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Sample records for methionine adenosyltransferase 1a

  1. Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.

    PubMed

    Martínez-Chantar, Maria L; García-Trevijano, Elena R; Latasa, M Ujue; Martín-Duce, Antonio; Fortes, Puri; Caballería, Juan; Avila, Matías A; Mato, José M

    2003-04-01

    Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.

  2. Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis.

    PubMed

    Berger, Bradley J; Knodel, Marvin H

    2003-06-16

    Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM. The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity

  3. Transsulfuration in an adult with hepatic methionine adenosyltransferase deficiency.

    PubMed Central

    Gahl, W A; Bernardini, I; Finkelstein, J D; Tangerman, A; Martin, J J; Blom, H J; Mullen, K D; Mudd, S H

    1988-01-01

    We investigated sulfur and methyl group metabolism in a 31-yr-old man with partial hepatic methionine adenosyltransferase (MAT) deficiency. The patient's cultured fibroblasts and erythrocytes had normal MAT activity. Hepatic S-adenosylmethionine (SAM) was slightly decreased. This clinically normal individual lives with a 20-30-fold elevation of plasma methionine (0.72 mM). He excretes in his urine methionine and L-methionine-d-sulfoxide (2.7 mmol/d), a mixed disulfide of methanethiol and a thiol bound to an unidentified group X, which we abbreviate CH3S-SX (2.1 mmol/d), and smaller quantities of 4-methylthio-2-oxobutyrate and 3-methylthiopropionate. His breath contains 17-fold normal concentrations of dimethylsulfide. He converts only 6-7 mmol/d of methionine sulfur to inorganic sulfate. This abnormally low rate is due not to a decreased flux through the primarily defective enzyme, MAT, since SAM is produced at an essentially normal rate of 18 mmol/d, but rather to a rate of homocysteine methylation which is abnormally high in the face of the very elevated methionine concentrations demonstrated in this patient. These findings support the view that SAM (which is marginally low in this patient) is an important regulator that helps to determine the partitioning of homocysteine between degradation via cystathionine and conservation by reformation of methionine. In addition, these studies demonstrate that the methionine transamination pathway operates in the presence of an elevated body load of that amino acid in human beings, but is not sufficient to maintain methionine levels in a normal range. PMID:3339126

  4. Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.

    PubMed

    Torres, L; Avila, M A; Carretero, M V; Latasa, M U; Caballería, J; López-Rodas, G; Boukaba, A; Lu, S C; Franco, L; Mato, J M

    2000-01-01

    Methionine adenosyltransferase (MAT) is the enzyme that catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main donor of methyl groups in the cell. In mammals MAT is the product of two genes, MAT1A and MAT2A. MAT1A is expressed only in the mature liver whereas fetal hepatocytes, extrahepatic tissues and liver cancer cells express MAT2A. The mechanisms behind the tissue and differentiation state specific MAT1A expression are not known. In the present work we examined MAT1A promoter methylation status by means of methylation sensitive restriction enzyme analysis. Our data indicate that MAT1A promoter is hypomethylated in liver and hypermethylated in kidney and fetal rat hepatocytes, indicating that this modification is tissue specific and developmentally regulated. Immunoprecipitation of mononucleosomes from liver and kidney tissues with antibodies mainly specific to acetylated histone H4 and subsequent Southern blot analysis with a MAT1A promoter probe demonstrated that MAT1A expression is linked to elevated levels of chromatin acetylation. Early changes in MAT1A methylation are already observed in the precancerous cirrhotic livers from rats, which show reduced MAT1A expression. Human hepatoma cell lines in which MAT1A is not expressed were also hypermethylated at this locus. Finally we demonstrate that MAT1A expression is reactivated in the human hepatoma cell line HepG2 treated with 5-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin, suggesting a role for DNA hypermethylation and histone deacetylation in MAT1A silencing.

  5. Short-chain ceramide regulates hepatic methionine adenosyltransferase expression.

    PubMed

    Frago, L M; Pañeda, C; Fabregat, I; Varela-Nieto, I

    2001-02-01

    The metabolism of methionine plays an important role in regulating hepatic cellular function. Methionine adenosyltransferase (MAT) is the enzyme that catalyses the biosynthesis of S-adenosylmethionine (AdoMet) from ATP and methionine. Liver-specific MAT I/III levels are down-regulated in the regenerating rat liver after partial hepatectomy. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are two cytokines fundamental for liver regeneration. TNF-alpha stimulates sphingomyelin metabolism and ceramide generation in a variety of cell systems. The role of exogenous cell-permeable ceramide in modifying MAT I/III mRNA levels and its association with TNF-alpha and IL-6 actions were investigated in rat hepatocytes and H35 hepatoma cells. C2-ceramide (N-acetylsphingosine) at 1-10 microM decreased MAT I/III expression. The effect was maximum after 2 h of treatment and it was maintained up to 24 h. MAT I/III protein levels also decreased. IL-6 (1-10 ng/ml) potentiated C2-ceramide effects in cultured hepatocytes while decreasing by itself MAT I/III levels with a similar time-response curve in both cell types. C2-ceramide actions were not associated with an increase in cell death. TNF-alpha was also a potent antagonist for MAT I/III expression, at 1-20 ng/ml decreased MAT I/III levels and induced endogenous ceramide generation. The decrease of MAT I/III mRNA levels (in all the cases) was not due to a decrease in mRNA half-life which suggests a regulation at the transcriptional level. Finally, the decrease in MAT I/III mRNA levels correlated to a decrease in MAT activity. This work demonstrates that short-chain ceramide can be used as a novel exogenous agonist that can modulate hepatic methionine metabolism in association with cytokines.

  6. Refolding and characterization of methionine adenosyltransferase from Euglena gracilis.

    PubMed

    Garrido, Francisco; Estrela, Sylvie; Alves, Claudia; Sánchez-Pérez, Gabino F; Sillero, Antonio; Pajares, María A

    2011-09-01

    Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 μmol/min/mg and K(m) values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.

  7. Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis

    PubMed Central

    2013-01-01

    Background Methionine adenosyltransferases catalyse the synthesis of S-adenosylmethionine, a cofactor abundant in all domains of life. In contrast to the enzymes from bacteria and eukarya that show high sequence similarity, methionine adenosyltransferases from archaea diverge on the amino acid sequence level and only few conserved residues are retained. Results We describe the initial characterisation and the crystal structure of the methionine adenosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis. As described for other archaeal methionine adenosyltransferases the enzyme is a dimer in solution and shows high temperature stability. The overall structure is very similar to that of the bacterial and eukaryotic enzymes described, with some additional features that might add to the stability of the enzyme. Compared to bacterial and eukaryotic structures, the active site architecture is largely conserved, with some variation in the substrate/product-binding residues. A flexible loop that was not fully ordered in previous structures without ligands in the active side is clearly visible and forms a helix that leaves an entrance to the active site open. Conclusions The similar three-dimensional structures of archaeal and bacterial or eukaryotic methionine adenosyltransferases support that these enzymes share an early common ancestor from which they evolved independently, explaining the low similarity in their amino acid sequences. Furthermore, methionine adenosyltransferase from T. kodakarensis is the first structure without any ligands bound in the active site where the flexible loop covering the entrance to the active site is fully ordered, supporting a mechanism postulated earlier for the methionine adenosyltransferase from E. coli. The structure will serve as a starting point for further mechanistic studies and permit the generation of enzyme variants with different characteristics by rational design. PMID:24134203

  8. Neurologically normal development of a patient with severe methionine adenosyltransferase I/III deficiency after continuing dietary methionine restriction.

    PubMed

    Hirabayashi, Koichi; Shiohara, Masaaki; Yamada, Kazuhiro; Sueki, Akane; Ide, Yuichiro; Takeuchi, Koichi; Hagimoto, Rokuro; Kinoshita, Tatsuya; Yabuhara, Akihiko; Mudd, S Harvey; Koike, Kenichi

    2013-11-01

    There is not much information on established standard therapy for patients with severe methionine adenosyltransferase (MAT) I/III deficiency. We report a boy with MAT I/III deficiency, in whom plasma methionine and total homocysteine, and urinary homocystine were elevated. Molecular genetic studies showed him to have novel compound heterozygous mutations of the MAT1A gene: c.191T>A (p.M64K) and c.589delC (p.P197LfsX26). A low methionine milk diet was started at 31 days of age, and during continuing dietary methionine restriction plasma methionine levels have been maintained at less than 750 μmol/L. He is now 5 years old, and has had entirely normal physical growth and psychomotor development. Although some severely MAT I/III deficient patients have developed neurologic abnormalities, we report here the case of a boy who has remained neurologically and otherwise normal for 5 years during methionine restriction, suggesting that perhaps such management, started in early infancy, may help prevent neurological complications. © 2013 Elsevier B.V. All rights reserved.

  9. L-methionine availability regulates expression of the methionine adenosyltransferase 2A gene in human hepatocarcinoma cells: role of S-adenosylmethionine.

    PubMed

    Martínez-Chantar, Maria L; Latasa, M Ujue; Varela-Rey, Marta; Lu, Shelly C; García-Trevijano, Elena R; Mato, José M; Avila, Matías A

    2003-05-30

    In mammals, methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (AdoMet) synthesis, is encoded by two genes, MAT1A and MAT2A. In liver, MAT1A expression is associated with high AdoMet levels and a differentiated phenotype, whereas MAT2A expression is associated with lower AdoMet levels and a dedifferentiated phenotype. In the current study, we examined regulation of MAT2A gene expression by l-methionine availability using HepG2 cells. In l-methionine-deficient cells, MAT2A gene expression is rapidly induced, and methionine adenosyltransferase activity is increased. Restoration of l-methionine rapidly down-regulates MAT2A mRNA levels; for this effect, l-methionine needs to be converted into AdoMet. This novel action of AdoMet is not mediated through a methyl transfer reaction. MAT2A gene expression was also regulated by 5'-methylthioadenosine, but this was dependent on 5'-methylthioadenosine conversion to methionine through the salvage pathway. The transcription rate of the MAT2A gene remained unchanged during l-methionine starvation; however, its mRNA half-life was significantly increased (from 100 min to more than 3 h). The effect of l-methionine withdrawal on MAT2A mRNA stabilization requires both gene transcription and protein synthesis. We conclude that MAT2A gene expression is modulated as an adaptive response of the cell to l-methionine availability through its conversion to AdoMet.

  10. The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

    PubMed Central

    Garrido, Francisco; Reytor, Edel; Portillo, Francisco; Pajares, María A.

    2016-01-01

    Methionine adenosyltransferases MAT I and MAT III (encoded by Mat1a) catalyze S-adenosylmethionine synthesis in normal liver. Major hepatic diseases concur with reduced levels of this essential methyl donor, which are primarily due to an expression switch from Mat1a towards Mat2a. Additional changes in the association state and even in subcellular localization of these isoenzymes are also detected. All these alterations result in a reduced content of the moderate (MAT I) and high Vmax (MAT III) isoenzymes, whereas the low Vmax (MAT II) isoenzyme increases and nuclear accumulation of MAT I is observed. These changes derive in a reduced availability of cytoplasmic S-adenosylmethionine, together with an effort to meet its needs in the nucleus of damaged cells, rendering enhanced levels of certain epigenetic modifications. In this context, the putative role of protein-protein interactions in the control of S-adenosylmethionine synthesis has been scarcely studied. Using yeast two hybrid and a rat liver library we identified PDRG1 as an interaction target for MATα1 (catalytic subunit of MAT I and MAT III), further confirmation being obtained by immunoprecipitation and pull-down assays. Nuclear MATα interacts physically and functionally with the PDRG1 oncogene, resulting in reduced DNA methylation levels. Increased Pdrg1 expression is detected in acute liver injury and hepatoma cells, together with decreased Mat1a expression and nuclear accumulation of MATα1. Silencing of Pdrg1 expression in hepatoma cells alters their steady-state expression profile on microarrays, downregulating genes associated with tumor progression according to GO pathway analysis. Altogether, the results unveil the role of PDRG1 in the control of the nuclear methylation status through methionine adenosyltransferase binding and its putative collaboration in the progression of hepatic diseases. PMID:27548429

  11. Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III☆

    PubMed Central

    Schröder, Paul C.; Fernández-Irigoyen, Joaquín; Bigaud, Emilie; Serna, Antonio; Renández-Alcoceba, Rubén; Lu, Shelly C.; Mato, José M.; Prieto, Jesús; Corrales, Fernando J.

    2015-01-01

    Methionine adenosyltransferase I/III (MATI/III) synthesizes S-adenosylmethionine (SAM) in quiescent hepatocytes. Its activity is compromised in most liver diseases including liver cancer. Since SAM is a driver of hepatocytes fate we have studied the effect of re-expressing MAT1A in hepatoma Huh7 cells using proteomics. MAT1A expression leads to SAM levels close to those found in quiescent hepatocytes and induced apoptosis. Normalization of intracellular SAM induced alteration of 128 proteins identified by 2D-DIGE and gel-free methods, accounting for deregulation of central cellular functions including apoptosis, cell proliferation and survival. Human Dead-box protein 3 (DDX3X), a RNA helicase regulating RNA splicing, export, transcription and translation was down-regulated upon MAT1A expression. Our data support the regulation of DDX3X levels by SAM in a concentration and time dependent manner. Consistently, DDX3X arises as a primary target of SAM and a principal intermediate of its antitumoral effect. Based on the parallelism between SAM and DDX3X along the progression of liver disorders, and the results reported here, it is tempting to suggest that reduced SAM in the liver may lead to DDX3X up-regulation contributing to the pathogenic process and that replenishment of SAM might prove to have beneficial effects, at least in part by reducing DDX3X levels. This article is part of a Special Issue entitled: Proteomics: The clinical link. PMID:22270009

  12. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes

    PubMed Central

    Murray, Ben; Antonyuk, Svetlana V.; Marina, Alberto; Lu, Shelly C.; Mato, Jose M.; Hasnain, S. Samar; Rojas, Adriana L.

    2016-01-01

    The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a “structural movie” of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE. PMID:26858410

  13. Novel immunoassays to detect methionine adenosyltransferase activity and quantify S-adenosylmethionine.

    PubMed

    Hao, Xiujuan; Zhou, Min; Li, Huijun; Angres, Isaac A

    2017-04-01

    We present a novel real-time immunoassay to measure methionine adenosyltransferase (MAT) activity that integrates the MAT-catalyzed reaction of Met and adenosine triphosphate to produce S-adenosylmethionine (SAM) and a highly sensitive immunoassay to specifically quantify SAM simultaneously. The cellular localization of SAM and S-adenosylhomocysteine varies with cell proliferation status: in normal cells, they are found mostly in the cytoplasm, but localize to the nucleus in proliferating cells. MAT-I/III activity is stimulated by Met, but inhibited by S-nitrosoglutathione, and the methylation index (MI) increases after Met stimulation of L02 cells. Met and S-nitrosoglutathione inhibit MAT-II activity, and the MI decreases after Met stimulation of HepG2 cells. The method described provides a significant advancement in the field for the measurement of MAT activity under various conditions. © 2017 Federation of European Biochemical Societies.

  14. Understanding Molecular Recognition of Promiscuity of Thermophilic Methionine Adenosyltransferase, sMAT from Sulfolobus solfataricus

    PubMed Central

    Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.

    2014-01-01

    Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoMet) bound, and unbound forms. Notably, among these structures, the AdoEth-bound form offers the first MAT structure containing a non-native product and cumulatively, these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in E. coli and human. As a thermostable MAT structure from archaea, the structures herein also provide as a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed ‘alkylrandomization’ and/or the study of methylation in the context of biological processes. PMID:24649856

  15. Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer.

    PubMed

    Tomasi, Maria Lauda; Ryoo, Minjung; Skay, Anna; Tomasi, Ivan; Giordano, Pasquale; Mato, José M; Lu, Shelly C

    2013-07-15

    Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70-75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

    SciTech Connect

    Tomasi, Maria Lauda; Ryoo, Minjung; Skay, Anna; Tomasi, Ivan; Giordano, Pasquale; Mato, José M.; Lu, Shelly C.

    2013-07-15

    Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100 pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell. -- Highlights: • MAT2A knockdown depletes putrescine and leads to apoptosis. • Putrescine attenuates MAT2A knockdown-induced apoptosis and growth

  17. Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma.

    PubMed

    Ito, K; Ikeda, S; Kojima, N; Miura, M; Shimizu-Saito, K; Yamaguchi, I; Katsuyama, I; Sanada, K; Iwai, T; Senoo, H; Horikawa, S

    2000-01-01

    Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet) from ATP and L-methionine. AdoMet is the major methyl donor in most transmethylation reactions in vivo, and it is also the propylamino donor in the biosynthesis of polyamines. In the present study, we assessed MAT activity in human colons with colorectal carcinoma and the values were compared with those of morphologically normal adjacent mucosa. Higher levels of MAT activity were observed in the colorectal carcinoma than in the normal colon. The ratio of MAT activity in tumor tissue versus normal tissue seemed to be correlated well will the stage of the colorectal tumor. Furthermore, immunoblot analysis showed that the high levels of MAT activity observed in colorectal carcinoma were due to the increased amounts of MAT protein. Immunohistochemical analysis revealed that MAT was most abundant in goblet cells, particularly in granules in the supranuclear area of these cells. In the colorectal carcinoma tissues, MAT was strongly stained in the cancerous cells and localized in granules in the supranuclear region. The results of this preliminary study suggest that determination of the relative ratio of MAT activity in both normal and tumor regions in human colorectal carcinoma could be a clinically useful tool for determining the stage of malignancy of colorectal carcinomas.

  18. Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.

    PubMed

    Yao, Gaofeng; Qin, Xiulin; Chu, Ju; Wu, Xiaole; Qian, Jiangchao

    2014-02-01

    Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using L-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93%, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg(2+) and was slightly stimulated by the monovalent cation K(+). It showed an improved thermostability, about 50% of the enzyme activity was retained even after preincubation at 50 °C for 2 h.

