Intracellular S-adenosylmethionine Research Articles

Evidence of morphology-specific isozymes inCandida albicans

S-Adenosylmethionine (SAM) synthetase of yeast and hyphal-phase cells of the dimorphic fungusCandida albicans was characterized by kinetic analysis and response to inhibitors. The enzyme from yeast-phase cells has a Km of 0.17 mM for methionine, 0.14 mM for ATP, and is inhibited (in vitro) by dimethyl-sulfoxide, methionine sulfone, and methionine sulfoxide. The hyphal-phase SAM synthetase has a Km of 0.06 mM for methionine, 0.02 mM for ATP, and its activity (in vitro) is enhanced by the substances that inhibit the yeast-phase enzyme. These data strongly suggest that isozymes of SAM synthetase are present inC. albicans and that they are possibly morphology specific. In vivo studies revealed that synthesis of the enzyme is repressed by the addition of methionine to the growth medium and that specific activity of the enzyme increases when intracellular SAM levels are lowered. In addition, it was shown that the increase in specific activity seen during yeast → hypha morphogenesis and in yeast cells grown in a methionine-free medium involves de novo protein synthesis.

S-Adenosylmethionine and S-adenosylhomocysteine transitions in encysting Physarum flavicomum amoebae

Previous studies from this laboratory suggested that in Physarum flavicomum an S-adenosylmethionine (SAM) related metabolic flux is involved in the developmental control of microcyst formation. In the present study, this phenomenon was further analyzed by comparing the metabolic effects of compounds found to favor, stimulate, or inhibit the normal encystment process. The compounds utilized were adenine, cycloleucine, L-ethionine, DL-selenomethionine, cadaverine, cyclohexylammonium sulfate, papaverine, mycophenolic acid, decoyinine, putrescine, spermidine, and spermine. Following incubation with the compounds or combinations of the compounds, the intracellular levels of SAM and SAM metabolites were determined using high performance liquid chromatography. The actual level of the intracellular SAM pool varied considerably (notably after 15 h of incubation), depending on the compound(s) in the incubation solution, but was always highest during inhibition of encystment. SAM was undetectable or barely detectable during stimulation of encystment or under conditions favoring encystment. In cells exposed to L-ethionine, we detected intracellular S-adenosylethionine rather than SAM. The intracellular methionine pool in cells encysting normally for 15 h was found to be 1.3 mumol/pg DNA. Both SAM and S-adenosylhomocysteine (SAH) were present in growing amoebae at concentrations greater than that of cells undergoing normal encystment. The developmental transition from growing amoebae to dormant cysts in P. flavicomum is metabolically characterized by the adjustment of the intracellular concentration of SAM and SAH to a minimal critical level of 0.31 and 0.70 fmol/pg DNA, respectively.

Genetic mapping and physiological consequences of metE mutations of Bacillus subtilis.

Three metE mutations of Bacillus subtilis, which cause cells to have a 25- to 200-fold decrease in L-methionine S-adenosyltransferase (EC 2.5.1.6) activity, were mapped between bioB and thr. The corresponding three metE mutants contained three- to fourfold less intracellular S-adenosylmethionine (SAM) but at least sevenfold more methionine than the metE+ strain when grown in synthetic medium. This indicates a strong feedback control of SAM on its synthesis. However, only the metE2 strain, with the lowest SAM concentration, grew at a slightly lower rate than the parent, which showed that an intracellular concentration of about 25 microM SAM was critical for growth at the normal rate. Neither DNA methylation (measured by bacteriophage luminal diameter 105 restriction) nor sporulation was affected at this low SAM concentration. Addition of methionine to the growth medium caused an increase in the pool of SAM in some but not all metE mutants. Coaddition of adenine did not change this result. However, the extent of sporulation (induced by mycophenolic acid) was decreased 50-fold in all mutants by the addition of methionine and adenine. Therefore, the combination of methionine and adenine suppresses sporulation regardless of whether it causes an increase in the level of SAM.

