Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Inhibition of tRNA methylation in vitro and in whole cells by an oncostatic S-adenosyl-homocysteine (SAH) analogue: 5''-deoxy 5''-S-isobutyl adenosine (SIBA).

A high increase in the amount of methylated tRNA bases was found in vivo in Rous sarcoma virus infected and transformed chick embryo fibroblasts in comparison with normal cells, tRNA methylases extracted from transformed cells showed also higher activity in vitro with a heterologous substrate. 5'-deoxy-5'-S-isobutyl adenosine, (a structural analogue of S-adenosyl-L homocysteine), which inhibits virus-induced cell transformation, inhibits also the increase of incorporation of labelled methyl groups into tRNA in infected and transformed cells. When normal cells are grown in the presence of this inhibitor, undermethylated tRNAs are obtained. The effect of the drug is different in normal, infected and transformed cells. The methylation of the different bases is inhibited in vitro and in vivo to various extent. The effect of this substance on tRNA methylation may be the cause of its inhibitory effect on cell transformation.     

Induction of betaine: Homocysteine methyltransferase in some murine cells cultured in vitro

Betaine when present in the culture medium could induce the activity of betaine: homocysteine methyltransferase (EC 2.1.1.5) in mouse L-cells, and leukemic L1210 cells, as well as in mouse embryo fibroblasts grown in vitro. We found this process to be time- and concentration-dependent. A persisting contact of the cells with betaine was indispensible for expressing and maintaining the enzyme activity. The treatment of cells with cycloheximide or actinomycin D abolished the process of induction. Methionine as well as homocysteine, when present either in the culture medium or in the reaction mixture, strongly depressed the activity of this enzyme. The L-cells with the induced betaine: homocysteine methyltransferase survived but did not multiply in the methionine-deficient medium, therefore, they did not become prototrophs with respect to methionine.     

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.     

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).     

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.     

A change in growth potential of cells after conversion by Rous sarcoma virus

Chick embryo cells transformed by Rous sarcoma virus (RSV) were able to grow in suspension, either as colonies when trapped in nutrient agar, or in spinner cultures using liquid medium. Two strains of RSV, RSV (RAV-1) and Schmidt-Ruppin RSV, were able to increase the ability of chick embryo cells to grow in suspension but Rous-associated virus (RAV-1) and polyoma virus were not. Cells growing in suspension supported high levels of RSV production and a simple method for propagating large amounts of virus is suggested. Suspended noninfected cells, which do not grow extensively, lose their ability to be infected by RSV, suggesting that cellular divisions must be in progress for successful infection by RSV.     

Role of Methionine in Cephalosporin Synthesis

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.     

Atherogenic diet alters folate enzymes in mice: implications of folate deficient homocysteinemia.

The two key enzymes, methylenetetrahydrofolate reductase and methionine synthase involved in methionine synthesis from homocysteine were studied in atherogenic diet fed mice. Methylenetetrahydrofolate reductase activity was elevated while methionine synthase was impaired in atherogenic diet fed group. Impaired methionine synthase activity would adversely affect the methionine synthesis from homocysteine, resulting in a rise in the homocysteine levels, which are atherogenic. This is reflected by the increased levels of very low density and low density lipoprotein cholesterol values and a higher ratio for total cholesterol to high density lipoprotein cholesterol.     

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.     

Characteristics of MNNG induced repair synthesis and DNA synthesis induced by human cytomegalovirus

DNA synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse embryo and human embryo cells was compared with DNA synthesis induced in these cells by human cytomegalovirus. In virus infected human embryo cells grown in the medium depleted of arginine DNA synthesis showed resistance to hydroxyurea and arabinofuranosylcytosine, similarly as repair synthesis induced by MNNG. DNA synthesis induced by the virus in mouse embryo cells was partially sensitive to both inhibitors.     

Life history of the bird cherry-oat aphid, Rhopalosiphum padi (Homoptera: Aphididae), on transgenic and untransformed wheat challenged with barley yellow dwarf virus

The life history of Rhopalosiphum padi (L.) was monitored on transgenic and untransformed (soft white winter wheat plants that were infected with Barley yellow dwarf virus (BLDV), noninfected, or challenged with virus-free aphids under laboratory conditions. Two transgenic soft white winter wheat genotypes (103.1J and 126.02) derived from the parental variety Lambert and expressing the barley yellow dwarf virus coat protein gene, and two untransformed varieties, virus-susceptible Lambert and virus-tolerant Caldwell, were tested. B. padi nymphal development was significantly longer on the transgenic genotypes infected with BYDV, compared with noninfected transgenic plants. In contrast, nymphal development on Lambert was significantly shorter on BYDV-infected than on noninfected plants. Nymphal development on noninfected Lambert was significantly longer than on noninfected transgenics. No significant difference in nymphal development period was detected between virus-infected and noninfected Caldwell. Aphid total fecundity, length of reproductive period, and intrinsic rate of increase were significantly reduced on BYDV-infected transgenic plants compared with BYDV-infected Lambert. In contrast, reproductive period, total adult fecundity, and intrinsic rate of increase on noninfected Lambert were significantly reduced compared with noninfected transgenics. Transgenic plants infected with BYDV were inferior hosts for R. padi compared with infected Lambert. However, noninfected transgenics were superior hosts for aphids than noninfected Lambert. Moderate resistance to BYDV, as indicated by a significantly lower virus titer, was detected in the transgenic genotypes compared with the untransformed ones. Results show for the first time that transgenic virus resistance in wheat can indirectly influence R. padi life history.     

