A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Homocysteine and cysteine - albumin binding in homocystinuria: assessment of cysteine status and implications for glutathione synthesis?

Summary. Measurement of plasma total cysteine rather than free dimeric cystine gives a better indication of cysteine status in homocystinuric patients. This is the result of displacement of cysteine from albumin by homocysteine and is related to the plasma homocysteine concentration. In control subjects the free/bound cyst(e)ine ratio was independent of albumin and total cysteine concentrations. In homocystinuric (HCU) patients both free and total cyst(e)ine values differed significantly from control values (P < 0.001) but whilst free cystine considerably overlapped control values the total cysteine concentrations were almost invariably lower. The possible consequences of this on glutathione synthesis was explored by assay of plasma total glutathione but no evidence for glutathione deficiency was found. Measurement of total cysteine, rather than free cystine, provides a better indication of cysteine status in HCU. Received February 1, 2001 Accepted November 13, 2001     

Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [³⁵S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [³⁵S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.     

A method for measurement of free and bound plasma cyst(e)ine

A method for the measurement of free and bound cyst(e)ine and the sum thereof has been developed. In adult human blood, cyst(e)ine is distributed equally between that which is bound to plasma proteins and that which is free. Cyst(e)ine is bound predominantly to albumin, and this binding is not an in vitro artifact. Cysteine bound to plasma proteins may be displaced by homocysteine which competes for the available sulfhydryl groups of plasma proteins. Rats starved for 8 days had a significant decrease in both plasma free cyst(e)ine and bound cysteine. These data suggest that present methods for the determination of plasma cyst(e)ine under-estimate the quantity of cyst(e)ine in the plasma available for cellular metabolism.     

Formation and utilization of methionine by rat liver cells in culture

Summary The enzymeN ⁵-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells. Presented in part at the Conference on Differentiation and Carcinogenesis in Liver Cell Cultures sponsored by the New York Academy of Sciences, October 11, 1979 (see reference 1).     

Sulfate Uptake and Its Regulation in Lemna paucicostata Hegelm. 6746

The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down-regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2-) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly.     

A yeast with unusual sulphur amino acid metabolism

A yeast strain highly resistant to propargylglycine (an inhibitor of cystathionine gamma-lyase) was isolated from air. It was partially characterized, but it has not been identified with any known yeast species. Its sulphur amino acid metabolism differed from that of other fungi by the lack of the reverse transsulphuration pathway from methionine to cysteine, as no activity of cystathionine beta-synthase or cystathionine gamma-lyase was found. The functional lack of this pathway was confirmed by growth tests and by experiments with [35S]methionine. In contrast to Saccharomyces cerevisiae neither homocysteine synthase nor the sulphate assimilation pathway were repressible by methionine in the new strain; on the contrary, a regulatory effect of cysteine was observed.     

Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: the mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency.

Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from cells grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase.     

The formation of sulphur-containing amino acids in germinating seeds of rape (Brassica napus L.)

Sodium [(35)S]sulphide was fed to batches of germinating rapeseed, in some instances with the addition of unlabelled cysteine. Both the total radioactivity and specific radioactivity of the free sulphur-containing amino acids were examined. Cysteine and homocysteine were rapidly labelled; label subsequently appeared in cystathionine and methionine. The results obtained indicated that both the sulphydration and trans-sulphuration pathways were operating. This conclusion was reinforced by the results of experiments in which batches of rapeseed were incubated with l-[(14)C]homoserine. These showed the formation of labelled homocysteine, cystathione and methionine. It was thought the trans-sulphuration pathway was making the greater contribution to the biosynthesis of methionine in germinating rapeseed.     