  19. Impact of glutathione supplementation of parenteral nutrition on hepatic methionine adenosyltransferase activity

    PubMed Central

    Elremaly, Wesam; Mohamed, Ibrahim; Rouleau, Thérèse; Lavoie, Jean-Claude

    2015-01-01

    Background The oxidation of the methionine adenosyltransferase (MAT) by the combined impact of peroxides contaminating parenteral nutrition (PN) and oxidized redox potential of glutathione is suspected to explain its inhibition observed in animals. A modification of MAT activity is suspected to be at origin of the PN-associated liver disease as observed in newborns. We hypothesized that the correction of redox potential of glutathione by adding glutathione in PN protects the MAT activity. Aim To investigate whether the addition of glutathione to PN can reverse the inhibition of MAT observed in animal on PN. Methods Three days old guinea pigs received through a jugular vein catheter 2 series of solutions. First with methionine supplement, (1) Sham (no infusion); (2) PN: amino acids, dextrose, lipids and vitamins; (3) PN-GSSG: PN+10 μM GSSG. Second without methionine, (4) D: dextrose; (5) D+180 μM ascorbylperoxide; (6) D+350 μM H2O2. Four days later, liver was sampled for determination of redox potential of glutathione and MAT activity in the presence or absence of 1 mM DTT. Data were compared by ANOVA, p<0.05. Results MAT activity was 45±4% lower in animal infused with PN and 23±7% with peroxides generated in PN. The inhibition by peroxides was associated with oxidized redox potential and was reversible by DTT. Correction of redox potential (PN+GSSG) or DTT was without effect on the inhibition of MAT by PN. The slope of the linear relation between MAT activity and redox potential was two fold lower in animal infused with PN than in others groups. Conclusion The present study suggests that prevention of peroxide generation in PN and/or correction of the redox potential by adding glutathione in PN are not sufficient, at least in newborn guinea pigs, to restore normal MAT activity. PMID:26722840

  20. Cloning expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes.

    PubMed

    Reguera, Rosa M; Balaña-Fouce, Rafael; Pérez-Pertejo, Yolanda; Fernández, Francisco J; García-Estrada, Carlos; Cubría, Juan C; Ordóñez, César; Ordóñez, David

    2002-02-01

    Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis. In this study, genomic DNA from the protozoan parasite Leishmania infantum was cloned and characterized. L. infantum MAT, unlike mammalian MAT, is codified by two identical genes in a tandem arrangement and is only weakly regulated by AdoMet. L. infantum MAT mRNA is expressed as a single transcript, with the enzyme forming a homodimer with tripolyphosphatase in addition to MAT activity. Expression of L. infantum MAT in Escherichia coli proves that the MAT and tripolyphosphatase activities are functional in vivo. MAT shows sigmoidal behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoidal behavior and is strongly activated by AdoMet. Plasmids containing the regions flanking MAT2 were fused immediately upstream and downstream of the luciferase-coding region and transfected into L. infantum. Subsequent examination of luciferase activity showed that homologous expression in L. infantum promastigotes was dramatically dependent on the presence of polypyrimidine tracts and a spliced leader junction site upstream of the luciferase gene, whereas downstream sequences appeared to have no bearing on expression.

  1. Characterization of a methionine adenosyltransferase over-expressing strain in the trypanosomatid Leishmania donovani.

    PubMed

    Pérez-Pertejo, Yolanda; Reguera, Rosa M; Ordóñez, David; Balaña-Fouce, Rafael

    2006-01-01

    Methionine adenosyltransferase (MAT: EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (AdoMet) in two sequential steps, AdoMet formation and subsequent tripolyphosphate (PPPi) cleavage, induced by AdoMet. In pursuit of a better understanding of the biological function of the enzyme, the MAT gene was cloned into vector PX63NEO to induce episomal overexpression in leishmania parasites. Neomycin-selected clones originated a strain of such overexpressing parasites that accumulated more than 3-fold AdoMet than mock-transfected cells and showed over ten times the wild type MAT activity, concurring with a significant accumulation of the MAT protein during the early logarithmic phase and MAT transcripts throughout the growth cycle. The rate of AdoMet efflux, practically nil in the control promastigotes, was exceptionally high in the MAT-overexpressing parasites, whilst growth in this strain was comparable to development in control cells, i.e., it was not affected by deleterious hypermethylation. Moreover, the modified strain was 10-fold more resistant to sinefungin, a S-adenosylmethionine-like antibiotic, than control cells. The effects of overexpression on polyamine metabolism and transport were likewise studied.

  2. Leishmania donovani: proteasome-mediated down-regulation of methionine adenosyltransferase.

    PubMed

    Pérez-Pertejo, Yolanda; Alvarez-Velilla, Raquel; Estrada, Carlos García; Balaña-Fouce, Rafael; Reguera, Rosa M

    2011-08-01

    Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and 'mock-transfected' leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.

  3. Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits.

    PubMed

    Shafqat, Naeem; Muniz, Joao R C; Pilka, Ewa S; Papagrigoriou, Evangelos; von Delft, Frank; Oppermann, Udo; Yue, Wyatt W

    2013-05-15

    MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.

  4. Determination of Autosomal Dominant or Recessive Methionine Adenosyltransferase I/III Deficiencies Based on Clinical and Molecular Studies.

    PubMed

    Kim, Yoo-Mi; Kim, Ja Hye; Choi, Jin-Ho; Kim, Gu-Hwan; Kim, Jae-Min; Kang, Minji; Choi, In-Hee; Cheon, Chong Kun; Sohn, Young Bae; Maccarana, Marco; Yoo, Han-Wook; Lee, Beom Hee

    2016-02-18

    Methionine adenosyltransferase (MAT) I/III deficiency can be inherited as autosomal dominant (AD) or as recessive (AR) traits in which mono- or bi-allelic MAT1A mutations have been identified, respectively. Although most patients have benign clinical outcomes, some with the AR form have neurological deficits. Here we describe 16 Korean patients with MAT I/III deficiency from 15 unrelated families identified by newborn screening. Ten probands had the AD MAT I/III deficiency, while six had AR MAT I/III deficiency. Plasma methionine (145.7 μmol/L vs. 733.2 μmol/L, P < 0.05) and homocysteine levels (12.3 μmol/L vs. 18.6 μmol/L, P < 0.05) were lower in the AD type than in AR type. In addition to the only reported AD MAT1A mutation, p.Arg264His, we identified two novel AD mutations, p.Arg249Gln and p.Gly280Arg. In the AR type, four previously reported and two novel mutations, p.Arg163Trp and p.Tyr335*, were identified. No exonic deletions were found by quantitative genomic PCR. Three-dimensional structural prediction programs indicated that the AD type mutations were located on the dimer interface or in the substrate binding site, hindering MAT I/III dimerization or substrate binding, respectively, whereas the AR mutations were distant from the interface or substrate binding site. These results indicate that the AD or AR MAT I/III deficiency is correlated with clinical findings, substrate levels, and structural features of the mutant proteins, which is important for the neurological management and genetic counseling of the patients.

  5. Analysis of genetic elements regulating the methionine adenosyltransferase gene in Leishmania infantum.

    PubMed

    García-Estrada, Carlos; Pérez-Pertejo, Yolanda; Ordóñez, David; Balaña-Fouce, Rafael; Reguera, Rosa M

    2007-03-15

    Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, inasmuch as its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Our prior studies have shown that the Leishmania infantum genome contains two identical copies of the gene encoding MAT (MAT2 gene), arranged in head-to-tail configuration and alternating with another gene, called LORIEN that contains a zinc-finger motif. Both genes are constitutively expressed throughout the promastigote stage of the parasite cell cycle, and their flanking regions were detected by RT-PCR. Luciferase (luc) reporter assays indicated the presence of regulatory elements within the MAT2 3'UTR and intergenic region, and fragments responsible for such regulation were identified by deletional analysis. By site-directed mutagenesis of the wild-type -42 AG recognized in the trans-splicing of the MAT2 gene, the AG slightly downstream (position -36) was observed to be able to generate the same levels of luc expression, thus suggesting that potentially this gene has alternative spliced leader acceptor sites. The stability of MAT2 and LORIEN transcripts was very similar in both logarithmic and stationary phases. However, cycloheximide (CHX) inhibition of protein synthesis increased MAT2 and LORIEN mRNA levels in the logarithmic phase only, an indication that these genes are regulated in promastigotes at the post-transcriptional level by protein factors that targets both transcripts for degradation. However, during the stationary phase, another CHX-independent factor also led to MAT2 and LORIEN mRNAs degradation, indicating the existence of different mechanisms operating on the post-transcriptional regulation of these two genes.

  6. Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression.

    PubMed

    Kim, Jong-So; Coon, Steven L; Blackshaw, Seth; Cepko, Constance L; Møller, Morten; Mukda, Sujira; Zhao, Wan-Qian; Charlton, Clivel G; Klein, David C

    2005-01-07

    (S)-adenosylmethionine (SAM) is a critical element of melatonin synthesis as the methyl donor in the last step of the pathway, the O-methylation of N-acetyl 5-hydroxytryptamine by hydroxyindole-O-methyltransferase. The activity of the enzyme that synthesizes SAM, methionine adenosyltransferase (MAT), increases 2.5-fold at night in the pineal gland. In this study, we found that pineal MAT2A mRNA and the protein it encodes, MAT II, also increase at night, suggesting that the increase in MAT activity is caused by an increase in MAT II gene products. The night levels of MAT2A mRNA in the pineal gland were severalfold higher than in other neural and non-neural tissues examined, consistent with the requirement for SAM in melatonin synthesis. Related studies indicate that the nocturnal increase in MAT2A mRNA is caused by activation of a well described neural pathway that mediates photoneural-circadian regulation of the pineal gland. MAT2A mRNA and MAT II protein were increased in organ culture by treatment with norepinephrine (NE), the sympathetic neurotransmitter that stimulates the pineal gland at night. NE is known to markedly elevate pineal cAMP, and here it was found that cAMP agonists elevate MAT2A mRNA levels by increasing MAT2A mRNA synthesis and that drugs that block cAMP activation of cAMP dependent protein kinase block effects of NE. Therefore, the NE-cAMP dependent increase in pineal MAT activity seems to reflect an increase in MAT II protein, which occurs in response to cAMP-->protein kinase-dependent increased MAT2A expression. The existence of this MAT regulatory system underscores the importance that MAT plays in melatonin biogenesis. These studies also point to the possibility that SAM production in other tissues might be regulated through cAMP.

  7. Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation.

    PubMed

    Lindermayr, Christian; Saalbach, Gerhard; Bahnweg, Günther; Durner, Jörg

    2006-02-17

    In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling.

  8. Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation.

    PubMed

    Huang, Y; Gou, X; Hu, H; Xu, Q; Lu, Y; Cheng, J

    2012-04-01

    S-adenosyl-l-methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Through spaceflight culture, a SAM-accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one-factor-at-a-time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l(-1) of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l(-1) of SAM was produced in fermenter cultivation. A new SAM-accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l(-1) of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for

  9. Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.

    PubMed Central

    Gasset, María; Alfonso, Carlos; Neira, José L; Rivas, Germán; Pajares, María A

    2002-01-01

    The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established. PMID:11772402

  10. Characterization of methionine adenosyltransferase 2beta gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs.

    PubMed

    Fang, Qian; Yin, Jingdong; Li, Fengna; Zhang, Jinxiao; Watford, Malcolm

    2010-06-01

    Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit (MAT II beta) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2beta as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2beta cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2beta cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2beta is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2beta mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II beta protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II beta subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2beta gene in regulation of IMF deposition in skeletal muscle.

  11. S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells.

    PubMed

    Lozano-Sepulveda, Sonia Amelia; Bautista-Osorio, Eduardo; Merino-Mascorro, Jose Angel; Varela-Rey, Marta; Muñoz-Espinosa, Linda Elsa; Cordero-Perez, Paula; Martinez-Chantar, María Luz; Rivas-Estilla, Ana Maria

    2016-04-14

    To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM treatment at both the

  12. S-adenosyl-L-methionine modifies antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells

    PubMed Central

    Lozano-Sepulveda, Sonia Amelia; Bautista-Osorio, Eduardo; Merino-Mascorro, Jose Angel; Varela-Rey, Marta; Muñoz-Espinosa, Linda Elsa; Cordero-Perez, Paula; Martinez-Chantar, María Luz; Rivas-Estilla, Ana Maria

    2016-01-01

    AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine (SAM) decreases hepatitis C virus (HCV) expression. METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times (24-72 h), then total RNA and proteins were isolated. cDNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2 (SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A (MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1A, MAT2A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman’s recycling method (0-24 h). Reactive oxidative species (ROS) levels were quantified by the dichlorofluorescein assay (0-48 h); Pyrrolidin dithiocarbamate (PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent. RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls (24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression (SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2A, since MAT1A expression was increased (2.5 fold-times at 48 h) and MAT2A was diminished (from 24 h) upon SAM

  13. Deregulated Methionine Adenosyltransferase α1, c-Myc and Maf Proteins Interplay Promotes Cholangiocarcinoma Growth in Mice and Humans

    PubMed Central

    Yang, Heping; Liu, Ting; Wang, Jiaohong; Li, Tony W.H.; Fan, Wei; Peng, Hui; Krishnan, Anuradha; Gores, Gregory J.; Mato, Jose M.; Lu, Shelly C.

    2016-01-01

    We reported c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice. We also showed induction of Maf proteins (MafG and c-Maf) contributed to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration was protective. Here we determined whether there is interplay between c-Myc, Maf proteins and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms and functional outcomes. We found c-Myc, MATα1 (encoded by MAT1A), MafG and c-Maf interact with each other directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver but this switches to c-Maf, MafG and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG and c-Maf, but it negatively regulates MAT1A. MATα1 represses whereas c-Myc, MafG and c-Maf enhance E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. Conclusion We have uncovered a novel interplay between MATα1, c-Myc and Maf proteins and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. PMID:26969892

  14. NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

    PubMed Central

    Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A.

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells. PMID:23189196

  15. NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

    PubMed

    González, Beatriz; Garrido, Francisco; Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

  16. Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by P(GAP)-driven Pichia pastoris overexpressing methionine adenosyltransferase.

    PubMed

    Hu, Xiao-Qing; Chu, Ju; Zhang, Zhuo; Zhang, Si-Liang; Zhuang, Ying-Ping; Wang, Yong-Hong; Guo, Mei-Jin; Chen, Hua-Xin; Yuan, Zhong-Yi

    2008-10-10

    Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)), to biosynthesize S-adenosyl-l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A-C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2-0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A-E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05Ug(-1) dried biomass for strategy E, since the specific glycerol consumption rate (F(G)) ascended due to the elevated specific oxygen uptake rate (qO(2)). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth (C*) and volumetric oxygen transfer coefficient (k(L)a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058gg(-1) and 9.26gl(-1), respectively.

  17. Methionine adenosyltransferase II-dependent histone H3K9 methylation at the COX-2 gene locus.

    PubMed

    Kera, Yohei; Katoh, Yasutake; Ohta, Mineto; Matsumoto, Mitsuyo; Takano-Yamamoto, Teruko; Igarashi, Kazuhiko

    2013-05-10

    MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.

  18. Comparative protein modeling of methionine S-adenosyltransferase (MAT) enzyme from Mycobacterium tuberculosis: a potential target for antituberculosis drug discovery.

    PubMed

    Khedkar, Santosh A; Malde, Alpeshkumar K; Coutinho, Evans C

    2005-01-01

    Mycobacterium tuberculosis (Mtb) is a successful pathogen that overcomes the numerous challenges presented by the immune system of the host. In the last 40 years few anti-TB drugs have been developed, while the drug-resistance problem is increasing; there is thus a pressing need to develop new anti-TB drugs active against both the acute and chronic growth phases of the mycobacterium. Methionine S-adenosyltransferase (MAT) is an enzyme involved in the synthesis of S-adenosylmethionine (SAM), a methyl donor essential for mycolipid biosynthesis. As an anti-TB drug target, Mtb-MAT has been well validated. A homology model of MAT has been constructed using the X-ray structures of E. coli MAT (PDB code: 1MXA) and rat MAT (PDB code: 1QM4) as templates, by comparative protein modeling principles. The resulting model has the correct stereochemistry as gauged from the Ramachandran plot and good three-dimensional (3D) structure compatibility as assessed by the Profiles-3D score. The structurally and functionally important residues (active site) of Mtb-MAT have been identified using the E. coli and rat MAT crystal structures and the reported point mutation data. The homology model conserves the topological and active site features of the MAT family of proteins. The differences in the molecular electrostatic potentials (MEP) of Mtb and human MAT provide evidences that selective and specific Mtb-MAT inhibitors can be designed using the homology model, by the structure-based drug design approaches.

  19. Biosynthesis of S-Adenosylmethionine by Magnetically Immobilized Escherichia coli Cells Highly Expressing a Methionine Adenosyltransferase Variant.

    PubMed

    Yin, Chunli; Zheng, Tao; Chang, Xin

    2017-08-18

    S-Adenosylmethionine (SAM) is a natural metabolite having important uses in the treatment of various diseases. To develop a simple and effective way to produce SAM, immobilized Escherichia coli cells highly expressing an engineered variant of methionine adenosyltransferase (MAT) were employed to synthesize SAM. The recombinant I303V MAT variant was successfully produced at approximately 900 mg/L in a 10-L bioreactor and exhibited significantly less product inhibition and had a four-fold higher specific activity (14.2 U/mg) than the wild-type MAT (3.6 U/mg). To reduce the mass transfer resistance, the free whole-cells were permeabilized and immobilized using gellan gum gel as support in the presence of 100 mg/L Fe₃O₄ nanoparticles, and the highest activity (4152.4 U/L support) was obtained, with 78.2% of the activity recovery. The immobilized cells were more stable than the free cells under non-reactive conditions, with a half-life of 9.1 h at 50 °C. Furthermore, the magnetically immobilized cells were employed to produce SAM at a 40-mM scale. The residual activity of the immobilized cells was 67% of its initial activity after 10 reuses, and the conversion rate of ATP was ≥95% in all 10 batches. These results indicated that magnetically immobilized cells should be a promising biocatalyst for the biosynthesis of SAM.