Measurement of intracellular sulfur amino acid metabolism in humans

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.     

甲硫氨酸脑啡肽对猴免疫缺陷病毒早期感染引起CEM x1 74细胞凋亡的可能作用(英文)

探讨短期甲硫氨酸脑啡肽作用对SIV感染CEMx174细胞凋亡的可能作用 .用MTS法测定甲硫氨酸脑啡肽对感染CEMx174细胞存活率的影响 ,流式细胞仪分析SIV诱导细胞凋亡及其甲硫氨酸脑啡肽的作用 ,并测定了cAMP含量、PKA活性和组蛋白磷酸化的水平 .SIV能够显著减少CEMx174细胞数 ,甲硫氨酸脑啡肽可以提高感染细胞的存活率 .AnnexinⅤ结合实验显示 ,1μmol L甲硫氨酸脑啡肽可以增加存活细胞的比率 ,减少凋亡细胞数 .甲硫氨酸脑啡肽降低正常细胞和感染细胞cAMP的含量和PKA活性 ,但是在感染组较为明显 .在感染的情况下 ,磷酸化的组蛋白增加 ,甲硫氨酸脑啡肽可以减少其含量 .甲硫氨酸脑啡肽的作用可以被纳酪酮所拮抗 .研究结果提示 ,甲硫氨酸脑啡肽对SIV感染引起早期细胞凋亡的作用涉及cAMP PKA信号传递过程     

Suppression of methionine-induced hyperhomocysteinemia by dietary eritadenine in rats

The effect of dietary eritadenine on the plasma homocysteine concentration was investigated in methionine-induced hyperhomocysteinemic rats. The rats were fed on the control or eritadenine-supplemented (50 mg/kg) diet for 10 d. The animals were then injected with saline or methionine at a level of 100 or 300 mg/kg of body weight, and sacrificed 2 h or a more appropriate time after injection. The methionine injection increased the post-2 h concentration of plasma homocysteine in a dose-dependent manner in the control rats, this increase being significantly suppressed in the eritadenine-fed rats. This effect persisted up to 8 h after the methionine injection. The hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine were increased by eritadenine, whereas the hepatic homocysteine concentration was inversely decreased. The cystathionine beta-synthase activity in the liver was increased by eritadenine. It is suggested from these results that eritadenine might suppress the methionine-induced increase in plasma homocysteine concentration by dual mechanisms: slowing the homocysteine production from S-adenosylhomocysteine and increasing the removal of homocysteine due to the enhanced activity of cystathionine beta-synthase.     

Macromolecular Content of Inclusions Produced by a Canine Adenovirus

Early inclusions induced by a canine adenovirus in a canine cell line, appearing before the formation of infectious virus particles, were purified by differential centrifugation in sucrose followed by CsCl density gradient centrifugation. Chemical analysis of these inclusions revealed that they contained deoxyribonucleic acid (DNA), ribonucleic acid, and protein. On the basis of density gradient centrifugation, the DNA extracted from the inclusions was found to be viral DNA. Electron microscope autoradiography showed that these inclusions were the sites of DNA synthesis. In addition, association of DNA polymerase activity with the inclusions was detected by incorporation of radioactivity from (3)H-thymidine triphosphate into a DNA product. The in vitro product of the enzyme had a density equal to that of viral DNA rather than host DNA. The level of DNA polymerase activity in exponentially growing infected and uninfected whole cells was similar, but in cells in stationary phase the enzyme activity of infected cells was twice that in noninfected cells. Furthermore, nuclei isolated from infected cells showed a fourfold increase in DNA polymerase activity over the noninfected cells.     

Methionine metabolism in BHK cells: selection and characterization of ethionine resistant clones.

The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo-B12. Clones resistant to 30 mug/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 X 10(-5). An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone. Measurement of the specific activity of menadione reductase, B12 methyltransferase and ATP: L-methionine S-adenosyl-transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback-inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase. These results suggest that the pathway of re-use of S-adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.     

Methionine metabolism in mammals. Adaptation to methionine excess

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.     

Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.