Homocysteine metabolism in peripheral blood mononuclear cells: evidence for cystathionine beta-synthase activity in resting state

Activated peripheral blood mononuclear cells (PBMC) release homocysteine and possess cystathionine β-synthase (CBS) activity; however, it was thought that there is no CBS in resting state. Previously, we found that nickel decreased intracellular homocysteine concentration in un-stimulated (e.g. resting) PBMC, suggesting that resting PBMC might also have active homocysteine metabolism. Here, we demonstrated that un-stimulated PBMC synthesize (incorporate L-[methyl-14C]methionine to DNA, lipids and proteins), release (increase extracellular homocysteine), and metabolize homocysteine. Intracellular homocysteine concentration varied with incubation time, depending on extracellular concentrations of methionine, homocysteine, and glutathione. Methionine synthase activity was constant and independent of thiol concentrations. In Western blot, CBS protein was clearly identified in freshly isolated PBMC. CBS protein level and activity increased with incubation time, upon stimulation, and similar to intracellular homocysteine, depending on intra- and extracellular homocysteine and glutathione concentrations. According to our knowledge, this is the first evidence that certifies homocysteine metabolism and regulatory role of CBS activity to keep balanced intracellular homocysteine level in resting PBMC. Homocysteine, released by PBMC, in turn can modulate its functions contributing to the development of hyperhomocysteinemia-induced diseases.     

Sulfur-containing Compounds in Lemna perpusilla 6746 Grown at a Range of Sulfate Concentrations

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 μm, was labeled to radioisotopic equilibrium with ³⁵SO₄²⁻. Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. γ-Glutamylcyst(e)ine, erythro- and threo-thiothreonine, and S-methylcysteine were not detected. No volatile ³⁵S compounds were formed during plant growth at 1,000 μm sulfate, nor were significant amounts of ³⁵S compounds excreted into the medium.     

Downregulation of Cdc6 and pre-replication complexes in response to methionine stress in breast cancer cells

Methionine and homocysteine are metabolites in the transmethylation pathway leading to synthesis of the methyl-donor S-adenosylmethionine (SAM). Most cancer cells stop proliferating during methionine stress conditions, when methionine is replaced in the growth media by its immediate metabolic precursor homocysteine (Met-Hcy+). Non-transformed cells proliferate in Met-Hcy+ media, making the methionine metabolic requirement of cancer cells an attractive target for therapy, yet there is relatively little known about the molecular mechanisms governing the methionine stress response in cancer cells. To study this phenomenon in breast cancer cells, we selected methionine-independent-resistant cell lines derived from MDAMB468 breast cancer cells. Resistant cells grew normally in Met-Hcy+ media, whereas their parental MDAMB468 cells rapidly arrest in the G₁ phase. Remarkably, supplementing Met-Hcy+ growth media with S-adenosylmethionine suppressed the cell proliferation defects, indicating that methionine stress is a consequence of SAM limitation rather than low amino acid concentrations. Accordingly, mTORC1 activity, the primary effector responding to amino acid limitation, remained high. However, we found that levels of the replication factor Cdc6 decreased and pre-replication complexes were destabilized in methionine-stressed MDAMB468 but not resistant cells. Our study characterizes metabolite requirements and cell cycle responses that occur during methionine stress in breast cancer cells and helps explain the metabolic uniqueness of cancer cells.     

Downregulation of Cdc6 and pre-replication complexes in response to methionine stress in breast cancer cells

Methionine and homocysteine are metabolites in the transmethylation pathway leading to synthesis of the methyl-donor S-adenosylmethionine (SAM). Most cancer cells stop proliferating during methionine stress conditions, when methionine is replaced in the growth media by its immediate metabolic precursor homocysteine (Met-Hcy+). Non-transformed cells proliferate in Met-Hcy+ media, making the methionine metabolic requirement of cancer cells an attractive target for therapy, yet there is relatively little known about the molecular mechanisms governing the methionine stress response in cancer cells. To study this phenomenon in breast cancer cells, we selected methionine-independent-resistant cell lines derived from MDAMB468 breast cancer cells. Resistant cells grew normally in Met-Hcy+ media, whereas their parental MDAMB468 cells rapidly arrest in the G₁ phase. Remarkably, supplementing Met-Hcy+ growth media with S-adenosylmethionine suppressed the cell proliferation defects, indicating that methionine stress is a consequence of SAM limitation rather than low amino acid concentrations. Accordingly, mTORC1 activity, the primary effector responding to amino acid limitation, remained high. However, we found that levels of the replication factor Cdc6 decreased and pre-replication complexes were destabilized in methionine-stressed MDAMB468 but not resistant cells. Our study characterizes metabolite requirements and cell cycle responses that occur during methionine stress in breast cancer cells and helps explain the metabolic uniqueness of cancer cells.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