  20. ROLE OF METHIONINE ADENOSYLTRANSFERASE α2 AND β PHOSPHORYLATION AND STABILIZATION IN HUMAN HEPATIC STELLATE CELL TRANS-DIFFERENTIATION

    PubMed Central

    Ramani, Komal; Donoyan, Shant; Tomasi, Maria Lauda; Park, Sunhee

    2016-01-01

    Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members [ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (B-Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation. PMID:25294683

  1. Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.

    PubMed

    Ramani, Komal; Donoyan, Shant; Tomasi, Maria Lauda; Park, Sunhee

    2015-05-01

    Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation. © 2014 Wiley Periodicals, Inc.

  2. Temporal study of acetaminophen (APAP) and S-adenosyl-L-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity.

    PubMed

    Brown, J Michael; Ball, John G; Hogsett, Amy; Williams, Tierra; Valentovic, Monica

    2010-08-15

    Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 g) were treated with vehicle (Veh; water 15 ml/kg ip injections), 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered 1h after APAP injection (SAMe and SAMe+APAP). Hepatic tissue was collected 2, 4, and 6h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4h by APAP overdose, but not at 2 or 6h. APAP depressed mitochondrial SAMe levels at 4 and 6h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4h following APAP administration. SAMe administration following APAP (SAMe+APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity. (c) 2010 Elsevier Inc. All rights reserved.

  3. Temporal study of acetaminophen (APAP) and S-adenosyl-L-methionine (SAMe) effects on subcellular hepatic SAMe levels and methionine adenosyltransferase (MAT) expression and activity

    SciTech Connect

    Brown, J. Michael; Ball, John G.; Hogsett, Amy; Williams, Tierra; Valentovic, Monica

    2010-08-15

    Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 g) were treated with vehicle (Veh; water 15 ml/kg ip injections), 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered 1 h after APAP injection (SAMe and SAMe + APAP). Hepatic tissue was collected 2, 4, and 6 h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4 h by APAP overdose, but not at 2 or 6 h. APAP depressed mitochondrial SAMe levels at 4 and 6 h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4 h following APAP administration. SAMe administration following APAP (SAMe + APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity.

  4. Temporal Study of Acetaminophen (APAP) and S-Adenosyl-L-methionine (SAMe) Effects on Subcellular Hepatic SAMe Levels and Methionine Adenosyltransferase (MAT) Expression and Activity

    PubMed Central

    Brown, J. Michael; Ball, John G.; Hogsett, Amy; Williams, Tierra; Valentovic, Monica

    2010-01-01

    Acetaminophen (APAP) is the leading cause of drug induced liver failure in the United States. Previous studies in our laboratory have shown that S-adenosyl methionine (SAMe) is protective for APAP hepatic toxicity. SAMe is critical for glutathione synthesis and transmethylation of nucleic acids, proteins and phospholipids which would facilitate recovery from APAP toxicity. SAMe is synthesized in cells through the action of methionine adenosyltransferase (MAT). This study tested the hypothesis that total hepatic and subcellular SAMe levels are decreased by APAP toxicity. Studies further examined MAT expression and activity in response to APAP toxicity. Male C57BL/6 mice (16-22 grams) were treated with vehicle (Veh; water 15ml/kg ip injections). 250 mg/kg APAP (15 ml/kg, ip), SAMe (1.25 mmol/kg) or SAMe administered one h after APAP injection (SAMe and SAMe+APAP). Hepatic tissue was collected 2, 4, and 6 h after APAP administration. Levels of SAMe and its metabolite S-adenosylhomocysteine (SAH) were determined by HPLC analysis. MAT expression was examined by Western blot. MAT activity was determined by fluorescence assay. Total liver SAMe levels were depressed at 4 h by APAP overdose, but not at 2 or 6 h. APAP depressed mitochondrial SAMe levels at 4 and 6 h relative to the Veh group. In the nucleus, levels of SAMe were depressed below detectable limits 4 h following APAP administration. SAMe administration following APAP (SAMe+APAP) prevented APAP associated decline in mitochondrial and nuclear SAMe levels. In conclusion, the maintenance of SAMe may provide benefit in preventing damage associated with APAP toxicity. PMID:20450926

  5. Inhibition of hepatic methionine adenosyltransferase by peroxides contaminating parenteral nutrition leads to a lower level of glutathione in newborn Guinea pigs.

    PubMed

    Elremaly, Wesam; Rouleau, Thérèse; Lavoie, Jean-Claude

    2012-12-15

    Premature newborn infants on total parenteral nutrition (TPN) are at risk of oxidative stress because of peroxides contaminating TPN and low glutathione level. Low cysteine availability limits glutathione synthesis. In this population, the main source of cysteine derives from the hepatic conversion of methionine. The first enzyme of this conversion, methionine adenosyltransferase (MAT), contains redox-sensitive cysteinyl residues. We hypothesize that inhibition of MAT by peroxides contaminating TPN leads to a lower availability of cysteine for glutathione synthesis. At 3 days of life, animals were fitted with a jugular catheter for intravenous infusion. Four groups were compared by ANOVA (P<0.05): (1) Control, without surgery, fed regular chow; (2) Sham, fitted with an obstructed catheter, fed orally regular chow; (3) TPN, fed exclusively TPN (dextrose, amino acids, fat, vitamins) containing 350 μM peroxides; (4) H2O2, fed regular chow orally and infused with 350 μM H2O2. Four days later, MAT activity and glutathione in liver and blood were lower in TPN and H2O2 groups. The redox potential was more oxidized in blood and liver of the TPN group. In conclusion, peroxides generated in TPN inhibit methionine adenosyltransferase activity with, among consequences, a low level of glutathione and a more oxidized redox potential.

  6. Regulation of the human MAT2B gene encoding the regulatory beta subunit of methionine adenosyltransferase, MAT II.

    PubMed

    LeGros, L; Halim, A B; Chamberlin, M E; Geller, A; Kotb, M

    2001-07-06

    Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5'-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between -4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position -115 provided full activity; this was attributed to a TATA at -32. The Sp1 site at position +9 was key for the formation of protein.DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

  7. Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis.

    PubMed

    Frau, Maddalena; Feo, Francesco; Pascale, Rosa M

    2013-10-01

    Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients' survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC. Copyright © 2013 European Association for the Study of the Liver

  8. The Improvement of SAM Accumulation by Integrating the Endogenous Methionine Adenosyltransferase Gene SAM2 in Genome of the Industrial Saccharomyces cerevisiae Strain.

    PubMed

    Zhao, Weijun; Shi, Feng; Hang, Baojian; Huang, Lei; Cai, Jin; Xu, Zhinan

    2016-03-01

    S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.

  9. Inhibition of methionine adenosyltransferase II induces FasL expression, Fas-DISC formation and caspase-8-dependent apoptotic death in T leukemic cells.

    PubMed

    Jani, Tanvi S; Gobejishvili, Leila; Hote, Prachi T; Barve, Aditya S; Joshi-Barve, Swati; Kharebava, Giorgi; Suttles, Jill; Chen, Theresa; McClain, Craig J; Barve, Shirish

    2009-03-01

    Methionine adenosyltransferase II (MAT II) is a key enzyme in cellular metabolism and catalyzes the formation of S-adenosylmethionine (SAMe) from L-methionine and ATP. Normal resting T lymphocytes have minimal MAT II activity, whereas activated proliferating T lymphocytes and transformed T leukemic cells show significantly enhanced MAT II activity. This work was carried out to examine the role of MAT II activity and SAMe biosynthesis in the survival of leukemic T cells. Inhibition of MAT II and the resultant decrease in SAMe levels enhanced expression of FasL mRNA and protein, and induced DISC (Death Inducing Signaling Complex) formation with FADD (Fas-associated Death Domain) and procaspase-8 recruitment, as well as concomitant increase in caspase-8 activation and decrease in c-FLIP(s) levels. Fas-initiated signaling induced by MAT II inhibition was observed to link to the mitochondrial pathway via Bid cleavage and to ultimately lead to increased caspase-3 activation and DNA fragmentation in these cells. Furthermore, blocking MAT 2A mRNA expression, which encodes the catalytic subunits of MAT II, using a small-interfering RNA approach enhanced FasL expression and cell death, validating the essential nature of MAT II activity in the survival of T leukemic cells.

  10. Calcium-dependent protein kinase CPK28 targets the methionine adenosyltransferases for degradation by the 26S proteasome and affects ethylene biosynthesis and lignin deposition in Arabidopsis.

    PubMed

    Jin, Yu; Ye, Nenghui; Zhu, Fuyuan; Li, Haoxuan; Wang, Juan; Jiang, Liwen; Zhang, Jianhua

    2017-04-01

    S-adenosylmethionine (AdoMet) is synthesized by methionine adenosyltransferase (MAT), and plays an essential role in ethylene biosynthesis and other methylation reactions. Despite increasing knowledge of MAT regulation at transcriptional levels, how MAT is post-translationally regulated remains unknown in plant cells. Phosphorylation is an important post-translational modification for regulating the activity of enzymes, protein function and signaling transduction. Using molecular and biochemical approaches, we have identified the phosphorylation of MAT proteins by calcium-dependent protein kinase (CPK28). Phenotypically, both MAT2-overexpressing transgenic plants and cpk28 mutants display short hypocotyls and ectopic lignifications. Their shortened hypocotyl phenotypes are caused by ethylene overproduction and rescued by ethylene biosynthesis inhibitor aminoethoxyvinylglycine treatment. Genetic evidence reveals that MAT2 mutation restores the phenotype of ectopic lignification in CPK28-deficient plants. We find that total MAT proteins and AdoMet are increased in cpk28 mutants, but decreased in CPK28-overexpressing seedlings. We also find that MATs in OE::CPK28 are degraded through the 26S proteasome pathway. Our work suggests that CPK28 targets MATs (MAT1, MAT2 and MAT3) for degradation by the 26S proteasome pathway, and thus affects ethylene biosynthesis and lignin deposition in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  11. Ethanol inhibits methionine adenosyltransferase II activity and S-adenosylmethionine biosynthesis and enhances caspase-3-dependent cell death in T lymphocytes: relevance to alcohol-induced immunosuppression.

    PubMed

    Hote, Prachi T; Sahoo, Rashmita; Jani, Tanvi S; Ghare, Smita S; Chen, Theresa; Joshi-Barve, Swati; McClain, Craig J; Barve, Shirish S

    2008-06-01

    An important aspect in alcohol abuse-associated immune suppression is the loss of T helper CD4(+) lymphocytes, leading to impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4(+) T lymphocytes to caspase-3-dependent activation-induced cell death (AICD). It has been demonstrated that the formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase (MAT) II is essential for CD4(+) T-cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4(+) T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25 mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Furthermore, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3-dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data show that MAT II and SAMe are critical molecular components essential for CD4(+) T-cell survival that are affected by ethanol, leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse.

  12. MicroRNA-21-3p, a berberine-induced miRNA, directly down-regulates human methionine adenosyltransferases 2A and 2B and inhibits hepatoma cell growth.

    PubMed

    Lo, Ting-Fang; Tsai, Wei-Chung; Chen, Shui-Tein

    2013-01-01

    Methionine adenosyltransferase (MAT) is the cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM), the principal biological methyl donor and a key regulator of hepatocyte proliferation, death and differentiation. Two genes, MAT1A and MAT2A, encode 2 distinct catalytic MAT isoforms. A third gene, MAT2B, encodes a MAT2A regulatory subunit. In hepatocellular carcinoma (HCC), MAT1A downregulation and MAT2A upregulation occur, known as the MAT1A:MAT2A switch. The switch is accompanied with an increasing expression of MAT2B, which results in decreased SAM levels and facilitates cancer cell growth. Berberine, an isoquinoline alkaloid isolated from many medicinal herbs such as Coptis chinensis, has a wide range of pharmacological effects including anti-cancer effects. Because drug-induced microRNAs have recently emerged as key regulators in guiding their pharmacological effects, we examined whether microRNA expression is differentially altered by berberine treatment in HCC. In this study, we used microRNA microarrays to find that the expression level of miR-21-3p (previously named miR-21*) increased after berberine treatment in the HepG2 human hepatoma cell line. To predict the putative targets of miR-21-3p, we integrated the gene expression profiles of HepG2 cells after berberine treatment by comparing with a gene list generated from sequence-based microRNA target prediction software. We then confirmed these predictions through transfection of microRNA mimics and a 3' UTR reporter assay. Our findings provide the first evidence that miR-21-3p directly reduces the expression of MAT2A and MAT2B by targeting their 3' UTRs. In addition, an overexpression of miR-21-3p increased intracellular SAM contents, which have been proven to be a growth disadvantage for hepatoma cells. The overexpression of miR-21-3p suppresses growth and induces apoptosis in HepG2 cells. Overall, our results demonstrate that miR-21-3p functions as a tumor suppressor by directly

  13. ETHANOL INHIBITS METHIONINE ADENOSYLTRANSFERASE II (MAT II) ACTIVITY AND S-ADENOSYLMETHIONINE (SAMe) BIOSYNTHESIS AND ENHANCES CASPASE-3 DEPENDENT CELL DEATH IN T LYMHOCYTES: RELEVANCE TO ALCOHOL INDUCED IMMUNOSUPPRESSION

    PubMed Central

    Hote, Prachi T.; Sahoo, Rashmita; Jani, Tanvi S.; Ghare, Smita S.; Chen, Theresa; Joshi-Barve, Swati; McClain, Craig J.; Barve, Shirish S.

    2015-01-01

    An important aspect in alcohol abuse associated immune suppression is the loss of T helper CD4+ lymphocytes leading to an impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4+ T lymphocytes to activation-induced, caspase-3 dependent cell death (AICD). It has been demonstrated that formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase II (MAT II) is essential for CD4+ T cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4+ T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Further, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3 dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data shows that MAT II and SAMe are critical molecular components essential for CD4+ T cell survival which are affected by ethanol leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of the much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse. PMID:17869084

  14. MAT1A variants modulate the effect of dietary fatty acids on plasma homocysteine concentrations and DNA damage

    USDA-ARS?s Scientific Manuscript database

    Dietary n-3 polyunsaturated fatty acids (PUFA) are associated with decreased plasma homocysteine (Hcy), an important biomarker for cardiovascular disease. Methionine adenosyltransferase (MAT1A) is an enzyme involved in formation of form S-adenosylmethionine during methionine metabolism. The objectiv...

  15. Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease.

    PubMed

    Pacana, Tommy; Cazanave, Sophie; Verdianelli, Aurora; Patel, Vaishali; Min, Hae-Ki; Mirshahi, Faridoddin; Quinlivan, Eoin; Sanyal, Arun J

    2015-01-01

    Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA

  16. Methionine and methionine sulfoxide treatment induces M1/classical macrophage polarization and modulates oxidative stress and purinergic signaling parameters.

    PubMed

    Dos Santos, Lien M; da Silva, Tatiane M; Azambuja, Juliana H; Ramos, Priscila T; Oliveira, Pathise S; da Silveira, Elita F; Pedra, Nathalia S; Galdino, Kennia; do Couto, Carlus A T; Soares, Mayara S P; Tavares, Rejane G; Spanevello, Roselia M; Stefanello, Francieli M; Braganhol, Elizandra

    2017-01-01

    Methionine is an essential amino acid involved in critical metabolic process, and regulation of methionine flux through metabolism is important to supply this amino acid for cell needs. Elevation in plasma methionine commonly occurs due to mutations in methionine-metabolizing enzymes, such as methionine adenosyltransferase. Hypermethioninemic patients exhibit clinical manifestations, including neuronal and liver disorders involving inflammation and tissue injury, which pathophysiology is not completely established. Here, we hypothesize that alterations in macrophage inflammatory response may contribute to deleterious effects of hypermethioninemia. To this end, macrophage primary cultures were exposed to methionine (1 mM) and/or its metabolite methionine sulfoxide (0.5 mM), and M1/proinflammatory or M2/anti-inflammatory macrophage polarization was evaluated. In addition, inflammation-related pathways including oxidative stress parameters, as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities; reactive oxygen species (ROS) production, and purinergic signaling, as ATP/ADP/AMPase activities, were investigated. Methionine and/or methionine sulfoxide induced M1/classical macrophage activation, which is related to proinflammatory responses characterized by increased iNOS activity and TNF-α release. Further experiments showed that treatments promoted alterations on redox state of macrophages by differentially modulated SOD and CAT activities and ROS levels. Finally, methionine and/or methionine sulfoxide treatment also altered the extracellular nucleotide metabolism, promoting an increase of ATPase/ADPase activities in macrophages. In conclusion, these findings contribute to better understand the participation of proinflammatory responses in cell injury observed in hypermethioninemic patients.

  17. Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

    PubMed Central

    Allu, Praveen Kumar; Marada, Adinarayana; Boggula, Yerranna; Karri, Srinivasu; Krishnamoorthy, Thanuja; Sepuri, Naresh Babu V.

    2015-01-01

    Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway. PMID:25428986

  18. Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes

    PubMed Central

    Chandler, Tawny L.

