Production of methanethiol from methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Translational capacity of sheep oocytes microinjected with messenger RNA

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.     

The inhibition of accumulation of [1-14C]glycine and its incorporation into protein of Ehrlich ascites-carcinoma cells by dl-methionine

1. By incubation of Ehrlich ascites-carcinoma cells in vitro with [1-(14)C]glycine the relation between the uptake of glycine and its incorporation into protein was examined. 2. With dl-methionine as a competitive inhibitor, there was not only a decrease in uptake of this amino acid, but also inhibition of its incorporation into protein. 3. It is only in its initial stage that the increase in incorporation is accompanied by increase in intracellular concentration of free glycine. Further increase in the amino acid pool has no effect on protein synthesis. 4. Even with a high cell concentration of glycine, methionine produces a decrease both in the uptake and its incorporation. This suggests that the inhibition of incorporation of glycine by methionine is due, not only to decrease in its intracellular concentration, but also to changes in other processes responsible for protein synthesis.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.     

Formation of methanethiol from methionine by leaf tissue

Leaf discs, but not detached leaves, exposed to L-methionine or S-methyl-L-cysteine emitted a volatile sulphur compound identified as methanethiol by different trapping systems and by GC. Methanethiol emission was analyzed using pumpkin (Cucurbita pepo) leaf discs. Emission was observed in darkness or light, however methanethiol emission was greately stimulated by light. Light-dependent emission started after a lag-time of 5–6 hr with an emission peak after 36–40 hr. Maximum rates obtained were in the range of 200 pmol methanethiol/min/cm² leaf area. After a period of 42 hr about 60–80% of total methionine sulphur added was released as methanethiol. Addition of chloramphenicol did not alter the induction period nor the maximum emission rate of methanethiol in response to L-methionine. Emission was also observed in response to S-methyl-L-cysteine; however, the shorter lag-period for methanethiol formation suggests metabolism via a different enzyme system. In a cell-free system of pumpkin leaves methanethiol formation occured in response to L-methionine. Feeding experiments with L-[³⁵S]methionine to leaf discs showed that more than 80% of methanethiol emitted was derived from the labelled methionine fed. These findings suggest that plants have the capacity to degrade L-methionine to methanethiol. Whole leaves fed L-methionine by the petiole system do not emit methanethiol, but this compound is formed and transported into the feeding solution. Thus, methanethiol is also produced by the intact leaf, but, in contrast to sulphide, is not released into the atmosphere. It is suggested that translocation of methanethiol may function as a signal for the regulation of sulphate uptake.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Use of 13C Nuclear Magnetic Resonance and Gas Chromatography To Examine Methionine Catabolism by Lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, ¹³C nuclear magnetic resonance (NMR) performed with uniformly enriched [¹³C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the ¹³C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

Abscisic Acid Effects on Growth and Metabolism in the Roots ofLemna minor

The effects of abscisic acid (ABA) on individual plants of Lemna minor L. were studied. The effects on growth and metabolism of the roots were the most noticeable and the most desirable to measure. Two mg/1 of ABA inhibited the root growth rate by 60% and this was accompanied by a 60% deceleration in the rate of uridine incorporation. The uptake of uridine and leucine and the incorporation of leucine were not affected by ABA. The latent period of root growth inhibition was 1 hour, whereas the inhibition of ribonucleic acid (RNA) synthesis occurred 2 to 4 hours after application. The growth inhibition caused an accumulation of starch in the peripheral, differentiated cell layer of the cortex. Apparently, the growth inhibition by ABA was not entirely due to an inhibition of RNA synthesis, and other plausible mechanisms of growth inhibition are discussed.     

Pathways of Assimilative Sulfur Metabolism in Pseudomonas putida

Cysteine and methionine biosynthesis was studied in Pseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine gamma-synthase and cystathionine beta-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine gamma-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine gamma-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.     

Effect of ethionine on the in vitro synthesis and degradation of mitochondrial translation products in yeast

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.     

Relative changes in incorporation rates of leucine and methionine during starvation survival of two bacteria isolated from marine waters

Abstract Two copiotrophic Gram-negative bacteria isolated from marine waters, S14 and Vibrio sp. DW1, were examined for changes in the rate of protein synthesis in the initial phase of energy and nutrient deprivation. The incorporation of [³H] leucine into the trichloroacetic acid (TCA)-insoluble material was examined as a method for estimating rates of protein synthesis. The incorporation of methionine was measured and compared with the results of leucine incorporation. Protein synthesis was demonstrated throughout a period of 120 h of starvation. The incorporation rate was related to the time of starvation and decreased subsequent to an initial increase during the first few hours of dormancy. Control experiments with proteinase K and chloramphenicol demonstrated that the labelled amino acids were preferentially incorporated into proteins. It was also demonstrated that the uptake of amino acids was not a rate-limiting step. During the first hours of starvation the ratio of the protein to the dry weight of the S14 cells increased parallel to the increase in the amino acid incorporation rate. The increased activity of the protein-synthesising system during the first hours of nutrient and energy depletion indicates the presence of an active cellular response to the downshift conditions. Furthermore, these findings are consistent with the increased respiratory activity during the first hours of starvation, which has previously been observed for the bacteria examined in this study.     

Regulation of cellular growth by sodium pump activity.

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178-Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short-term adaptation (within one generation) occured; i.e., neither growth rate nor (Na+ + K+)-ATPase activity increased in cells cultured for 1-2 days in ouabain. Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H-leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course or the dissipation of the sodium gradient. On the other hand, 3H-thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H-alanine, a neutral amino acid thought to be transported via a Na+-dependent carri-r, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+-dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation. A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.     

Uptake of (14C) leucine and protein synthesis in potato tuber buds and effect of abscisic acid on these processes]

The uptake of [14C] leucine and its incorporation into proteins of dormant and growing potato tuber buds were studied. It was found that the label uptake was increased at the beginning of the growth period, whereas the dynamics of this process were not changed in comparison with the dormant buds samples. The rate of [14C] leucine incorporation into proteins was increased in the growing buds; this increase was not, however, due to the increase in the uptake of the labelled precursor and was probably caused by activation of the protein synthesis. In contrast, the activation of protein synthesis was accompanied by changses is the dynamic incorporation of [14C] leucine into the protein at the end of dormancy. The effect of abscisic acid (10(-7) M) on the protein synthesis was not connected with its action of the uptake of labelled precursor and depended on the physiological state of buds and incubation time. A possible mechanism of regulatory effect of abscisic acid on protein synthesis in potato tuber buds is discussed.     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway

Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B(12)) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [(14)C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [(14)C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

Xylem wall deposition

Summary Tritiated leucine, tyrosine, phenylalanine, methyllabelled methionine, and cinnamic acid were used to study xylem wall deposition and lignin formation with radioautography. Leucine did not specifically label xylem thickenings; tyrosine, phenylalanine and methionine were quite good precursors in this regard. Cinnamic acid was also readily taken up by the tissues and was very markedly concentrated in the xylem thickenings; the labelling of thickenings also occurred in empty tracheids. In developing xylem cells, labelling of the cytoplasm indicated that both the endoplasmic reticulum and Golgi bodies were associated with the wall incorporation. Vesicles probably derived from the Golgi bodies, were generally observed to aggregate in the cytoplasm near the bands of wall microtubules (even if secondary wall thickening had not commenced). Simple biochemical analyses showed that incorporation of cinnamic acid into amino acids and proteins was negligible, but some lignin oxidation products were heavily labelled. The results are related to the biochemistry of lignin synthesis, and confirm that cinnamic acid is a highly specific marker for some forms of wall synthesis.