A mutant ofEscherichia colitemperature sensitive in the biosynthesis ofS-adenosylmethionine

S-Adenosylmethionine (SAM) is synthesized in vitro by SAM synthetase, which is coded for by the metK gene of Escherichia coli. Since E. coli cells are impermeable to SAM it has been impossible to obtain mutants which require SAM for growth. The finding that SAM is required for regulating methionine biosynthesis was used to select metK mutants which have the phenotype of resistance to methionine analogues. These mutants show reduced SAM synthesis in vitro, and a lower level of intracellular SAM. However, as all these mutants can grow, without added SAM, the block in SAM synthesis must be incomplete. To try to block SAM synthesis more completely we combined temperature-sensitive metK mutation with a block in the synthesis of cystathionine (metA), a precursor that supports only slow growth because of slow entry. At the non-permissive temperature for metK these double mutants could grow on methionine but not on cystathionine, in correlation with the greatly reduced intracellular level of SAM. This result indicates that it is possible to reduced intracellular SAM concentrations to levels which are low enough to prevent growth of E. coli. Moreover, since the mutation responsible for the reduction in SAM level is in the metK gene, it is suggested that SAM synthetase is the enzyme which catalyses SAM biosynthesis in E. coli cells.

S-adenosylmethionine may not be essential for signal transduction during bacterial chemotaxis.

We previously showed that a mutant strain of Salmonella typhimurium completely deficient in both the chemoreceptor methylating (CheR) and demethylating (CheB) enzymes can still exhibit chemotaxis to aspartate and other attractants (J. Stock, A. Borczuk, F. Chiou, and J. E. B. Burchenal, Proc. Natl. Acad. Sci. USA 82:8364-8368, 1985). We used this cheR cheB mutant to examine the possibility of an additional requirement for S-adenosylmethionine in chemotaxis besides its role in chemoreceptor methylation. A metE mutation was transduced into a cheR cheB double mutant, and the cells were starved for methionine. Despite the fact that intracellular S-adenosylmethionine dropped from approximately 100 microM to less than 0.2 microM, chemotaxis was largely unaffected. In contrast, a corresponding cheR+ cheB+ metE mutant completely lost its chemotaxis ability after being starved for methionine. We conclude from this observation that the primary requirement for S-adenosylmethionine during bacterial chemotaxis is in the methylation of receptor proteins.

S-Adenosylmethionine and transmethylation reactions in trichomonads

S-Adenosylmethionine (SAM) levels in trichomonads, a range of trypanosomatids and mouse liver were measured using HPLC techniques. The concentrations were found to be similar in each with the exception of Herpetomonas muscarum ingenoplastis, which contained approximately ten-fold more. Living trichomonads were found to incorporate exogenous L-methionine into intracellular SAM and its methyl carbon was also detected in lipids and nucleic acids, presumably through its involvement in transmethylation reactions. Norleucine and cycloleucine inhibited L-methionine uptake and incorporation into living Trichomonas vaginalis. Both the rates of incorporation of exogenous L-methionine into intracellular SAM and its involvement in transmethylation reactions were greater for Trichomonas vaginalis than for Tritrichomonas foetus. The results suggest that Trichomonas vaginalis and other trichomonads contain enzymes equivalent to SAM synthetase (EC 2.5.1.6) and SAM-dependent methyltransferases (EC 2.1.1).

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.

Restoration of aerial mycelium and antibiotic production in a Streptomyces griseoflavus arginine auxotroph.

An arginine auxotrophic mutant was obtained from Streptomyces griseoflavus (bicozamycin-producing strain). The mutant grew on synthetic agar supplemented with either arginine, ornithine, citrulline or argininosuccinate, but produced massive aerial mycelium and bicozamycin only with citrulline. In liquid culture, citrulline also completely restored the ability of the mutant to produce bicozamycin. Culture with arginine or ornithine markedly changed intracellular pools of these ornithine-cycle amino acids, but did not affect the other amino acid pools. The ability to produce antibiotic (but not that to form aerial mycelium) was partially restored by certain mutations to ethionine resistance (Eth-1 and Eth-2). These mutations caused decreased or increased S-adenosylmethionine synthetase activity, but both resulted in a 4.5-8-fold increase in the intracellular S-adenosylmethionine pool. Exogenous addition of S-adenosylmethionine (0.5-3 mM) also partially restored the antibiotic-producing ability of the arginine auxotroph. No difference in the S-adenosylmethionine pool was observed in organisms grown with arginine and citrulline. It was suggested that citrulline and S-adenosylmethionine are somehow involved in the initiation of differentiation and secondary metabolism of S. griseoflavus.