    2017-01-01

    Intersections in hepatic methyl group metabolism pathways highlights potential competition or compensation of methyl donors. The objective of this experiment was to examine the expression of genes related to methyl group transfer and lipid metabolism in response to increasing concentrations of choline chloride (CC) and DL-methionine (DLM) in primary neonatal hepatocytes that were or were not exposed to fatty acids (FA). Primary hepatocytes isolated from 4 neonatal Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (61, 128, 2028, and 4528 μmol/L) and DLM (16, 30, 100, 300 μmol/L), with or without a 1 mmol/L FA cocktail in a factorial arrangement. After 24 h of treatment, media was collected for quantification of reactive oxygen species (ROS) and very low-density lipoprotein (VLDL), and cell lysates were collected for quantification of gene expression. No interactions were detected between CC, DLM, or FA. Both CC and DLM decreased the expression of methionine adenosyltransferase 1A (MAT1A). Increasing CC did not alter betaine-homocysteine S-methyltranferase (BHMT) but did increase 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylenetetrahydrofolate reductase (MTHFR) expression. Increasing DLM decreased expression of BHMT and MTR, but did not affect MTHFR. Expression of both phosphatidylethanolamine N-methyltransferase (PEMT) and microsomal triglyceride transfer protein (MTTP) were decreased by increasing CC and DLM, while carnitine palmitoyltransferase 1A (CPT1A) was unaffected by either. Treatment with FA decreased the expression of MAT1A, MTR, MTHFR and tended to decrease PEMT but did not affect BHMT and MTTP. Treatment with FA increased CPT1A expression. Increasing CC increased secretion of VLDL and decreased the accumulation of ROS in media. Within neonatal bovine hepatocytes, choline and methionine differentially regulate methyl carbon pathways and suggest that choline may play a critical role in

  19. Computational Analysis Reveals the Association of Threonine 118 Methionine Mutation in PMP22 Resulting in CMT-1A.

    PubMed

    Kumar, Chundi Vinay; Swetha, Rayapadi G; Anbarasu, Anand; Ramaiah, Sudha

    2014-01-01

    The T118M mutation in PMP22 gene is associated with Charcot Marie Tooth, type 1A (CMT1A). CMT1A is a form of Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. Mutations in CMT related disorder are seen to increase the stability of the protein resulting in the diseased state. We performed SNP analysis for all the nsSNPs of PMP22 protein and carried out molecular dynamics simulation for T118M mutation to compare the stability difference between the wild type protein structure and the mutant protein structure. The mutation T118M resulted in the overall increase in the stability of the mutant protein. The superimposed structure shows marked structural variation between the wild type and the mutant protein structures.

  20. Dietary L-methionine supplementation mitigates gamma-radiation induced global DNA hypomethylation: enhanced metabolic flux towards S-adenosyl-L-methionine (SAM) biosynthesis increases genomic methylation potential.

    PubMed

    Batra, Vipen; Verma, Poonam

    2014-07-01

    The objective of this study was to examine the effect of (60)Co-gamma (γ) radiation on modulation of genomic DNA methylation, if any, of mice maintained (6 weeks) on normal control diet (NCD) and L-methionine supplemented diet (MSD). To elucidate the possible underlying mechanism(s), we exposed the animals to γ-radiation (2, 3 and 4 Gy) and investigated the profile of downstream metabolites and enzymes involved in S-adenosyl-L-methionine (SAM) generation. Liver samples were also subjected to histopathological examinations. Compared to NCD fed and irradiated animals, hepatic folate, choline and L-methionine levels decreased moderately, while hepatic SAM levels increased in MSD fed and irradiated animals. Under these conditions, a marked modulation of methionine adenosyltransferase (MAT) and L-methionine synthase (MSase) activities was observed. Concomitant with increase in liver SAM pool, increased DNA methyltransferase (dnmt) activity in MSD fed mice indicated enhanced metabolic flux towards DNA methylation. Further results showed that genomic DNA methylation and 5-methyl-2'-deoxy cytidine residues were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals. In conclusion, our results suggest that increasing supply of preformed methyl groups, via dietary L-methionine supplementation might significantly increase methylation potential of radiation stress compromised DNA methylation cycle. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  2. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  3. The effect of increasing concentrations of dl-methionine and 2-hydroxy-4-(methylthio) butanoic acid on hepatic genes controlling methionine regeneration and gluconeogenesis.

    PubMed

    Zhang, Qian; Bertics, Sandra J; Luchini, N Daniel; White, Heather M

    2016-10-01

    Metabolizable methionine (Met) concentrations can be increased by feeding rumen-protected dl-Met or the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (HMBi). Hepatic responses to increasing concentrations of metabolizable Met as a result of supplementation of different Met sources have not been comparatively examined. The objective of this experiment was to examine the regulation of key genes for Met metabolism, gluconeogenesis, and fatty acid oxidation in response to increasing concentrations of dl-Met or 2-hydroxy-4-(methylthio) butanoic acid (HMB) in bovine primary hepatocytes. Hepatocytes isolated from 4 Holstein calves less than 7d old were maintained as monolayer cultures for 24h before addition of treatments. Cells were then exposed to treatments of dl-Met or HMB (0, 10, 20, 40, or 60 µM) in Met-free medium for 24h and collected for RNA isolation and quantification of gene expression by quantitative PCR. Expression of betaine-homocysteine methyltransferase (BHMT), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and 5,10 methylenetetrahydrofolate reductase (MTHFR) genes, which catalyze regeneration of Met from betaine and homocysteine, decreased linearly with increasing dl-Met concentration. We observed similar effects with increasing HMB concentration, except expression of MTHFR, which was not altered. Expression of Met adenosyltransferase 1A (MAT1A), which catalyzes the first step of Met metabolism to generate S-adenosylmethionine (SAM), a primary methyl donor, was decreased with increasing dl-Met or HMB concentration. Expression of S-adenosylhomocysteine hydrolase (SAHH) was decreased linearly with increasing HMB concentration, but not altered by dl-Met. Increasing concentrations of dl-Met and HMB decreased cytosolic phosphoenolpyruvate carboxykinase (PCK1) expression, but did not alter the expression of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) or pyruvate carboxylase (PC). Expression of glucose-6-phosphatase(G6PC

  4. L-Methionine Production.

    PubMed

    Shim, Jihyun; Shin, Yonguk; Lee, Imsang; Kim, So Young

    2016-11-22

    L-Methionine has been used in various industrial applications such as the production of feed and food additives and has been used as a raw material for medical supplies and drugs. It functions not only as an essential amino acid but also as a physiological effector, for example, by inhibiting fat accumulation and enhancing immune response. Producing methionine from fermentation is beneficial in that microorganisms can produce L-methionine selectively using eco-sustainable processes. Nevertheless, the fermentative method has not been used on an industrial scale because it is not competitive economically compared with chemical synthesis methods. Presented are efforts to develop suitable strains, engineered enzymes, and alternative process of producing L-methionine that overcomes problems of conventional fermentation methods. One of the alternative processes is a two-step process in which the L-methionine precursor is produced by fermentation and then converted to L-methionine by enzymes. Directed efforts toward strain development and enhanced enzyme engineering will advance industrial production of L-methionine based on fermentation.

  5. Restricted Role for Methionine Synthase Reductase Defined by Subcellular Localization

    PubMed Central

    Froese, DS; Wu, X; Zhang, J; Dumas, R; Schoel, WM; Amrein, M; Gravel, RA

    2009-01-01

    Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target to the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target to mitochondria was also suggested by the work of Leal et al. (2004) who showed that it can act as the reducing enzyme in combination with MMAB (ATP:cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin. PMID:18221906

  6. Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.

    PubMed

    Chen, Hailong; Wang, Zhou; Wang, Zhilai; Dou, Jie; Zhou, Changlin

    2016-04-01

    S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.

  7. Photooxidation of Methionine

    ERIC Educational Resources Information Center

    Lewis, Catherine; Scouten, William H.

    1976-01-01

    Describes an experiment in which the photooxidation of methionine using free methylene blue as the sensitizer is applied to the isolated amino acid or to the methionyl residues of a complex polypeptide. (MLH)

  8. Photooxidation of Methionine

    ERIC Educational Resources Information Center

    Lewis, Catherine; Scouten, William H.

    1976-01-01

    Describes an experiment in which the photooxidation of methionine using free methylene blue as the sensitizer is applied to the isolated amino acid or to the methionyl residues of a complex polypeptide. (MLH)

  9. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  10. Prebiotic synthesis of methionine.

    PubMed

    Van Trump, J E; Miller, S L

    1972-11-24

    Methionine has been shown to be a product of the action of a spark discharge on a simulated primitive earth atmosphere containing CH(4), N(2), NH(3), H(2)O, and H(2)S or CH(3)SH. Acrolein has also been shown to be a product of the discharge and is proposed as an intermediate in the prebiotic synthesis of methionine and of glutamic acid, homocysteine, homoserine, and alpha,gamma-diaminobutyric acid.

  11. PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen

    PubMed Central

    Saichana, Natsaran; Tanizawa, Katsuyuki; Pechoušek, Jiří; Novák, Petr; Yakushi, Toshiharu; Toyama, Hirohide; Frébortová, Jitka

    2016-01-01

    Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S]2+ cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S]2+ and [2Fe-2S]2+ clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions. PMID:26188050

  12. N-Terminal Methionine Processing.

    PubMed

    Wingfield, Paul T

    2017-04-03

    Protein synthesis is initiated by methionine in eukaryotes and by formylmethionine in prokaryotes. N-terminal methionine can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). When recombinant proteins are expressed in bacterial and mammalian expression systems, there is a simple universal rule that predicts whether the initiating methionine will be processed by MAP based on the size of the residue adjacent (penultimate) to the N-methionine. In general, if the side chains of the penultimate residues have a radius of gyration of 1.29 Å or less, methionine is cleaved. © 2017 by John Wiley & Sons, Inc.

  13. Salt stress enhances xylem development and expression of S-adenosyl-L-methionine synthase in lignifying tissues of tomato plants.

    PubMed

    Sánchez-Aguayo, Inmaculada; Rodríguez-Galán, José Manuel; García, Remedios; Torreblanca, José; Pardo, José Manuel

    2004-12-01

    S-Adenosyl-L-methionine synthase (SAM; ATP: L-methionine adenosyltransferase, EC 2.5.1.6) catalyzes the biosynthesis of S-adenosyl-L-methionine (AdoMet), a universal methyl-group donor. This enzyme is induced by salinity stress in tomato (Lycopersicon esculentum Mill.). To elucidate the role of SAM and AdoMet in the adaptation of plants to a saline environment, the expression pattern and histological distribution of SAM was investigated in control and salt-stressed tomato plants. Immunohistochemical analysis showed that SAM proteins were expressed in all cell types and plant organs, albeit with preferential accumulation in lignified tissues. Lignin deposition was estimated by histochemical tests and the extent of tissue lignification in response to salinity was quantified by image analysis. The average number of lignified cells in vascular bundles was significantly greater in plants under salt stress, with a maximal expansion of the lignified area found in the root vasculature. Accordingly, the greatest abundance of SAM gene transcripts and proteins occurred in roots. These results indicate that increased SAM activity correlated with a greater deposition of lignin in the vascular tissues of plants under salinity stress. A model is proposed in which an increased number of lignified tracheary elements in tomato roots under salt stress may enhance the cell-to-cell pathway for water transport, which would impart greater selectivity and reduced ion uptake, and compensate for diminished bulk flow of water and solutes along the apoplastic pathway.

  14. Methionine requirements in healthy adolescents

    USDA-ARS?s Scientific Manuscript database

    To investigate methionine requirements in healthy adolescents, we conducted an experiment in 28 children age 15.6+2.1 years, wt. 57.8+9.7 kg, using the intravenous indicator amino acid oxidation and balance (IV-IAAOB) technique. Children were randomly assigned to 7 different levels of methionine int...

  15. Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves.

    PubMed

    Jacometo, C B; Zhou, Z; Luchini, D; Corrêa, M N; Loor, J J

    2017-02-01

    An important mechanism of nutritional "programming" induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from -21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 μM) at -10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-β-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ∼50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless

  16. Effects of chronic dietary selenomethionine exposure on repeat swimming performance, aerobic metabolism and methionine catabolism in adult zebrafish (Danio rerio).

    PubMed

    Thomas, Jith K; Wiseman, Steve; Giesy, John P; Janz, David M

    2013-04-15

    swimming resulted in lesser concentrations of glycogen in the body, exposure to SeMet in the diet had no significant effect on glycogen content. Exposure to SeMet significantly down-regulated mRNA abundance of protein tyrosine phosphatase 1B (PTP 1B) in muscle, and β-hydroxyacyl coenzyme A dehydrogenase (HOAD), sterol regulatory element binding protein 1 (SREBP 1) and methionine adenosyltransferase 1 alpha (MAT 1A) in liver of adult zebrafish. Overall the results of this study suggest chronic exposure of adult zebrafish to SeMet in the diet can cause both cellular and organismal effects that could affect fitness and survivability of fish.

  17. Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

    PubMed Central

    Delgado, Miguel; Garrido, Francisco; Pérez-Miguelsanz, Juliana; Pacheco, María; Partearroyo, Teresa; Pérez-Sala, Dolores

    2014-01-01

    Abstract Aims: The discovery of methionine metabolism enzymes in the cell nucleus, together with their association with key nuclear processes, suggested a putative relationship between alterations in their subcellular distribution and disease. Results: Using the rat model of d-galactosamine intoxication, severe changes in hepatic steady-state mRNA levels were found; the largest decreases corresponded to enzymes exhibiting the highest expression in normal tissue. Cytoplasmic protein levels, activities, and metabolite concentrations suffered more moderate changes following a similar trend. Interestingly, galactosamine treatment induced hepatic nuclear accumulation of methionine adenosyltransferase (MAT) α1 and S-adenosylhomocysteine hydrolase tetramers, their active assemblies. In fact, galactosamine-treated livers showed enhanced nuclear MAT activity. Acetaminophen (APAP) intoxication mimicked most galactosamine effects on hepatic MATα1, including accumulation of nuclear tetramers. H35 cells that overexpress tagged-MATα1 reproduced the subcellular distribution observed in liver, and the changes induced by galactosamine and APAP that were also observed upon glutathione depletion by buthionine sulfoximine. The H35 nuclear accumulation of tagged-MATα1 induced by these agents correlated with decreased glutathione reduced form/glutathione oxidized form ratios and was prevented by N-acetylcysteine (NAC) and glutathione ethyl ester. However, the changes in epigenetic modifications associated with tagged-MATα1 nuclear accumulation were only prevented by NAC in galactosamine-treated cells. Innovation: Cytoplasmic and nuclear changes in proteins that regulate the methylation index follow opposite trends in acute liver injury, their nuclear accumulation showing potential as disease marker. Conclusion: Altogether these results demonstrate galactosamine- and APAP-induced nuclear accumulation of methionine metabolism enzymes as active oligomers and unveil the implication of

  18. The eutT gene of Salmonella enterica Encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme.

    PubMed

    Buan, Nicole R; Suh, Sang-Jin; Escalante-Semerena, Jorge C

    2004-09-01

    The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.

  19. 63 FR 41290 - Synthetic Methionine From Japan

    Federal Register 2010, 2011, 2012, 2013, 2014

    1998-08-03

    ... COMMISSION Synthetic Methionine From Japan AGENCY: United States International Trade Commission. ACTION: Institution of a five-year review concerning the antidumping duty order on synthetic methionine from Japan... antidumping duty order on synthetic methionine from Japan would be likely to lead to continuation...

  20. Methionine requirements for the preimplantation bovine embryo.

    PubMed

    BONILLA, Luciano; LUCHINI, Daniel; DEVILLARD, Estelle; HANSEN, Peter J

    2010-10-01

    The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 µmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 µmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 µmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 µmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 µmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of

  1. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... source of L-methionine to improve significantly the biological quality of the total protein in a food containing naturally occurring primarily intact vegetable protein that is considered a significant dietary protein source, provided that: (1) A reasonable daily adult intake of the finished food furnishes at...

  2. Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level.

    PubMed

    Li, Ying; Cong, Hua; Liu, Bingnan; Song, Jinzhu; Sun, Xueying; Zhang, Junzheng; Yang, Qian

    2016-09-01

    Relieving the feedback inhibition of key enzymes in a metabolic pathway is frequently the first step of producer-strain construction by genetic engineering. However, the strict feedback regulation exercised by microorganisms in methionine biosynthesis often makes it difficult to produce methionine at a high level. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for methionine production. First, the metD gene encoding the methionine uptake system was deleted to achieve extracellular accumulation of methionine. Then, random mutagenesis was performed to remove feedback inhibition by metabolic end-products. The resulting strain C. glutamicum ENM-16 was further engineered to block or decrease competitive branch pathways by deleting the thrB gene and changing the start codon of the dapA gene, followed by point mutations of lysC (C932T) and pyc (G1A, C1372T) to increase methionine precursor supply. To enrich the NADPH pool, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway were mutated to reduce their sensitivity to inhibition by intracellular metabolites. The resultant strain C. glutamicum LY-5 produced 6.85 ± 0.23 g methionine l(-1) with substrate-specific yield (Y P/S) of 0.08 mol per mol of glucose after 72 h fed-batch fermentation. The strategies described here will be useful for construction of methionine engineering strains.

  3. Methionine sulfoximine intensifies cancer anorexia.

    PubMed

    Chance, W T; Zhang, F S; Fischer, J E

    1991-05-01

    Consistent anorexia was first observed 33 days after inoculating Fischer 344 rats with methylcholanthrene-induced sarcoma. Daily treatment of a similar group of rats with the glutamine synthetase inhibitor, methionine sulfoximine, elicited significant reductions of feeding by day 29 at a dose that had no effect on nontumor-bearing rats. Blood concentrations of ammonia were elevated in both groups of tumor-bearing rats and brain ammonia level was increased in the methionine sulfoximine-treated tumor-bearing rats. Forebrain concentrations of tyrosine, tryptophan, DOPAC and 5-HIAA were elevated in both groups of tumor-bearing rats. Since ammonia is detoxified through the glutamine synthetase reaction, these results suggest that blood and brain ammonia concentrations are more important than the neurochemical consequences of ammonia detoxification for the etiology of cancer anorexia.

  4. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  5. The physiological role of reversible methionine oxidation.

