Methionine Pool Research Articles

The S-Methylmethionine Cycle in Lemna paucicostata

The metabolism of S-methylmethionine has been studied in cultures of plants of Lemna paucicostata and of cells of carrot (Daucus carota) and soybean (Glycine max). In each system, radiolabeled S-methylmethionine was rapidly formed from labeled l-methionine, consistent with the action of S-adenosyl-l-methionine:methionine S-methyltransferase, an enzyme which was demonstrated during these studies in Lemna homogenates. In Lemna plants and carrot cells radiolabel disappeared rapidly from S-methylmethionine during chase incubations in nonradioactive media. The results of pulse-chase experiments with Lemna strongly suggest that administered radiolabeled S-methylmethionine is metabolized initially to soluble methionine, then to the variety of compounds formed from soluble methionine. An enzyme catalyzing the transfer of a methyl group from S-methylmethionine to homocysteine to form methionine was demonstrated in homogenates of Lemna. The net result of these reactions, together with the hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine, is to convert S-adenosylmethionine to methionine and adenosine. A physiological advantage is postulated for this sequence in that it provides the plant with a means of sustaining the pool of soluble methionine even when overshoot occurs in the conversion of soluble methionine to S-adenosylmethionine. The facts that the pool of soluble methionine is normally very small relative to the flux into S-adenosylmethionine and that the demand for the latter compound may change very markedly under different growth conditions make it plausible that such overshoot may occur unless the rate of synthesis of S-adenosylmethionine is regulated with exquisite precision. The metabolic cost of this apparent safeguard is the consumption of ATP. This S-methylmethionine cycle may well function in plants other than Lemna, but further substantiating evidence is neeeded.

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.

Isolation and incubation conditions to study heart mitochondrial protein synthesis

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

SV LM21 , a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase

The replication of Sindbis virus (SV STD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5′ cap of the viral mRNAs. SV LM21, a strain of Sindbis virus derived in our laboratory from SV STD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SV STD and SV LM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SV LM21, generated a methyltransferase with a K m for ado met 10-fold lower than that generated by either SV STD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SV LM21, generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SV STD and SV LM21, enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SV LM21, phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SV LM21, to methionine deprivation.

Plasma and muscle free amino acids during continuous ambulatory peritoneal dialysis

Free amino acids (AA) were determined in plasma and in muscle tissue of 29 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) for 2 to 38 months. Muscle biopsies were taken in the morning after an overnight dwell with 1.36% glucose dialysis fluid. Muscle intracellular water was calculated using the chloride method. The intracellular (ic) and extracellular (ec) concentration and the ic/ec gradient for each AA was calculated and compared with values in matched healthy controls. Most of the essential and several non-essential AA were low in plasma. By contrast, none of the essential AA were low in muscle, and methionine was increased as were ornithine, asparagine, and aspartic acid; however, muscle taurine was markedly reduced. The ic/ec gradient was increased for most essential and several non-essential AA. In plasma, taurine precursors, methionine and cysteine, were not reduced and the ratios taurine/cysteine and taurine/methionine were low. Muscle taurine/methionine was also low. Thus, during CAPD muscle free AA are, in general, well maintained, suggesting that marked reductions of plasma AA levels in CAPD patients may reflect an ec to ic shift rather than depletion. The finding of low muscle taurine, but normal or increased cysteine and methionine pools, suggests that taurine depletion during CAPD is caused by blocked synthesis or low intake of taurine.

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast, Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation. The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg· l −1·h −1 at a dilution rate of 0.05·h −1. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.

Isolation of l-methionine-enriched mutant of a methylotrophic yeast, Candida biodinii No. 2201

Six strains of methylotrophic yeast were examined for production of l-methionine-enriched cells. Candida biodinii ( Kloeckera sp.) No. 2201, which accumulated 0.54 mg/g-dry cell weight (DCW) of free l-methionine (pool methionine), was selected as the parental strain for breeding l-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradation. A mutant strain, E500-78, which was resistant to 500 μg/ml of dl-ethionine, accumated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were o-ptimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.