Disturbance of methyl group metabolism in alloxan-diabetic sheep

Alloxan-induced diabetes results in changes in the activities of a number of enzymes related to methyl group metabolism in sheep. Decreases in the activities of phospholipid methyltransferase and betaine-homocysteine methyltransferase in diabetic sheep liver indicate a reduced rate of choline synthesis and oxidation. A 65-fold increase in the activity of glycine methyltransferase and a 4-fold rise in the activity of gamma-cystathionase in diabetic sheep liver with elevated urinary excretion of cyst(e)ine suggest that catabolism of the methyl group of methionine and homocysteine was enhanced in the diabetic state.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.     

Homocysteine export from cells cultured in the presence of physiological or superfluous levels of methionine: methionine loading of non-transformed, transformed, proliferating, and quiescent cells in culture

Determination of the transient increase in plasma homocysteine following administration of excess methionine is an established procedure for the diagnosis of defects in homocysteine metabolism in patients. This so-called methionine loading test has been used for 25 years, but the knowledge of the response of various cell types to excess methionine is limited. In the present paper we investigated homocysteine export from various cell types cultured in the presence of increasing concentrations (15-1,000 microM) of methionine. For comparison of homocysteine export, the export rates per million cells were plotted versus cell density for proliferating cells, and versus time for quiescent cells. The homocysteine export from growing cells was greatest during early to mid-exponential growth phase, and then decreased as a function of cell density. The export rate was higher from phytohemagglutinin-stimulated than non-stimulated lymphocytes, and higher from proliferating than from quiescent fibroblasts. The hepatocytes showed highest export rate among the cell types investigated. The enhancement of homocysteine export by excess methionine ranged from no stimulation to marked enhancement, depending on cell type investigated, and three different response patterns could be distinguished: 1) quiescent fibroblasts and growing murine lymphoma cell showed no significant increase in homocysteine export following methionine loading; export from human lymphocytes was only slightly enhanced in the presence of excess methionine; 2) the homocysteine export from proliferating hepatoma cells and benign and transformed fibroblasts was stimulated three to eightfold by increasing the methionine concentration in the medium from 15 to 1,000 microM; and 3) the response to methionine loading was particularly increased (about 15-fold) in non-transformed primary hepatocytes in stationary culture. The results outline a potentially useful procedure for the comparison of homocysteine export during cell growth in the presence of various concentrations of methionine. The results are discussed in relation to the special feature of homocysteine metabolism in various cell types and tissues including liver, and to the possible source of plasma homocysteine following methionine loading in vivo.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

The effect of folic acid and/or methionine deficiency on deoxyribonucleotide pools and cell cycle distribution in mitogen-stimulated rat lymphocytes

Abstract. Folate deficiency will induce abnormal deoxynucleoside triphosphate (dNTP) metabolism because folate-derived one-carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Under conditions of methionine deprivation, a functional folate deficiency for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous methionine resynthesis from homocysteine. The purpose of the present study was to examine the effect of nutritional folate and/or methionine deprivation in vitro on intracellular dNTP pools as related to DNA synthesis activity and cell cycle progression. Primary cultures of mitogen-stimulated rat splenic T-cells were incubated in complete RPMI 1640 medium or in custom-prepared RPMI 1640 medium lacking in folic acid and/or methionine. Parallel cultures, initiated from the same cell suspension, were analysed for deoxyribonucleotide pool levels and for cell proliferation. The distribution of cells within the cell cycle was quantified by dual parameter flow cytometric bromodeoxyuridine/propidium iodide DNA analysis which allows more accurate definition of DNA synthesizing S-phase cells than the traditional DNA-specific staining with propidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool and an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels were significantly reduced with single or combined deficiencies of folate and methionine. These nucleotide pool alterations were associated with a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G₂+ M phase of the cell cycle. Folate deprivation in the presence of adequate methionine produced a moderate decrease in DNA synthesizing cells over the 68 h incubation. However, methionine deprivation, in the presence or absence of folate, severely compromised DNA synthesis activity. These results are consistent with the established ‘methyl trap’ diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide synthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the availability of folate one-carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression and DNA synthesis.     

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 Mg²⁺ 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.