    PubMed

    Drazic, Adrian; Winter, Jeannette

    2014-08-01

    Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

  6. Influence of copper supplementation on the relationship between dietary methionine and free plasma methionine.

    PubMed

    Ekperigin, H E; Vohra, P

    1981-09-01

    One-day-old broiler chicks were adapted to a basal, isolated soyprotein-cornstarch diet containing 20% protein, 0.59% methionine and 3,300 kcal metabolizable energy (ME)/kg. They were then fed experimental diets consisting of three levels (0, 500, 1,000 ppm) of copper added to each of the basal diet plus four levels (0, 0.4, 0.8, or 1.5%) of L-methionine for 1 or 4 weeks. Growth was retarded after 1 week by all levels of supplementary methionine, and by 500 or 1,000 ppm excess copper. The plasma concentrations of free methionine, serine, alpha-aminobutyric acid and cystathionine were increased by excess dietary methionine. Excess copper prevented the increase in plasma methionine. After 4 weeks, the plasma methionine concentration and rate of growth of chicks fed 0.4% excess L-methionine did not differ significantly from basal values. The growth retardation caused by 500 ppm excess copper was alleviated by 0.4% supplemental methionine, and the elevations in plasma methionine and liver and spleen iron concentrations observed in chicks fed 1.5% excess methionine were reduced by 1,000 ppm excess copper. The patterns of the relationship between dietary methionine and liver or spleen iron, in the presence or absence of supplementary copper, were similar to those between dietary and free plasma methionine.

  7. Sex-specific dysregulation of cysteine oxidation and the methionine and folate cycles in female cystathionine gamma-lyase null mice: a serendipitous model of the methylfolate trap.

    PubMed

    Jiang, Hua; Hurt, K Joseph; Breen, Kelsey; Stabler, Sally P; Allen, Robert H; Orlicky, David J; Maclean, Kenneth N

    2015-08-14

    In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL) is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap.

  8. Sex-specific dysregulation of cysteine oxidation and the methionine and folate cycles in female cystathionine gamma-lyase null mice: a serendipitous model of the methylfolate trap

    PubMed Central

    Jiang, Hua; Hurt, K. Joseph; Breen, Kelsey; Stabler, Sally P.; Allen, Robert H.; Orlicky, David J.; Maclean, Kenneth N.

    2015-01-01

    ABSTRACT In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL) is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap. PMID:26276101

  9. Methionine Metabolism of the Myxomycete Physarum polycephalum

    PubMed Central

    Daniel, John W.; Babcock, Karlee

    1966-01-01

    Daniel, John W. (University of Wisconsin, Madison), and Karlee Babcock. Methionine metabolism of the myxomycete Physarum polycephalum. J. Bacteriol. 92:1028–1035. 1966.—Previous studies have shown that Physarum polycephalum requires exogenous methionine for growth, but not cysteine, folic acid, or vitamin B12. Methionine can also serve as the sole source of sulfur for all cellular requirements, without limiting the growth rate. S-methyl-l-cysteine, 2-hydroxy-4-methiol butyric acid, S-adenosyl-l-methionine, and methionine peptides were the only compounds supporting growth, when substituted for methionine. Other methionine analogues, methyl donors in combination with homocysteine, and intermediates of the cystathionine pathway were not active. Ethionine and S-ethyl cysteine were good methionine antagonists. This myxomycete is apparently unable to synthesize the methyl or S-methyl group, although it still appears able to transmethylate, at least from S-methyl cysteine, and probably from S-adenosyl methionine, which can also serve as a source of adenine. PMID:5951320

  10. Depletion of S-adenosyl-L-methionine with cycloleucine potentiates cytochrome P450 2E1 toxicity in primary rat hepatocytes

    PubMed Central

    Zhuge, Jian; Cederbaum, Arthur I.

    2007-01-01

    S-Adenosyl-L-methionine (SAM) is the principal biological methyl donor. Methionine adenosyltransferase (MAT) catalyzes the only reaction that generates SAM. Hepatocytes were treated with cycloleucine, an inhibitor of MAT, to evaluate whether hepatocytes enriched in cytochrome P450 2E1(CYP2E1) were more sensitive to a decline in SAM. Cycloleucine decreased SAM and glutathione (GSH) levels and induced cytotoxicity in hepatocytes from pyrazole-treated rats (with an increased content of CYP2E1) to a greater extent as compared to hepatocytes from saline-treated rats. Apoptosis caused by cycloleucine in pyrazole hepatocytes appeared earlier and was more pronounced than control hepatocytes and could be prevented by incubation with SAM, glutathione reduced ethyl ester and antioxidants. The cytotoxicity was prevented by treating rats with chlormethiazole, a specific inhibitor of CYP2E1. Cycloleucine induced greater production of reactive oxygen species (ROS) in pyrazole hepatocytes than in control hepatocytes, and treatment with SAM, Trolox and chlormethiazole lowered ROS formation. In conclusion, lowering of hepatic SAM levels produced greater toxicity and apoptosis in hepatocytes enriched in CYP2E1. This is due to elevated ROS production by CYP2E1 coupled to lower levels of hepatoprotective SAM and GSH. We speculate that such interactions e.g. induction of CYP2E1, decline in SAM and GSH may contribute to alcohol liver toxicity. PMID:17640612

  11. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

    PubMed

    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  12. Crystallization and preliminary X-ray crystallographic studies of a PduO-type ATP:cob(I)alamin adenosyltransferase from Bacillus cereus

    SciTech Connect

    Park, Ae Kyung; Moon, Jin Ho; Lee, Sung Haeng; Chi, Young Min

    2008-07-01

    Orthorhombic crystals of a PduO-type ATP:cob(I)alamin adenosyltransferase from B. cereus were obtained both as an apoenzyme and in the presence of Mg{sup 2+} and ATP. Cobalamin adenosyltransferases transfer a 5′-deoxyadenosyl moiety from ATP and covalently attach it to the cobalt(I) ion of the corrin ring of cobalamin to generate adenosylcobalamin. The PduO-type adenosyltransferase from Bacillus cereus was overexpressed in Escherichia coli, purified and crystallized as the apoenzyme as well as in complex with Mg{sup 2+} and ATP (MgATP). Diffraction data were collected to 1.9 Å resolution for the native crystals and 2.0 Å resolution for the complexed crystals. Both crystals belonged to the orthorhombic space group C222{sub 1}; the native crystals have unit-cell parameters a = 64.93, b = 137.08, c = 158.55 Å. The asymmetric unit contained one trimer, with a corresponding V{sub M} of 2.69 Å{sup 3} Da{sup −1}.

  13. Efficiency of methionine retention in ducks.

    PubMed

    Adeola, Olayiwola

    2007-03-01

    The accretion of methionine and protein as a function of methionine intake was assessed in growing ducks between 22 and 42 d post-hatching. Four graded doses of dl-methionine at 0, 0 x 5, 1 x 0 or 1 x 5 g/kg diet were added to a methionine-limiting basal diet and fed to four replicate groups of four ducks each. The growth and efficiency of food use for growth increased linearly (P<0 x 05) as a function of methionine intake. The accretion of body protein increased (P<0 x 001) from 87 x 5 to 182 x 2 g, and that of methionine from 1616 to 3125 mg, over the 21 d period as dietary methionine increased. The accretion rate of methionine in the body (y, mg/d) as a function of methionine intake (x, mg/d) of ducks fed diets containing supplemental methionine at 0, 0 x 5, 1 x 0 or 1 x 5 g/kg diet from day 22 to day 42 post-hatching gave the regression equation: y=-148 x 86 (se 32 x 558)+0 x 312 (se 0 x 0384)X, r2=0 x 8253. For protein accretion rate in the body (y, mg/d) as a function of methionine intake (x, mg/d), the regression equation was: y= -9782 (se 2204)+19 x 505 (se 2 x 5994)x, r2=0 x 8009. There was a linear relationship between methionine (y, mg/d) and protein (x, mg/d) accretion in ducks that was described by the equation y=12 x 757 (se 7 x 4019)+0 x 01 525 (se 0 x 00 107)x, r2=0 x 9355. The results of these studies suggest a constant utilisation of methionine over the range 2 x 4-3 x 9 g digestible methionine/kg diet, with an efficiency of 31 %. Furthermore, the results suggest a quantitative relationship of 15 mg methionine for every gram of protein accretion.

  14. Increasing levels of dietary crystalline methionine affect plasma methionine profiles, ammonia excretion, and the expression of genes related to the hepatic intermediary metabolism in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Rolland, Marine; Skov, Peter V; Larsen, Bodil K; Holm, Jørgen; Gómez-Requeni, Pedro; Dalsgaard, Johanne

    2016-08-01

    Strictly carnivorous fish with high requirements for dietary protein, such as rainbow trout (Oncorhynchus mykiss) are interesting models for studying the role of amino acids as key regulators of intermediary metabolism. Methionine is an essential amino acid for rainbow trout, and works as a signalling factor in different metabolic pathways. The study investigated the effect of increasing dietary methionine intake on the intermediary metabolism in the liver of juvenile rainbow trout. For this purpose, five diets were formulated with increasing methionine levels from 0.60 to 1.29% dry matter. The diets were fed in excess for six weeks before three sampling campaigns carried out successively to elucidate (i) the hepatic expression of selected genes involved in lipid, glucose and amino acid metabolism; (ii) the postprandial ammonia excretion; and (iii) the postprandial plasma methionine concentrations. The transcript levels of enzymes involved in lipid metabolism (fatty acid synthase, glucose 6 phosphate dehydrogenase and carnitine palmitoyl transferase 1 a), gluconeogenesis (fructose-1,6-biphosphatase) and amino acid catabolism (alanine amino transferase and glutamate dehydrogenase) were significantly affected by the increase in dietary methionine. Changes in gene expression reflected to some extent the decrease in ammonia excretion (P=0.022) and in the hepatosomatic index (HSI; P<0.001) when dietary methionine increased. Postprandial plasma methionine concentrations correlated positively with the dietary level (P<0.001) at the different sampling points. The study shows that the expression of several genes related to the hepatic intermediary metabolism in rainbow trout responded in a dose-dependent manner to increasing levels of dietary methionine.

  15. Methionine metabolism in Yucatan miniature swine.

    PubMed

    McBreairty, Laura E

    2016-06-01

    Methionine is an essential amino acid which when not incorporated into protein, can be converted to S-adenosylmethionine, the universal methyl donor in over 200 transmethylation reactions, which include creatine and phosphatidylcholine (PC) synthesis, as well as deoxyribonucleic acid (DNA) methylation. Following transmethylation, homocysteine is formed, which can be converted to cysteine via transsulfuration or remethylated to methionine by receiving a methyl group from folate or betaine. Changes to methyl group availability in utero can lead to permanent changes in epigenetic patterns of DNA methylation, which has been implicated in "fetal programming", a phenomenon associated with poor nutrition during fetal development that results in low birth weight and disease in later life. It has been shown that programming can also occur in the neonate. Our global objective was to understand how the variability of nutrients involved in methionine metabolism can affect methionine and methyl group availability. We hypothesize that nutrients that converge on methionine metabolism can affect methionine availability for its various functions. In this thesis, we used intrauterine growth restricted (IUGR) piglets to investigate whether a global nutritional insult in utero can lead to a perturbed methionine metabolism. Our results demonstrate that IUGR piglets have a lower capacity to dispose of homocysteine via both transsulfuration and remethylation pathways, as well as a lower incorporation of methyl groups into PC. The second objective of this thesis was to determine whether variation in methionine supply and demand can affect methionine availability. We demonstrated that stimulating either acute or chronic creatine synthesis leads to lower methyl incorporation into protein and PC in pigs. Furthermore, when methionine is limiting, supplementation with either folate or betaine leads to higher methionine availability for protein synthesis. Finally, because creatine is

  16. Interaction of genetic mechanisms regulating methionine concentration in maize grain

    USDA-ARS?s Scientific Manuscript database

    Methionine is a limiting amino acid in poultry diets so methionine supplementation is typically required to meet nutritional demands. Maize varieties with increased methionine levels have been developed utilizing three different approaches: i.) increased levels of the methionine-rich 10 kDa zein, ii...

  17. Metabolism of 5-methylthioribose to methionine

    SciTech Connect

    Miyazaki, J.H.; Yang, S.F.

    1987-06-01

    During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine is released as 5'-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, (/sup 14/C)MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, L-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O/sub 2/ in the conversion of MTR to methionine is discussed.

  18. l-Methionine Placental Uptake

    PubMed Central

    Araújo, João R.; Correia-Branco, Ana; Ramalho, Carla; Gonçalves, Pedro; Pinho, Maria J.; Keating, Elisa

    2013-01-01

    Our aim was to investigate the influence of gestational diabetes mellitus (GDM) and GDM-associated conditions upon the placental uptake of 14C-l-methionine (14C-l-Met). The 14C-l-Met uptake by human trophoblasts (TBs) obtained from normal pregnancies (normal trophoblast [NTB] cells) is mainly system l-type amino acid transporter 1 (LAT1 [L])-mediated, although a small contribution of system y+LAT2 is also present. Comparison of 14C-l-Met uptake by NTB and by human TBs obtained from GDM pregnancies (diabetic trophoblast [DTB] cells) reveals similar kinetics, but a contribution of systems A, LAT2, and b0+ and a greater contribution of system y+LAT1 appears to exist in DTB cells. Short-term exposure to insulin and long-term exposure to high glucose, tumor necrosis factor-α, and leptin decrease 14C-l-Met uptake in a human TB (Bewo) cell line. The effect of leptin was dependent upon phosphoinositide 3-kinase, extracellular-signal-regulated kinase 1/2 (ERK/MEK 1/2), and p38 mitogen-activated protein kinase. In conclusion, GDM does not quantitatively alter 14C-l-Met placental uptake, although it changes the nature of transporters involved in that process. PMID:23653387

  19. Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase

    PubMed Central

    Elmore, C. Lee; Wu, Xuchu; Leclerc, Daniel; Watson, Erica D.; Bottiglieri, Teodoro; Krupenko, Natalia I.; Krupenko, Sergey A.; Cross, James C.; Rozen, Rima; Gravel, Roy A.; Matthews, Rowena G.

    2007-01-01

    Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrrgt/gt mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrrgt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocysteinemia may be useful in understanding biological effects of elevated homocyst(e)ine. PMID:17369066

  20. The catabolic enzyme methionine gamma-lyase limits methionine accumulation in potato tubers.

    PubMed

    Huang, Tengfang; Joshi, Vijay; Jander, Georg

    2014-09-01

    Increasing methionine in potato tubers is desirable, both to increase the availability of this limiting essential amino acid and to enhance the aroma of baked and fried potatoes. Previous attempts to elevate potato methionine content using transgenic approaches have focused on increasing methionine biosynthesis. Higher isoleucine accumulation in these transgenic tubers suggested that the potatoes compensate for increased methionine biosynthesis with enhanced catabolism via methionine gamma-lyase (MGL), thereby producing 2-ketybutyrate for isoleucine biosynthesis. In the current study, we show that potato StMGL1 encodes a functional MGL in potato tubers. In planta silencing of StMGL1 results in an increased methionine to isoleucine ratio in the free amino acid profile of potato tubers and, in some transgenic lines, elevated accumulation of free methionine. In both wild-type and transgenic tubers, the ratio of methionine to isoleucine is negatively correlated with the level of StMGL1 transcript. A three-dimensional distribution of free amino acids in potato tubers is also described. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Sulfur amino acids in methionine-restricted rats: hyperhomocysteinemia.

    PubMed

    Elshorbagy, Amany K; Valdivia-Garcia, Maria; Refsum, Helga; Smith, A David; Mattocks, Dwight A L; Perrone, Carmen E

    2010-01-01

    Dietary methionine restriction in Fischer-344 rats favorably influences visceral fat mass, insulin sensitivity, metabolic parameters, and longevity. However, little is known about the effects of methionine restriction on serum methionine and its downstream sulfur amino acids. We investigated the serum sulfur amino acid profile of male Fischer-344 rats fed a methionine-restricted diet for 3 mo. Using tandem mass spectrometry, we observed marked reduction in serum concentrations of methionine, cystathionine, cysteine, and taurine in methionine-restricted rats compared with control (P<0.001) and a 2.5-fold elevation of homocysteine (P<0.001). This suggests that homocysteine trans-sulfuration may be inhibited by methionine restriction, and that some of the effects of methionine restriction may be mediated by changes in sulfur amino acids downstream of methionine. Published by Elsevier Inc.

  2. Modulation of Potassium Channel Function by Methionine Oxidation and Reduction

    NASA Astrophysics Data System (ADS)

    Ciorba, Matthew A.; Heinemann, Stefan H.; Weissbach, Herbert; Brot, Nathan; Hoshi, Toshinori

    1997-09-01

    Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.

  3. Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins

    PubMed Central

    2012-01-01

    Background Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative. Results We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity. Conclusion Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation. PMID:23088625

  4. Dissimilation of Methionine by Achromobacter starkeyi1

    PubMed Central

    Ruiz-Herrera, José; Starkey, Robert L.

    1970-01-01

    Methionine was decomposed by some bacteria which were isolated from soil. The sulfur of the methionine was liberated as methanethiol, and part of this became oxidized to dimethyl disulfide. Detailed studies with one of these cultures, Achromobacter starkeyi, indicated that the first step in methionine decomposition was its oxidadative deamination to α-keto-γ-methyl mercaptobutyrate by a constitutive amino acid oxidase. The following steps were carried out by inducible enzymes, the synthesis of which was inhibited by chloramphenicol. α-Keto-γ-methyl mercaptobutyrate was split producing methanethiol and α-keto butyrate which was oxidized to propionate. The metabolism of propionate was similar to that described for animal tissues; the propionate was carboxylated to succinate via methyl malonyl coenzyme A, and the succinate was metabolized through the Krebs cycle. PMID:16559105

  5. A review of methionine dependency and the role of methionine restriction in cancer growth control and life-span extension.