Absence of antiviral activity of the broad-spectrum picornavirus inhibitor disoxaril (WIN 51711) against hepatitis A virus : Anders Widell, Laboratory of Clinical Virology, University of Lund, Malmö General Hospital, Malmö, Sweden

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5′ cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21, generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21, generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21, enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21, phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21, to methionine deprivation.

Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth

The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU. Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again. These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity. The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E. coli cells.

Na + -Stimulated Transport of l -Methionine in Brevibacterium linens CNRZ 918

The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium levels of its niche.

Methionine metabolism after portacaval shunt in the rat.

The effect of portacaval shunt (PCS) on methionine metabolism in the rat was investigated. Male Sprague-Dawley rats were subjected to PCS and maintained on an 18% casein diet. Growth curves of operated rats were similar to controls. PCS rats excreted more urinary 35SO4 and less [35S]taurine than controls after intraperitoneal injection of 0.3 mmol/100 g [35S]methionine or [35S]cysteine. Total urinary taurine excretion was similar in PCS and control rats after a methionine or cysteine load; however, under basal conditions PCS rats had higher urinary taurine levels than controls, indicating that PCS may cause the taurine pool to be expanded. Hepatic methionine, S-adenosylmethionine, and cysteine pools were significantly decreased in PCS rats, while S-adenosylhomocysteine levels were unchanged. Relative rates of transsulfuration in PCS and control rats were studied by following the decrease in the 3H-to-35S ratio in liver protein after injection of [methyl-3H]methionine and [35S]methionine, and no difference in flux of 35S from [35S]methionine to [35S]cysteine was found. Similarly, total hepatic activities of methionine adenosyltransferase, cystathionine synthase, and cystathionine gamma-lyase were unchanged in PCS rats. These results indicate that altered methionine metabolism in PCS rats is not explained by changes in conversion of methionine to cysteine via the transsulfuration pathway.

Theoretical mechanisms for synthesis of carcinogen-induced embryonic proteins: XII mutational and non-mutational mechanism as subsets of a more general mechanism. Part A — Ethionine

The ethionine-induced genic derepression mechanism is visualized as a secondary process that occurs after the S-adenosyl-L methionine pool concentrations are lowered to critical levels. Although DNA methylation has been shown to be correlated with genic activity, the times observed for inducement (3 days) of the alpha-fetoprotein gene and its reversibility (within 7 days) does not make it likely that alterations in the methylated status of DNA is involved. The specific mechanism is theorized to be as follows: the adenine moiety of S-adenosyl-L-methionine base-pairs with thymine of a specific structural area of the alpha-fetoprotein gene. The process is visualized as a frequent event during moments of structural relaxation of an otherwise hyperspiralized condition of the chromatin. This weak hydrogen bonding situation allows the methylation by protein methylases of a precursor chromatin protein that after methylation by the S-adenosyl-L methionine which is base-paired to the specific DNA site, conformationally is set or locked into place and acts as a specific repressor for the alpha-fetoprotein gene. This subsequently disallows RNA polymerase activity of the region. During turnover of this chromatin protein the replacement of the methylated repressor is normally maintained. But if the S-adenosyl-L-methionine pool concentration is lowered to a level below that required for base-pairing by the adenine moiety, then the repressed conformational condition of the alpha-fetoprotein gene is altered allowing transcription. In this manner the correlation between low S-adenosyl-L-methionine and alpha-fetoprotein synthesis can be made.

Kinetics of Methionine and Choline and Their Incorporation into Plasma Lipids and Milk Components in Lactating Goats

Rates of entry of L-methionine and choline and their incorporations into lipids of blood plasma and milk components were studied in lactating goats fed a concentrate-roughage ration with primed constant infusion of L-[methyl-carbon-14] methionine and [methyl-carbon-14] choline. Entry rates of methionine and choline were 30 and 47 µmol/h/ kg body weight. Of the methionine pool, 10 and 28% were recovered in carbon dioxide and choline of blood plasma, but the contribution of methionine to the total carbon dioxide production was negligible. Two percent of serine was derived from methionine. Six percent of the choline pool was derived from methionine, whereas labeled choline was not recovered in methionine. Methionine and choline contributed to at least 4 and 19% of the phospholipid pool. Of the methionine dose, .2 and 3.7% appeared in milk lactose and protein. Little (.3%) labeled choline was recovered in milk; however, its contribution to milk fat was 10 times as high as that of methionine. The lipogenic effect of methionine in the liver is proposed as one mechanism among others stimulating production of milk and milk fat.

