein methylase I (EC 2.1.1.23) [9,10] and on SAH hydrohse [ 111. These inhibition studies have been performed in view of a hy~the~ed similarity between SIBA and SAH [9] (see scheme 1). In our opinion, however, the structure of SIBA resembles more 5’.methylthioadenosine, a natural product of S-adenosylmethionine metabolism, than

Effects of exogenous L-dopa on the metabolism of methionine and S-adenosylmethionine in the brain.

The catecholamines dopamine, norepinephrine, and epinephrine are metabolized by oxidative deamination and by O-methylation. This latter pathway is catalyzed by the enzyme catechol-O-methyl-transferase (1); it utilizes S-adenosyl-methionine (SAMe) as the methyl donor (Fig. 1), and its products are, for the most part, methylated on the metahydroxyl group. The catecholamines themselves are formed within only a few specific tissues in the body (21). These include a relatively small number of brain neurons, postganglionic sympathetic neurons, adrenomedullary chromaffin cells, and interneurons within sympathetic ganglia. The catechol-amines can be O-methylated within their cells of origin, within synapses, or, after secretion into the blood stream, within the liver, kidneys, and other organs.KeywordsMethionine AdenosyltransferaseMethionine ConcentrationPostganglionic Sympathetic NeuronHomocysteine MethyltransferaseMethyl Group MetabolismThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

S-adenosylmethionine metabolism and its relation to polyamine synthesis in rat liver. Effect of nutritional state, adrenal function, some drugs and partial hepatectomy.

S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.

A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.

S-adenosylmethionine metabolism by members of the genus Candida.

S-Adenosylmethionine and S-methylmethionine: homocysteine methyltransferase activity was measured in cell-free extracts from seven different species (15 strains) of the genus Candida. All strains were able to form methionine via a transmethylation reaction with S-adenosylmethionine and homocysteine. However, some strains were not able to use S-methylmethionine as a methyl donor. Cell-free extracts from two strains of C. guilliermondii formed no methionine when S-methylmethionine was the methyl donor and one strain (C. guilliermondii, CDC) showed methionine-forming activity with S-adenosylmethionine and homocysteine only when the growth medium was supplemented with L-methionine. In general, greater methyltransferase activity was observed in the cell-free extracts from the more pathogenic members of the genus (i.e., C. albicans and C. tropicalis). Supplementation of the growth medium with L-methionine induced homocysteine methyltransferase activity. Ten of the 15 strains had the capacity to synthesize and store S-adenosylmethionine in their vacuole and all 15 strains had S-adenosylmethionine-degrading enzymes. The activity of the latter enzyme was not increased when cells were grown in L-methionine-supplemented medium.

Metabolism of S-adenosylmethionine in germinating pea seeds: Turnover and possible relationships between recycling of sulfur and transmethylation reactions

Tissue slices of pea cotyledons incubated with methionine labeled in the methyl group with 3H and 14C and with methionine 35S, rapidly incorporated radioactivity into S-adenosylmethionine. Such slices transferred to unlabeled methionine solutions showed a rapid turnover of labeled S-adenosylmethionine. Kinetic studies showed that sulfur-labeled and methyl-labeled S-adenosylmethionine had quite different rates of turnover. Such differences were related to the possible recycling of sulfur. Attempts to demonstrate direct methylation of S-adenosylhomocysteine in vitro were not successful. Experiments with S-adenosyl-8- 14C- l-homocysteine demonstrated conversion to adenosine- 14C and cell-free extracts catalyzed the synthesis of methionine from homocysteine, in the presence of S-adenosylmethionine, S-methylmethionine, or 5-methyltetrahydrofolate. It is concluded that in the metabolic turnover of S-adenosylmethionine the resulting S-adenosylhomocysteine is rapidly cleaved to give rise to adenosine and homocysteine. Homocysteine is then methylated by 5-methyltetrahydrofolate and the resulting methionine is utilized for regeneration of S-adenosylmethionine.