    PubMed

    Cavuoto, Paul; Fenech, Michael F

    2012-10-01

    Methionine is an essential amino acid with many key roles in mammalian metabolism such as protein synthesis, methylation of DNA and polyamine synthesis. Restriction of methionine may be an important strategy in cancer growth control particularly in cancers that exhibit dependence on methionine for survival and proliferation. Methionine dependence in cancer may be due to one or a combination of deletions, polymorphisms or alterations in expression of genes in the methionine de novo and salvage pathways. Cancer cells with these defects are unable to regenerate methionine via these pathways. Defects in the metabolism of folate may also contribute to the methionine dependence phenotype in cancer. Selective killing of methionine dependent cancer cells in co-culture with normal cells has been demonstrated using culture media deficient in methionine. Several animal studies utilizing a methionine restricted diet have reported inhibition of cancer growth and extension of a healthy life-span. In humans, vegan diets, which can be low in methionine, may prove to be a useful nutritional strategy in cancer growth control. The development of methioninase which depletes circulating levels of methionine may be another useful strategy in limiting cancer growth. The application of nutritional methionine restriction and methioninase in combination with chemotherapeutic regimens is the current focus of clinical studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and DL...

  7. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  8. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  9. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  10. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  11. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  12. Methionine acts as a "magnet" in photoaffinity crosslinking experiments.

    PubMed

    Wittelsberger, Angela; Thomas, Beena E; Mierke, Dale F; Rosenblatt, Michael

    2006-03-20

    Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.

  13. Local flexibility facilitates oxidization of buried methionine residues.

    PubMed

    Xu, Kuiran; Uversky, Vladimir N; Xue, Bin

    2012-06-01

    In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.

  14. Physiological and biochemical aspects of methionine isomers and a methionine analogue in broilers.

    PubMed

    Zhang, Shuai; Saremi, Behnam; Gilbert, Elizabeth R; Wong, Eric A

    2017-02-01

    Methionine is the first limiting amino acid in all poultry corn-soybean based diets. The objective of this study was to determine the effect of supplementation of L-methionine (L-Met), DL-methionine (DL-Met), and the methionine analogue, DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA), on biochemical and physiological parameters of broiler chickens. Male Cobb-500 broilers were fed from day of hatch (d 0) to d 35 posthatch using a basal diet deficient in methionine plus cysteine (Met + Cys) (control), or the basal diet supplemented with 0.22% DL-Met, 0.22% L-Met, or 0.31% DL-HMTBA to meet the Met + Cys requirements. Tissue (liver, duodenum, jejunum, and ileum) and blood samples were collected at various ages, from d 0 to d 35. Performance of the birds, blood parameters (e.g., acute phase proteins, white blood cell counts), mRNA expression of intestinal nutrient transporters and DNA methylation properties of liver tissues were examined. Both body weight and feed efficiency were improved in methionine supplemented groups compared to the control group. No significant differences were observed among DL-Met, L-Met, and DL-HMTBA for growth performance parameters. L-Met and DL-Met supplementation decreased the acute phase protein, serum amyloid A, while DL-HMTBA had no effect. Methionine supplementation had no effect on white blood cell differentiation count, hepatic total DNA methylation, or DNA methyltransferase activity. L-Met and DL-Met, but not DL-HMTBA, supplementation, resulted in enhanced expression of the ATB(0,+) and B(0)AT transporters in various small intestinal segments. All methionine sources increased expression of MCT1 in the jejunum. In conclusion, methionine supplementation improved growth performance of male broilers. Methionine supplementation was also associated with changes in intestinal nutrient transporter gene expression in certain segments and ages, suggesting that intestinal amino acid absorptive function can be regulated by the source

  15. Factors influencing methionine toxicity in young bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1981-01-01

    Young Bobwhite quail (Colinus virginianus) were fed low and adequate protein purified diets with and without excess methionine to evaluate factors affecting methionine toxicity. Growth of quail fed an adequate protein (27%) diet, without supplemental glycine, was depressed by 1.75% and 2.25% excess methionine. Supplemental glycine (.3%) alleviated growth depression caused by 2.25% excess methionine. Quail fed 1.75% and 2.25% excess methionine developed signs of toxicity characterized by weakness, a lowered, outstretched neck when moving, and ataxia. In addition, quail would fall on their sides when disturbed and spin with their heads retracted. These conditions were transient in nature. Growth of quail fed a low protein (18.9%) diet was depressed by 1% and 1.5% excess methionine and DL-homocystine. Quail fed 1% and 1.5% excess methionine in this diet also developed signs of toxicity, the incidence of which was greater and the duration longer than occurred with quail fed adequate protein. Supplementing a low protein (20.15%) diet with .3% or .6% glycine or threonine or a combination of these amino acids did not alleviate growth depression caused by 1.5% excess methionine; however, 2% and 3% supplemental glycine were somewhat effective. Supplements of glycine (2%, 3%) and threonine (1%) completely reversed growth depression from 1% excess methionine but did not influence growth of controls, indicating that both amino acids counteract methionine toxicity. Both glycine and threonine alone improved growth by about the same extent in diets with 1% or 1.5% excess methionine; however, these amino acids alleviated less than 30% of the growth depression resulting from 1.5% excess methionine. The effectiveness of glycine in alleviating methionine toxicity in a low protein diet was decreased, and hemoglobin levels were depressed with 1.5% excess methionine compared to less amounts.

  16. Methionine peptide formation under primordial earth conditions.

    PubMed

    Li, Feng; Fitz, Daniel; Fraser, Donald G; Rode, Bernd M

    2008-01-01

    According to recent research on the origin of life it seems more and more likely that amino acids and peptides were among the first biomolecules formed on earth and that a peptide/protein world was thus a key starting point in evolution towards life. Salt-induced Peptide Formation (SIPF) has repeatedly been shown to be the most universal and plausible peptide-forming reaction currently known under prebiotic conditions and forms peptides from amino acids with the help of copper ions and sodium chloride. In this paper we present experimental results for salt-induced peptide formation from methionine. This is the first time that a sulphur-containing amino acid was investigated in this reaction. The possible catalytic effects of glycine and L-histidine in this reaction were also investigated and a possible distinction between the L- and D-forms of methionine was studied as well.

  17. Characterization of isolated yeast growth response to methionine analogs.

    PubMed

    Saengkerdsub, Suwat; Lingbeck, Jody M; Wilkinson, Heather H; O'Bryan, Corliss A; Crandall, Philip G; Muthaiyan, Arunachalam; Biswas, Debabrata; Ricke, Steven C

    2013-01-01

    Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.

  18. Methionine restriction beyond life-span extension.

    PubMed

    Ables, Gene P; Hens, Julie R; Nichenametla, Sailendra N

    2016-01-01

    Dietary methionine restriction (MR) extends life span across species via various intracellular regulatory mechanisms. In rodents, MR induces resistance against adiposity, improves hepatic glucose metabolism, preserves cardiac function, and reduces body size, all of which can affect the onset of age-related diseases. Recent studies have shown that MR-affected biomarkers, such as fibroblast growth factor 21, adiponectin, leptin, cystathionine β synthase, and insulin-like growth factor 1, can potentially alter physiology. The beneficial effects of MR could be explained in part by its ability to reduce mitochondrial oxidative stress. Studies have revealed that MR can reduce reactive oxygen species that damage cells and promote cancer progression. It has been demonstrated that either MR or the targeting of specific genes in the methionine cycle could induce cell apoptosis while decreasing proliferation in several cancer models. The complete mechanism underlying the actions of MR on the cell cycle during cancer has not been fully elucidated. Epigenetic mechanisms, such as methylation and noncoding RNAs, are also possible downstream effectors of MR; future studies should help to elucidate some of these mechanisms. Despite evidence that changes in dietary methionine can affect epigenetics, it remains unknown whether epigenetics is a mechanism in MR. This review summarizes research on MR and its involvement in metabolism, cancer, and epigenetics.

  19. Decomposition of methionine by low energy electrons.

    PubMed

    Kopyra, Janina; Szamrej, Iwona; Abdoul-Carime, Hassan; Farizon, Bernadette; Farizon, Michel

    2012-06-14

    In this work, we present the results from low energy (<12 eV) electron impact on isolated methionine, Met. We show that dissociative electron attachment is the operative mechanism for the sulfur content amino-acid fragmentation. The two most dominant fragments are attributed to the (Met-H)(-) and (C(4)NOH(5))(-) ions that are formed at energy below 2 eV. The formation of the latter anion is accompanied by the loss of neutral counterparts, which are most likely a water molecule and highly toxic methanethiol, CH(3)SH. Further fragments are associated with the damage at the sulfur end of the amino acid, producing the methyl sulfide anion CH(3)S(-) or sulfur containing neutrals. In the context of radiation induced damage to biological material at the nano-scale level, the present interest of methionine arises from the implication of the molecule in biological processes (e.g., S-adenosyl methionine for the stimulation of DNA methyltransferase reactions or protein synthesis).

  20. Comparison of the effects of seleno-l-methionine, seleno-dl-methionine, and selenized yeast on reproduction of mallards

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.

    1996-01-01

    The toxicities of seleno-L-methionine, seleno-DL-methionine, and selenized yeast were compared. Ten pairs of mallards were fed a control diet and 15 pairs were fed diets containing 10 ppm selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. Hatching of fertile eggs was significantly lower for females fed 10 ppm selenium as seleno-DL-methionine (7.6%) and seleno-L-methionine (6.4%) than for controls (41.3%). Survival of ducklings was lower when their parents had been fed 10 ppm selenium as seleno-L-methionine (20.0%) than for controls (98.4%). The number of 6-day-old ducklings produced per female was significantly lower for mallards fed 10 ppm selenium as seleno-DL-methionine (0.47) or selenized yeast (2.67) than for controls (6.10), and was significantly lower for mallards fed seleno-L-methionine (0.13) than for mallards fed selenized yeast. The eighth eggs of females fed the DL or L forms of selenomethionine contained means of 9.2 and 8.9 ppm selenium, wet weight; these means were higher than the mean (6.6 ppm) for females fed selenized yeast. Among embryos that died at 7 days of age or older, the percentage of embryos that were deformed was 1.3% for controls, 24.6% for seleno-DL-methionine, 28.2% for seleno-L-methionine, and 11.0% for selenized yeast. The results suggested that seleno-DL-methionine and seleno-L-methionine were of similar toxicity and were both more toxic than selenium from selenized yeast.

  1. Stimulation of Ethylene Production in Apple Tissue Slices by Methionine

    PubMed Central

    Lieberman, Morris; Kunishi, Alice; Mapson, L. W.; Wardale, D. A.

    1966-01-01

    Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase. PMID:16656267

  2. Comparative dynamics of methionine side-chain in FMOC-methionine and in amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Vugmeyster, Liliya; Ostrovsky, Dmitry

    2017-04-01

    We compared the dynamics of key methionine methyl groups in the water-accessible hydrophobic cavity of amyloid fibrils and Fluorenylmethyloxycarbonyl-Methionine (FMOC-Met), which renders general hydrophobicity to the environment without the complexity of the protein. Met35 in the hydrated cavity was recently found to undergo a dynamical cross-over from the dominance of methyl rotations at low temperatures to the dominance of diffusive motion of methyl axis at high temperatures. Current results indicate that in FMOC-Met this cross-over is suppressed, similar to what was observed for the dry fibrils, indicating that hydration of the cavity is driving the onset of the dynamical transition.

  3. Methionine-to-Cysteine Recycling in Klebsiella aerogenes

    PubMed Central

    Seiflein, Thomas A.; Lawrence, Jeffrey G.

    2001-01-01

    In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. PMID:11114934

  4. Interference by methionine on valine uptake in Acremonium chrysogenum.

    PubMed Central

    Alonso, M J; Luengo, J M

    1987-01-01

    The incorporation of L-[U-14C]valine into delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a direct biosynthetic precursor of penicillins and cephalosporins, was studied. When DL-methionine was added to Acremonium chrysogenum culture broths, no labeled ACV was found, while a large amount of radioactive ACV was detected when methionine was not present. DL-Norleucine, a nonsulfur analog of methionine, also inhibited the synthesis of radioactive ACV to some degree. This effect was due to the inhibition of valine transport by methionine and norleucine. PMID:3566258

  5. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

  6. Dietary methionine requirement of the Chinese egg-laying duck.

    PubMed

    He, J H; Li, J B; Gao, F X; Liu, Q H; Shu, J C; Liu, D J

    2003-12-01

    1. The dietary methionine requirement of egg-laying ducks was assessed by feeding diets supplemented with graded levels of DL-methionine (0, 4, 8, 12, 16 g/kg dietary protein) for 8 weeks. The basal diet contained 175 g protein and 2.6 g methionine per kg feed (or 14.9 g/kg protein) and an estimated ME of 11.5 MJ/kg. 2. A total of 800 Shaoxin laying ducks (420 d old) were randomly divided into 5 groups of 160 each and fed in 4 separate pens. 3. Dietary supplementation of methionine significantly increased egg production and feed conversion efficiency. 4. Dietary methionine requirement for optimum egg production was estimated to be 25.7 g/kg of dietary protein or 4.5 g/kg of the diet or 380 mg/bird-d. 5. Methionine supplementation increased the methionine level in plasma, and the free glutamic acid and aspartic acid concentrations in plasma were quadratically related to dietary methionine levels. Increasing dietary methionine had little effect on egg quality characteristics.

  7. Regulation of thrombosis and vascular function by protein methionine oxidation

    PubMed Central

    Gu, Sean X.; Stevens, Jeff W.

    2015-01-01

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  8. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  9. Methionine as a Precursor of Ethylene—Commentary

    USDA-ARS?s Scientific Manuscript database

    Lieberman et al. showed in a 1966 publication of Plant Physiology that methionine is a precursor of ethylene. It was the first paper that showed ethylene carbons are derived from carbons 3 and 4 of methionine. This paper catalyzed remarkable interest among plant biologists to elucidate the biosynth...

  10. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  11. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  12. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in any...

  13. High Methionine Diet Poses Cardiac Threat: A Molecular Insight.

    PubMed

    Chaturvedi, Pankaj; Kamat, Pradip K; Kalani, Anuradha; Familtseva, Anastasia; Tyagi, Suresh C

    2016-07-01

    High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress; (2) by inflammatory manifestations; and (3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice: (1) WT; (2) WT + methionine; (3) CBS(+/-) ; (4) CBS(+/-) +methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS(+/-) and CBS(+/-) +methionine. Higher oxidative stress was indicated by high levels of SOD-1 in methionine fed mouse hearts whereas IL-1β, IL-6, TNFα, and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion, our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling, and decreased cardiac function.

  14. The distribution of methionine-enkephalin and leucine-enkephalin in the brain and peripheral tissues

    PubMed Central

    Hughes, J.; Kosterlitz, H.W.; Smith, T.W.

    1977-01-01

    1 A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD-2 resin. Elution with methanol separates the enkephalins and α-endorphin from β-endorphin. 2 Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine- and leucine-enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins. 3 An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed ε-endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts. 4 A differential assay of methionine- and leucine-enkephalin was made either by destroying methionine-enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography. 5 The ratio of methionine-enkephalin to leucine-enkephalin varied greatly in different brain regions. The highest proportions of leucine-enkephalin were found in the cerebral cortex and hippocampus. 6 Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides. 7 It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine- and leucine-enkephalin may subserve separate neuronal functions. PMID:597668

  15. Detection and Measurement of Methionine Oxidation in Proteins.

    PubMed

    Sen, K Ilker; Hepler, Robert; Nanda, Hirsh

    2017-02-02

    Methionine oxidation is a prevalent modification found in proteins both in biological settings and in the manufacturing of biotherapeutic molecules. In cells, the oxidation of specific methionine sites can modulate protein function or promote interactions that trigger signaling pathways. In biotherapeutic development, the formation of oxidative species could be detrimental to the efficacy or safety of the drug product. Thus, methionine oxidation is a critical quality attribute that needs to be monitored throughout development. Here we describe a method using LC/MS/MS to identify site-specific methionine modifications in proteins. Antibodies are stressed with hydrogen peroxide, and the level of Met oxidation is compared to that of reference molecules. The protocols presented here are not specific to methionine and can be used more generally to identify other PTM risk sites in molecules after various types of treatments. © 2017 by John Wiley & Sons, Inc.

  16. Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co(I)rrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co(II)rrinoids.

    PubMed

    Park, Kiyoung; Mera, Paola E; Moore, Theodore C; Escalante-Semerena, Jorge C; Brunold, Thomas C

    2015-06-08

    Three distinct families of ATP:corrinoid adenosyltransferases (ACATs) exist that are capable of converting vitamin B12 derivatives into coenzyme B12 by catalyzing the thermodynamically challenging reduction of Co(II) rrinoids to form "supernucleophilic" Co(I) intermediates. While the structures and mechanisms of two of the ACAT families have been studied extensively, little is known about the EutT enzymes beyond the fact that they exhibit a unique requirement for a divalent metal cofactor for enzymatic activity. In this study we have obtained compelling evidence that EutT converts cob(II)alamin into an effectively four-coordinate Co(II) species so as to facilitate Co(II)→Co(I) reduction. Intriguingly, EutT fails to promote axial ligand dissociation from the substrate analogue cob(II)inamide, a natural precursor of cob(II)alamin. This unique substrate specificity of EutT has important physiological implications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Structural Characterization of a Human-Type Corrinoid Adenosyltransferase Confirms That Coenzyme B[subscript 12] Is Synthesized through a Four-Coordinate Intermediate

    SciTech Connect

    St. Maurice, Martin; Mera, Paola; Park, Kiyoung; Brunold, Thomas C.; Escalante-Semerena, Jorge C.; Rayment, Ivan

    2008-11-18

    ATP:cob(I)alamin adenosyltransferases (ACAs) catalyze the transfer of the 5{prime}-deoxyadenosyl moiety from ATP to the upper axial ligand position of cobalamin in the synthesis of coenzyme B{sub 12}. For the ACA-catalyzed reaction to proceed, cob(II)alamin must be reduced to cob(I)alamin in the enzyme active site. This reduction is facilitated through the generation of a four-coordinate cob(II)alamin intermediate on the enzyme. We have determined the high-resolution crystal structure of a human-type ACA from Lactobacillus reuteri with a four-coordinate cob(II)alamin bound in the enzyme active site and with the product, adenosylcobalamin, partially occupied in the active site. The assembled structures represent snapshots of the steps in the ACA-catalyzed formation of the cobalt-carbon bond of coenzyme B{sub 12}. The structures define the corrinoid binding site and provide visual evidence for a base-off, four-coordinate cob(II)alamin intermediate. The complete structural description of ACA-mediated catalysis reveals the molecular features of four-coordinate cob(II)alamin stabilization and provides additional insights into the molecular basis for dysfunction in human patients suffering from methylmalonic aciduria.