Effect of intravenous administration of D-lysergic acid diethylamide on initiation of protein synthesis in a cell-free system derived from brain.

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.

Reduced free-methionine in methionine-dependent SV40-transformed human fibroblasts synthesizing apparently normal amounts of methionine.

Many different types of cancer cells have been shown to be methionine-dependent. These cells, unlike normal cells, grow poorly or not at all when methionine is replaced by its immediate precursor homocysteine in the growth medium (Met- Hcy+ medium). We have previously shown that apparently normal total amounts of methionine are synthesized by methionine-dependent SV40-transformed human fibroblasts. However, methionine-dependent cells in Met- Hcy+ medium accumulate reduced amounts of S-adenosylmethionine (AdoMet) and elevated amounts of S-adenosylhomocysteine (AdoHcy) that together probably limit growth. In this report, we demonstrate that the amount of free methionine is low in methionine-dependent SV40-transformed human fibroblasts in Met- Hcy+ medium compared to normal human diploid fibroblasts. In contrast, in Met+ Hcy- medium, the amount of free methionine is comparable in both cell types. The deficient pool of free methionine in methionine-dependent cells in Met- Hcy+ medium allows only low amounts of AdoMet to be formed. However, large amounts of the biosynthesized methionine are channeled into protein synthesis. Possible mechanisms are discussed to explain this cancer-associated metabolic defect.

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a K m for methionine of about 3–12 μM. In selected lines we have demonstrated a requirement for Mg 2+ in addition to that needed to form the Mg·ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.

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.

Measurement of protein synthesis in brain subcellular fractions in vivo by local flooding of methionine pools

Measurement of protein synthesis in brain subcellular fractions <i>in vivo</i> by local flooding of methionine pools

Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase.

Barley (Hordeum vulgare L.) mutants altered in the regulation of synthesis of aspartate-derived amino acids were sought by screening embryos for growth on a medium containing lysine plus threonine. One mutant, Rothamsted 2501, was selected with good growth. From the segregation of resistance in the following generations, it was concluded that the resistance was conferred by a dominant gene, Lt1. No homozygous Lt1/Lt1 fertile plants have been recovered. Partially purified aspartate kinase preparations from resistant and sensitive plants were separated on DEAE-cellulose chromatography into three peaks of activity (I, II, III) and the feedback regulatory properties of these peaks determined. These peaks are considered to be three isozymic forms of aspartate kinase, one predominantly sensitive to threonine and two sensitive to lysine or lysine plus S-adenosyl methionine. The feedback characteristics of one of the peaks of aspartate kinase activity from resistant plants were changed such that lysine was half-maximally inhibitory at 10 rather than 0.4 mM. Increases in te concentrations of the free pools of threonine (4x) and methionine (2x) were measured in young plants grown on a basal medium. Threonine in the soluble fraction of mature seeds from resistant plants was increased from 0.8 to 9.6% of the total threonine content. The total content of both threonine and methionine of the seeds was increased by 6% compared with grain of similar nitrogen content.

Complications in Interpreting Precursor/Product Relationships by Labeling Experiments Methionine as the Precursor of Ethylene in Tobacco Leaves

Summary The role of methionine as the precursor of ethylene in healthy and tobacco mosaic virus-infected tobacco leaves was studied by comparison of the specific radioactivities of methionine and ethylene after labelling leaves with L-[U-14C] methionine by petiolar uptake. The specific activity of the ethylene produced was far less than expected on the basis of the methionine pool size within the leaves and the amount of label applied. This was caused, on the one hand, by an irreversible binding of the applied methionine to the cell wall fraction and, on the other hand, its rapid metabolization. The rise in ethylene production due to virus infection was associated with a corresponding decrease in specific activity, suggesting a second precursor or another (unlabeled) methionine pool to be used for virus-induced ethylene production. However, virus-induced ethylene production occured only in the interveinal leaf parts where the specific radioactivity of methionine was (relatively) low. Hence, the decrease in the specific radioactivity of the ethylene resulted from the nonhomogeneous distribution of the applied 14C-labeled methionine within the leaf rather than from another precusor.