S-adenosylmethionine and morphogenesis inMucor racemosus

The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.

Methyl group transfer from exogenous S-adenosylmethionine on to plasma-membrane phospholipids without cellular uptake in isolated hepatocytes

At external concentration of 50 microM, L-methionine was rapidly taken up by hepatocytes, whereas almost no S-adenosylmethionine (SAM) was removed from the incubation medium. SAM did not enter the intracellular water space but equilibrated with a very small pool, which was most likely to be situated on the external side of the plasma membrane. Methyl groups from external L-methionine, but not from external SAM, were incorporated into total and nuclear RNA. A significant incorporation of methyl groups into phospholipids occurred not only with methionine but also with SAM. After subfractionation of hepatocytes it became evident that methyl groups from SAM were mainly incorporated into plasma-membrane phospholipids, and that phospholipid methylation in other cellular compartments resulted from contamination with plasma membrane. The pattern of methylation of the various phospholipid species with SAM as precursor was different from that obtained with L-methionine. In contrast with external L-methionine, external SAM did not enter the intracellular SAM pool. According to these results a transport system for SAM does not exist in rat hepatocytes, although methyl groups from external SAM can be incorporated into plasma-membrane phospholipids from the outside.

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8- 14C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.

Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U- 14C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U- 14C)-labeled adenine of 5′-methylthio[U- 14C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.

Methionine biosynthesis and S-adenosylmethionine degradation during an induced morphogenesis of Candida albicans.

Seven strains of Candida albicans were used in studies designed to delineate altered biochemical activity during an induced yeast to filamentous morphogenesis. Four of the strains studied underwent a rapid (1–2 h) yeast to filamentous transformation in a defined medium with glycine as the main nitrogen source; three other strains of C. albicans remained yeast-like under the same growth conditions. Cell-free extracts, prepared from cells harvested after various time intervals of growth in glycine medium, were used to measure enzymic capacity to form methionine, via the enzyme S-adenosylmethionine (or methylmethionine) homocysteine methyltransferase, and degrade S-adenosylmethionine. Increased enzymic capacity to synthesize methionine (with S-adenosylmethionine as methyl donor) and degrade S-adenosylmethionine appeared to correlate best with a yeast to filamentous transformation of C. albicans. Intracellular S-adenosylmethionine content also decreased with time, in all seven strains, when the cells were cultured in the glycine medium. S-adenosylmethionine may play a key role in the biochemical alterations that occur during the morphogenesis of C. albicans.

Methionine adenosyltransferase and ethionine resistance in Saccharomyces cerevisiae.

The methionine adenosyltransferase is repressed in Saccharomyces cerevisiae during growth in the presence of excess methionine. The relationship of this repression to the level of intracellular S-adenosylmethionine is discussed. In conjunction with these studies, an ethionine-resistant mutant has been investigated which has a low level of methionine adenosyltransferase under all conditions tested. The mechanism of ethionine resistance in the latter strain apparently depends on its inability to form large quantities of intracellular S-adenosylethionine. With respect to the methionine adenosyltransferase, there is no apparent interaction between ethionine-resistant and ethionine-sensitive alleles when both are present in the heterozygous diploid.

Observations on the metabolism of 5′-methylthioadenosine

The yeast Candida utilis assimilates 5′-methylthioadenosine from the culture medium and converts it into intracellular S-adenosylmethionine. Tracer experiments with labeled 5′-methylthioadenosine showed good recovery in S-adenosylmethionine when the label was in the adenine part (H 3) or in the sulfur atom (S 35). The specific activity recovered was lower when methyl-C 14-labeled material was used; this may be explained by loss of methyl groups in transmethylations and their regeneration from unlabeled precursors.