  18. Protein-borne methionine residues as structural antioxidants in mitochondria.

    PubMed

    Schindeldecker, Mario; Moosmann, Bernd

    2015-07-01

    Methionine is an oxidant-labile amino acid whose major oxidation products, methionine sulfoxides, can be readily repaired by various NADPH-dependent methionine sulfoxide reductases. Formally, the methionine oxidation-reduction circuit could act as a cellular antioxidant system, by providing a safe sink for oxidants that might cause much more damage if reacting otherwise. This concept is supported by focal experimental evidence; however, the global importance, scope and biochemical role of protein-borne methionine as an inbuilt macromolecular antioxidant have remained incompletely defined. In analyzing proteomic methionine usage on different levels of comparison, we find that protein methionine (i) is primarily an antioxidant of mitochondria, especially of the inner mitochondrial membrane, (ii) responds strongly to respiratory demands on an evolutionary timescale, (iii) acts locally, by selectively protecting its carrier protein, and (iv) might be utilized as a molecular predictor of aerobic metabolic rate in animals, to complement traditional markers like the presence of a respiratory pigment. Our data support the idea that proteins in need of a long lifespan or acting in dangerous environments may acquire massive structural alterations aimed at increasing their resistance to oxidation. Counterintuitively though, they sometimes do so by accumulating particularly labile rather than particularly stable building blocks, illustrating that the technical concept of cathodic protection is also employed by the animate nature.

  19. Efficacy of Methionine Against Ectoparasitic Nematodes on Golf Course Turf

    PubMed Central

    Cuda, James P.; Stevens, Bruce R.

    2009-01-01

    Plant-parasitic nematodes are important pathogens of intensely-managed turf used on golf courses. Two of these nematodes that are common in the southeastern US are Belonolaimus longicaudatus and Mesocriconema ornata. Currently, there is a lack of effective treatments that can be used to manage these important pests. Turfgrass field trials evaluated DL-methionine as a turfgrass nematicide against B. longicaudatus and M. ornata. One trial was on a bermudagrass putting green, the other was on zoysiagrass maintained under putting-green conditions. Two rates of methionine, 1120 kg/ha in a single application, and 112 kg/ha applied twice four weeks apart, were compared with untreated control and fenamiphos treatments. Measurements collected included soil nematode counts, turf density, and root lengths. In both trials, 1120 kg/ha of methionine reduced numbers of both nematode species (P ≤ 0.1), and 112 kg/ha of methionine reduced numbers of both nematode species after two applications. Bermudagrass turf density responded favorably to both methionine rates and root lengths were improved by the 1120 kg/ha rate. Zoysiagrass showed short-term phytotoxicity to methionine, but quickly recovered and treated plots were improved compared to the untreated controls by the end of the trial. These trials indicated that methionine has potential for development as a turfgrass nematicide, but further research is needed to determine how it can best be used. PMID:22736817

  20. Bioavailability of different dietary supplemental methionine sources in animals.

    PubMed

    Zhang, Shuai; Wong, Eric A; Gilbert, Elizabeth R

    2015-06-01

    Dietary methionine is indispensable for animal maintenance, growth and development. L-methionine (L-Met), and its synthetic forms DL-methionine (DL-Met) and 2-hydroxy-4 (methylthio) butanoic acid (HMTBA) are common supplemental methionine sources in animal diets. There are different characteristics for cellular absorption, transport, metabolism and bio-efficiency between these three dietary methionine sources. Moreover, there are differences in their utilization among various species such as chickens, pigs and ruminants. As a methionine precursor, HMTBA is efficacious in the promotion of growth in animals. It is absorbed mainly by monocarboxylate transporter 1 (MCT1), coupled with the activity of the Na(+)/H(+) exchanger (NHE3), while DL-Met uptake occurs via multiple carrier-mediated systems. Liver, kidney and small intestine can metabolize D-Met and HMTBA to L-Met through oxidation and transamination. In ruminants, the non-hepatic tissues act as major sites of HMTBA conversion, which are different from that in chickens and pigs. HMTBA also has additional benefits in anti-oxidation. Understanding the characteristics of uptake and metabolism of different methionine sources will greatly benefit the industry and bioscience research.

  1. A machine learning approach for predicting methionine oxidation sites.

    PubMed

    Aledo, Juan C; Cantón, Francisco R; Veredas, Francisco J

    2017-09-29

    The oxidation of protein-bound methionine to form methionine sulfoxide, has traditionally been regarded as an oxidative damage. However, recent evidences support the view of this reversible reaction as a regulatory post-translational modification. The perception that methionine sulfoxidation may provide a mechanism to the redox regulation of a wide range of cellular processes, has stimulated some proteomic studies. However, these experimental approaches are expensive and time-consuming. Therefore, computational methods designed to predict methionine oxidation sites are an attractive alternative. As a first approach to this matter, we have developed models based on random forests, support vector machines and neural networks, aimed at accurate prediction of sites of methionine oxidation. Starting from published proteomic data regarding oxidized methionines, we created a hand-curated dataset formed by 113 unique polypeptides of known structure, containing 975 methionyl residues, 122 of which were oxidation-prone (positive dataset) and 853 were oxidation-resistant (negative dataset). We use a machine learning approach to generate predictive models from these datasets. Among the multiple features used in the classification task, some of them contributed substantially to the performance of the predictive models. Thus, (i) the solvent accessible area of the methionine residue, (ii) the number of residues between the analyzed methionine and the next methionine found towards the N-terminus and (iii) the spatial distance between the atom of sulfur from the analyzed methionine and the closest aromatic residue, were among the most relevant features. Compared to the other classifiers we also evaluated, random forests provided the best performance, with accuracy, sensitivity and specificity of 0.7468±0.0567, 0.6817±0.0982 and 0.7557±0.0721, respectively (mean ± standard deviation). We present the first predictive models aimed to computationally detect methionine sites that

  2. Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidases by Bengamide Derivatives

    SciTech Connect

    Lu, Jing-Ping; Yuan, Xiu-Hua; Yuan, Hai; Wang, Wen-Long; Wan, Baojie; Franzblau, Scott G.; Ye, Qi-Zhuang

    2012-05-29

    Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.

  3. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional... reaction between equimolar amounts of zinc sulfate and DL-methionine in purified water. (b) The...

  4. Vitamin-dependent methionine metabolism and alcoholic liver disease.

    PubMed

    Halsted, Charles H; Medici, Valentina

    2011-09-01

    Emerging evidence indicates that ethanol-induced alterations in hepatic methionine metabolism play a central role in the pathogenesis of alcoholic liver disease (ALD). Because malnutrition is a universal clinical finding in this disease and hepatic methionine metabolism is dependent upon dietary folate and vitamins B-6 and B-12, ALD can be considered an induced nutritional disorder that is conditioned by alcohol abuse. The present review describes the etiologies of these 3 vitamin deficiencies in ALD and how they interact with chronic ethanol exposure to alter hepatic methionine metabolism. Subsequent sections focus on molecular mechanisms for the interactions of aberrant methionine metabolism with ethanol in the pathogenesis of ALD, in particular the role of S-adenosylmethionine (SAM) in regulating the epigenetic expressions of genes relevant to pathways of liver injury. The review will conclude with descriptions of studies on the efficacy of SAM in the treatment of ALD and with discussion of potentially fruitful future avenues of research.

  5. Vitamin-Dependent Methionine Metabolism and Alcoholic Liver Disease1

    PubMed Central

    Halsted, Charles H.; Medici, Valentina

    2011-01-01

    Emerging evidence indicates that ethanol-induced alterations in hepatic methionine metabolism play a central role in the pathogenesis of alcoholic liver disease (ALD). Because malnutrition is a universal clinical finding in this disease and hepatic methionine metabolism is dependent upon dietary folate and vitamins B-6 and B-12, ALD can be considered an induced nutritional disorder that is conditioned by alcohol abuse. The present review describes the etiologies of these 3 vitamin deficiencies in ALD and how they interact with chronic ethanol exposure to alter hepatic methionine metabolism. Subsequent sections focus on molecular mechanisms for the interactions of aberrant methionine metabolism with ethanol in the pathogenesis of ALD, in particular the role of S-adenosylmethionine (SAM) in regulating the epigenetic expressions of genes relevant to pathways of liver injury. The review will conclude with descriptions of studies on the efficacy of SAM in the treatment of ALD and with discussion of potentially fruitful future avenues of research. PMID:22332083

  6. S-Adenosyl-L-Methionine (SAMe): An Introduction

    MedlinePlus

    ... symptoms improved when they took SAMe. What the Science Says About the Effectiveness of SAMe SAMe has ... DJ. St. John’s wort and S-adenosyl methionine as “natural” alternatives to conventional antidepressants in the era of ...

  7. Growth rate of Enterobacteriaceae at elevated temperatures: limitation by methionine.

    PubMed

    Ron, E Z

    1975-10-01

    The effect of elevated temperatures on growth rate was studied in five strains of Enterobacteriaceae. In all the strains tested a shift to the elevated temperature resulted in an immediate decrease in growth rate which was due to limitation in the availability of endogenous methionine. The first biosynthetic enzyme of the methionine pathway-homoserine transsuccinylase-was studied in extracts of Aerobacter aerogenes, Salmonella typhimurium, and Escherichia coli and was shown to be temperature sensitive in all of them.

  8. Loss of conformational stability in calmodulin upon methionine oxidation.

    PubMed Central

    Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

    1998-01-01

    We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in

  9. Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri.

    PubMed

    Park, Kiyoung; Mera, Paola E; Escalante-Semerena, Jorge C; Brunold, Thomas C

    2016-09-01

    The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi(+)). This process requires the formation of "supernucleophilic" Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.

  10. the Eutt enzyme of Salmonella enterica is a unique ATP:Cob(I)alamin adenosyltransferase metalloprotein that requires ferrous ions for maximal activity.

    PubMed

    Moore, Theodore C; Mera, Paola E; Escalante-Semerena, Jorge C

    2014-02-01

    ATP:co(I)rrinoid adenosyltransferase (ACAT) enzymes convert vitamin B12 to coenzyme B12. EutT is the least understood ACAT. We report the purification of EutT to homogeneity and show that, in vitro, free dihydroflavins drive the adenosylation of cob(II)alamin bound to EutT. Results of chromatography analyses indicate that EutT is dimeric in solution, and unlike other ACATs, EutT catalyzes the reaction with sigmoidal kinetics indicative of positive cooperativity for cob(II)alamin. Maximal EutT activity was obtained after metalation with ferrous ions. EutT/Fe(II) protein lost all activity upon exposure to air and H2O2, consistent with previously reported results indicating that EutT was an oxygen-labile metalloprotein containing a redox-active metal. Results of in vivo and in vitro analyses of single-amino-acid variants affecting a HX11CCXXC(83) motif conserved in EutT proteins showed that residues His67, Cys80, and Cys83 were required for EutT function in vivo, while Cys79 was not. Unlike that of other variants, the activity of the EutT(C80A) variant was undetectable in vitro, suggesting that Cys80 was critical to EutT function. Results of circular dichroism studies indicate that the presence or absence of a metal ion does not affect protein folding. EutT can now be purified in the presence of oxygen and reactivated with ferrous ions for maximal activity.

  11. The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

    PubMed Central

    Moore, Theodore C.; Mera, Paola E.

    2014-01-01

    ATP:co(I)rrinoid adenosyltransferase (ACAT) enzymes convert vitamin B12 to coenzyme B12. EutT is the least understood ACAT. We report the purification of EutT to homogeneity and show that, in vitro, free dihydroflavins drive the adenosylation of cob(II)alamin bound to EutT. Results of chromatography analyses indicate that EutT is dimeric in solution, and unlike other ACATs, EutT catalyzes the reaction with sigmoidal kinetics indicative of positive cooperativity for cob(II)alamin. Maximal EutT activity was obtained after metalation with ferrous ions. EutT/Fe(II) protein lost all activity upon exposure to air and H2O2, consistent with previously reported results indicating that EutT was an oxygen-labile metalloprotein containing a redox-active metal. Results of in vivo and in vitro analyses of single-amino-acid variants affecting a HX11CCXXC83 motif conserved in EutT proteins showed that residues His67, Cys80, and Cys83 were required for EutT function in vivo, while Cys79 was not. Unlike that of other variants, the activity of the EutTC80A variant was undetectable in vitro, suggesting that Cys80 was critical to EutT function. Results of circular dichroism studies indicate that the presence or absence of a metal ion does not affect protein folding. EutT can now be purified in the presence of oxygen and reactivated with ferrous ions for maximal activity. PMID:24336938

  12. Methionine salvage pathway in relation to ethylene biosynthesis

    SciTech Connect

    Miyazaki, J.H.

    1987-01-01

    The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, (/sup 14/C)MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, with little evolution of other /sup 14/C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR.

  13. Modeling the oxidation of methionine residues by peroxides in proteins.

    PubMed

    Chennamsetty, Naresh; Quan, Yong; Nashine, Vishal; Sadineni, Vikram; Lyngberg, Olav; Krystek, Stanley

    2015-04-01

    We report the use of molecular modeling to predict the oxidation propensity of methionine residues in proteins. Oxidation of methionine to the sulfoxide form is one of the major degradation pathways for therapeutic proteins. Oxidation can occur during production, formulation, or storage of pharmaceuticals and it often reduces or eliminates biological activity. We use a molecular model based on atomistic simulations called 2-shell water coordination number to predict the oxidation rates for several model proteins and therapeutic candidates. In addition, we implement models that are based on static and simulation average of the solvent-accessible area (SAA) for either the side chain or the sulfur atom in the methionine residue. We then compare the results from the different models against the experimentally measured relative rates of methionine oxidation. We find that both the 2-shell model and the simulation-averaged SAA models are accurate in predicting the oxidation propensity of methionine residues for the proteins tested. We also find the appropriate parameter ranges where the models are most accurate. These models have significant predictive power and can be used to enable further protein engineering or to guide formulation approaches in stabilizing the unstable methionine residues.

  14. In vitro Characterization of Uptake Mechanism of L-[methyl-3H]-methionine in Hepatocellular Carcinoma

    PubMed Central

    Kuang, Yu; Wang, Fangjing; Corn, David J.; Tian, Haibin; Lee, Zhenghong

    2015-01-01

    Purpose Methionine (Met) could be a useful imaging biomarker for the diagnosis of hepatocellular carcinoma (HCC), as demonstrated by PET imaging with L-[methyl-11C]-Met. In HCC cells, protein synthesis mainly contributes to radiopharmaceutical uptake. In contrast, lipid synthesis via the phosphatidylethanolamine (PE) methylation pathway is the major metabolic route of L-[methyl-11C]-Met in normal hepatocytes, which contributes to the background contrast observed in PET images. However, the mechanisms of amino acid transport and the roles of the two key enzymes, methionine adenosyltransferase (MAT) and phosphatidylethanolamine N-methyltransferase (PEMT), are not yet completely understood. The aim of this study was to investigate the roles of the amino acid transporters and these two key enzymes in the uptake of L-[methyl-11C]-Met in HCC cells. Procedures A well-differentiated woodchuck HCC cell line, WCH17, was used for the study. The amino acid transporter of WCH17 cells was assayed to investigate the Met transport process in HCC. WCH17 cells were treated with 5 mM S-adenosylmethionine (SAM) for 8, 16, 24, and 48 h to downregulate MAT2A gene expression. Control or SAM-treated WCH17 cells were pulsed with L-[methyl-3H]-Met for 5 min and chased with cold media to mimic the rapid blood clearance of radiolabeled Met (pulse-chase experiment). In parallel, WCH17 cells were transfected with a mouse liver PEMT2 expression vector, and the pulse-chase experiment was performed to investigate the uptake of the radiolabeled Met in HCC cells. The water-soluble, protein, and lipid phases from the total uptake were subsequently extracted and measured, respectively. Results Met was transported into HCC cells via a facilitative transport process, which was characterized as system L and ASC-like, Na+ dependent, and low affinity with partial energy dependence. The total uptake of L-[methyl-3H]-Met was decreased in HCC cells with SAM treatment. This reduction pattern followed that of

  15. The low-methionine content of vegan diets may make methionine restriction feasible as a life extension strategy.

    PubMed

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2009-02-01

    Recent studies confirm that dietary methionine restriction increases both mean and maximal lifespan in rats and mice, achieving "aging retardant" effects very similar to those of caloric restriction, including a suppression of mitochondrial superoxide generation. Although voluntary caloric restriction is never likely to gain much popularity as a pro-longevity strategy for humans, it may be more feasible to achieve moderate methionine restriction, in light of the fact that vegan diets tend to be relatively low in this amino acid. Plant proteins - especially those derived from legumes or nuts - tend to be lower in methionine than animal proteins. Furthermore, the total protein content of vegan diets, as a function of calorie content, tends to be lower than that of omnivore diets, and plant protein has somewhat lower bioavailability than animal protein. Whole-food vegan diets that moderate bean and soy intake, while including ample amounts of fruit and wine or beer, can be quite low in methionine, while supplying abundant nutrition for health (assuming concurrent B12 supplementation). Furthermore, low-fat vegan diets, coupled with exercise training, can be expected to promote longevity by decreasing systemic levels of insulin and free IGF-I; the latter effect would be amplified by methionine restriction - though it is not clear whether IGF-I down-regulation is the sole basis for the impact of low-methionine diets on longevity in rodents.

  16. Toxicity of seleno-l-methionine, seleno-dl-methionine, high selenium wheat, and selenized yeast to mallard ducklings

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.; LeCaptain, L.J.

    1996-01-01

    The toxicity of four chemical forms of selenium (seleno-L-methionine, seleno-DL-methionine, selenized yeast, and high selenium wheat) was compared in day-old mallard ducklings (Anas platyrhynchos). In the first experiment, in which the basal diet was 75% wheat, survival after 2 weeks was lower for ducklings fed 30 ?g/g selenium as seleno-L-methionine (36%) than for ducklings fed 30 ?g/g selenium as seleno-DL-methionine (100%) or 30 ?g/g selenium from high selenium yeast (88%). In a second experiment, in which the basal diet was a commercial duck feed, survival after 2 weeks was 100% in ducklings fed 30 ?g/g selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. The greater toxicity of the L form of selenomethionine was probably related to the palatability or nutritional nature of the wheat-based diet used in experiment 1, but the exact reason for the difference between the DL and L forms is unknown. Biologically incorporated selenium, derived from high selenium wheat was no more toxic than selenium derived from the two purified forms of selenomethionine, and the selenium in selenized yeast was not as toxic as that in the two forms of selenomethionine.

  17. Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 5'-methylthioadenosine.

    PubMed

    Albers, Eva

    2009-12-01

    The methionine salvage pathway, also called the 5'-methylthioadenosine (MTA) cycle, recycles the sulfur of MTA, which is a by-product in the biosyntheses of polyamine and the plant hormone ethylene. MTA is first converted to 5'-methylthioribose-1-phosphate either by MTA phosphorylase or the combined action of MTA nucleosidase and 5'-methylthioribose kinase. Subsequently, five additional enzymatic steps, catalyzed by four or five proteins, will form 4-methylthio-2-oxobutyrate, the deaminated form of methionine. The final transamination is achieved by transaminases active in the amino acid biosynthesis. This pathway is present with some variations in all types of organisms and seems to be designed for a quick removal of MTA achieved by high affinities of the first enzymes. During evolution some enzymes have attained additional functions, like a proposed role in nuclear mRNA processing by the aci-reductone dioxygenase. For others the function seems to be lost due to conditions in specific ecological niches, such as, presence of sulfur and/or absence of oxygen resulting in that, for example, Escherichia coli is lacking a functional pathway. The pathway is regulated as response to sulfur availability and take part in the regulation of polyamine synthesis. Some of the enzymes in the pathway show separate specificities in different organisms and some others are unique for groups of bacteria and parasites. Thus, promising targets for antimicrobial agents have been identified. Other medical topics to which this pathway has connections are cancer, apoptosis, and inflammatory response.

  18. Transcriptional analysis of L-methionine catabolism in Brevibacterium linens ATCC9175.

    PubMed

    Cholet, Orianne; Hénaut, Alain; Bonnarme, Pascal

    2007-04-01

    The expression of genes possibly involved in L-methionine and lactate catabolic pathways were performed in Brevibacterium linens (ATCC9175) in the presence or absence of added L-methionine. The expression of 27 genes of 39 selected genes differed significantly in L-methionine-enriched cultures. The expression of the gene encoding L-methionine gamma-lyase (MGL) is high in L-methionine-enriched cultures and is accompanied by a dramatic increase in volatile sulfur compounds (VSC) biosynthesis. Several genes encoding alpha-ketoacid dehydrogenase and one gene encoding an acetolactate synthase were also up-regulated by L-methionine, and are probably involved in the catabolism of alpha-ketobutyrate, the primary degradation product of L-methionine to methanethiol. Gene expression profiles together with biochemical data were used to propose catabolic pathways for L-methionine in B. linens and their possible regulation by L-methionine.

  19. Response of rainbow trout to source and level of supplemental dietary methionine

    USGS Publications Warehouse

    Poston, H.A.

    1986-01-01

    1. Methionine and total sulfur amino acid (TSAA) requirements of rainbow trout (Salmo gairdneri) were investigated by feeding graded isosulfurous levels of l- and dl-methionine, l-cystine, and the free acid and calcium forms of methionine hydroxy analog (MHA).2. Added cystine did not promote growth, survival or prevent cataracts.3. l-methionine produced fastest growth, followed by dl-methionine, CaMHA and free acid MHA.4. Trout fed CaMHA gained 85.7 and 92.3% as much as those fed l-methionine and dl-methionine.5. Within each experiment, the level of L-methionine isomer that prevented cataracts was constant (1.86 g/100g protein in experiment (1), 1.45 in experiment (2) and was lower than for maximum growth (2.89 and 2.15 g) regardless of methionine source.

  20. Whole body methionine kinetics, transmethylation, transulfuration and remethylation during pregnancy.

    PubMed

    Kurpad, Anura V; Anand, Pauline; Dwarkanath, Pratibha; Hsu, Jean W; Thomas, Tinku; Devi, Sarita; Thomas, Annamma; Mhaskar, Rita; Jahoor, Farook

    2014-02-01

    There is evidence from a study of pregnant American women that methionine transmethylation (TM) and remethylation (RM) rates increases and transulfuration (TS) decreases as pregnancy progresses from trimester 1 to 3. To determine whether pregnant Indian women can make this adaptation successfully, methionine kinetics, TS, TM, and RM were measured in Indian women in early and late pregnancy. Measurements were made in the postabsorptive and fed states in the 1st and 3rd trimesters of pregnancy by infusing 1-(13)C,(2)H3-methionine in 24 women, 12 with low (≤150 pmol L(-1)) and 12 with normal (≥200 pmol L(-1)) vitamin B12 status at recruitment. From trimester 1 to 3, except RM which decreased significantly, there was no change in any weight-specific methionine kinetic parameter. When expressed per whole body, methionine flux from protein breakdown increased significantly from trimester 1 to 3 in the fed and postabsorptive states. Flux to protein synthesis also increased significantly in the fed state. Rates of TM, TS and RM did not change, regardless of vitamin B12 status at recruitment. Protein and methionine intakes correlated with TM and RM rates and the change in RM from trimester 1 to 3 correlated with the change in dietary protein intake. These results suggest that methionine flux and its utilization for protein synthesis increases in Indian women as pregnancy progresses from trimester 1 to 3. TM and RM rates do not increase however, possibly because of inadequate protein intake and not because of vitamin B12 deficiency at trimester 1. Copyright © 2013 Elsevier B.V. and NIPR. All rights reserved.

  1. Dissimilation of Methionine by a Demethiolase of Aspergillus Species1

    PubMed Central

    Ruiz-Herrera, José; Starkey, Robert L.

    1969-01-01

    Enzyme preparations obtained from the mycelium of Aspergillus species broke down methionine by co-dissimilation. The deaminase and demethiolase activities of crude extracts were increased 100-fold by precipitation with (NH4)2SO4 and column chromatography on diethylaminoethyl cellulose. The enzyme acted on d-methionine but not on l-methionine. The enzyme was labile: it was inactivated by oxygen and ascorbic acid but ethylenediaminetetraacetic acid and mercaptoethanol preserved its activity. Enzyme activity decreased even at 4 and −30 C and was lost rapidly above 45 C. It was most rapid at 35 C and at pH 8.0 to 9.0. For the following reasons, it was concluded that deamination and demethiolation of methionine were effected by the same enzyme: both activities increased equally at each stage of purification; ammonia, methanethiol, and α-keto butyric acid were formed in amounts equivalent to the amount of methionine dissimilated; the Km and optimal pH for formation of both keto acid and methanethiol were the same; both activities remained in the same fractions that were separated by electrophoresis and the activities were equivalent. The purified enzyme demethiolated α-keto methionine and α-hydroxy methionine and split the sulfur linkage of ethionine but did not cleave cystathionine. Few amino acids were deaminated. The enzyme was sensitive to some carbonyl and sulfhydryl reagents and was relatively insensitive to heavy metals other than Hg++. The Km was 1.3 × 10−3 to 1.5 × 10−3m at pH 7.0. No requirement for cofactors was noted, and attempts to dissociate the enzyme, including dialysis with hydroxylamine, were unsuccessful. PMID:5370277

  2. Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

    PubMed Central

    Rouf, Syed Fazle; Kitowski, Vera; Böhm, Oliver M.; Rhen, Mikael; Jäger, Timo; Bange, Franz-Christoph

    2011-01-01

    Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium. PMID:22073230

  3. Effect of excess methionine and methionine hydroxy analogue on growth performance and plasma homocysteine of growing Pekin ducks.

    PubMed

    Xie, M; Hou, S S; Huang, W; Fan, H P

    2007-09-01

    One experiment was conducted to study the effect of excess dl-methionine (DLM) and dl-2-hydroxy-4-methylthiobutanoic acid free acid (dl-HMB-FA) on duck growth. One-day-old male white Pekin ducklings were fed common starter diets from hatch to 21 d of age and then fed the experimental diets from 21 to 42 d of age. Three hundred twenty 21-d-old birds were allotted to 40 raised wire-floor pens with 8 birds per pen according to similar pen weight. There were 5 dietary treatments that included a methionine-adequate control diet and control diets supplemented with 2 levels of dry DLM (1 or 2%) or 2 equimolar levels of liquid dl-HMB-FA (1.13 or 2.26%). Each dietary treatment was replicated 8 times. At 42 d of age, weight gain, feed intake, and gain/feed were measured and plasma was collected to analyze homocysteine. Compared with ducks fed control diets, excess DLM or dl-HMB-FA supplementation reduced weight gain and feed intake of birds significantly. However, on the equimolar basis, at 1 or 2% supplemental methionine activity, dl-HMB-FA was less growth-depressing than DLM. According to the growth response to excess methionine, the tolerable upper limit of dietary methionine for growing ducks may be less than 1.38% when the methionine level of the control diet (0.38%) was considered. On the other hand, plasma homocysteine was elevated markedly when 2% DLM or 2.26% dl-HMB-FA was added to control diets, but plasma homocysteine of ducks fed 2.26% dl-HMB-FA supplemented diets was lower significantly than birds fed equimolar DLM-supplemented diets, which indicated the toxicity of excess methionine sources and less toxicity of dl-HMB-FA relative to DLM.

  4. Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ-Synthase and Silencing of Endogenous Methionine γ-Lyase Enhance Tuber Methionine Content in Solanum tuberosum.

    PubMed

    Kumar, Pavan; Jander, Georg

    2017-04-05

    Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line.

  5. A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum.

    PubMed

    Singh, Arvind Kumar; Syiem, Mayashree B; Singh, Rajkumar S; Adhikari, Samrat; Rai, Amar Nath

    2008-05-01

    We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues L-methionine-DL-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in 35S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.

  6. Production of methanethiol from methionine by Brevibacterium linens CNRZ 918.

    PubMed

    Ferchichi, M; Hemme, D; Nardi, M; Pamboukdjian, N

    1985-04-01

    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.

  7. Protein methionine oxidation augments reperfusion injury in acute ischemic stroke

    PubMed Central

    Gu, Sean X.; Blokhin, Ilya O.; Wilson, Katina M.; Dhanesha, Nirav; Doddapattar, Prakash; Grumbach, Isabella M.; Chauhan, Anil K.; Lentz, Steven R.

    2016-01-01

    Reperfusion injury can exacerbate tissue damage in ischemic stroke, but little is known about the mechanisms linking ROS to stroke severity. Here, we tested the hypothesis that protein methionine oxidation potentiates NF-κB activation and contributes to cerebral ischemia/reperfusion injury. We found that overexpression of methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that reverses protein methionine oxidation, attenuated ROS-augmented NF-κB activation in endothelial cells, in part, by protecting against the oxidation of methionine residues in the regulatory domain of calcium/calmodulin-dependent protein kinase II (CaMKII). In a murine model, MsrA deficiency resulted in increased NF-κB activation and neutrophil infiltration, larger infarct volumes, and more severe neurological impairment after transient cerebral ischemia/reperfusion injury. This phenotype was prevented by inhibition of NF-κB or CaMKII. MsrA-deficient mice also exhibited enhanced leukocyte rolling and upregulation of E-selectin, an endothelial NF-κB–dependent adhesion molecule known to contribute to neurovascular inflammation in ischemic stroke. Finally, bone marrow transplantation experiments demonstrated that the neuroprotective effect was mediated by MsrA expressed in nonhematopoietic cells. These findings suggest that protein methionine oxidation in nonmyeloid cells is a key mechanism of postischemic oxidative injury mediated by NF-κB activation, leading to neutrophil recruitment and neurovascular inflammation in acute ischemic stroke. PMID:27294204

  8. CHARACTERIZATION OF THE METHIONINE SULFOXIDE REDUCTASES OF SCHISTOSOMA MANSONI

    PubMed Central

    Oke, Tolulope T.; Moskovitz, Jackob; Williams, David L.

    2013-01-01

    Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, which is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic and zinc coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities towards these substrates. Using real-time PCR, both proteins were found to be expressed in all stages of the parasite’s life cycle with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite. PMID:19604033

  9. Oxidation increases the strength of the methionine-aromatic interaction

    PubMed Central

    Lewis, Andrew K.; Dunleavy, Katie; Senkow, Tiffany L.; Her, Cheng; Horn, Benjamin T.; Jersett, Mark A.; Mahling, Ryan; McCarthy, Megan R.; Perell, Gabriella T.; Valley, Christopher C.; Karim, Christine B.; Gao, Jiali; Pomerantz, William C. K.; Thomas, David D.; Cembran, Alessandro; Hinderliter, Anne; Sachs, Jonathan N.

    2016-01-01

    Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we show that oxidation increases the strength of the methionine-aromatic interaction motif—a driving force for protein folding and protein-protein interaction—by 0.5 – 1.4 kcal/mol. We find that non-hydrogen bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups are enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR experiments on model peptides demonstrate that oxidation of methionine stabilizes the interaction by 0.5–0.6 kcal/mol. We confirm the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on 1) calmodulin structure and dynamics and 2) lymphotoxin-α/TNFR1 binding. Thus, the methionine-aromatic motif is a determinant of protein structural and functional sensitivity to oxidative stress. PMID:27547920

  10. B-Vitamin dependent methionine metabolism and alcoholic liver disease.

    PubMed

    Halsted, Charles H

    2013-03-01

    Convincing evidence links aberrant B-vitamin dependent hepatic methionine metabolism to the pathogenesis of alcoholic liver disease (ALD). This review focuses on the essential roles of folate and vitamins B6 and B12 in hepatic methionine metabolism, the causes of their deficiencies among chronic alcoholic persons, and how their deficiencies together with chronic alcohol exposure impact on aberrant methionine metabolism in the pathogenesis of ALD. Folate is the dietary transmethylation donor for the production of S-adenosylmethionine (SAM), which is the substrate for all methyltransferases that regulate gene expressions in pathways of liver injury, as well as a regulator of the transsulfuration pathway that is essential for production of glutathione (GSH), the principal antioxidant for defense against oxidative liver injury. Vitamin B12 regulates transmethylation reactions for SAM production and vitamin B6 regulates transsulfuration reactions for GSH production. Folate deficiency accelerates the experimental development of ALD in ethanol-fed animals while reducing liver SAM levels with resultant abnormal gene expression and decreased production of antioxidant GSH. Through its effects on folate metabolism, reduced SAM also impairs nucleotide balance with resultant increased DNA strand breaks, oxidation, hepatocellular apoptosis, and risk of carcinogenesis. The review encompasses referenced studies on mechanisms for perturbations of methionine metabolism in ALD, evidence for altered gene expressions and their epigenetic regulation in the pathogenesis of ALD, and clinical studies on potential prevention and treatment of ALD by correction of methionine metabolism with SAM.

  11. Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation.

    PubMed

    Pallares, Ivan G; Moore, Theodore C; Escalante-Semerena, Jorge C; Brunold, Thomas C

    2016-03-23

    EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate "base-on" and "base-off" Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of "base-off" Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

  12. Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation.

    PubMed

    Pallares, Ivan G; Moore, Theodore C; Escalante-Semerena, Jorge C; Brunold, Thomas C

    2017-01-17

    The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin. Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutT(WT)/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutT(WT)/Co relative to that of the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data for EutT(WT)/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution of either His67, His75, Cys79, Cys80, or Cys83 indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.

  13. Propionylcarnitine and methionine concentrations in newborns with hypospadias.

    PubMed

    Kowal, Andrzej; Mydlak, Dariusz; Ołtarzewski, Mariusz; Bauer, Anna; Sawicka, Ewa; Hozyasz, Kamil K

    2013-01-01

    Of interest is if factors like maternal diet can influence the risk of hypospadias-affected pregnancy. Increased propionylcarnitine (C3) is regarded as a biomarker of vitamin B12 deficiency. The retrospective study was undertaken to determine whether increased propionylcarnitine and low methionine in newborns are associated with hypospadias. 41 newborns with hypospadias and 90 control newborns without congenital anomalies were investigated. Whole blood propionylcarnitine and methionine concentrations were measured using tandem mass spectrometry. The mean concentration of propionylcarnitine was higher in newborns with hypospadias compared with newborns without congenital anomalies (p = 0.026). The mean methionine level in cases was insignificantly lower than in controls. There appears to be an association between decreased vitamin B12, as indexed by an increase of propionylcarnitine, and hypospadias in the investigated group of patients.

  14. Propionylcarnitine and methionine concentrations in newborns with hypospadias

    PubMed Central

    Kowal, Andrzej; Mydlak, Dariusz; Ołtarzewski, Mariusz; Bauer, Anna; Sawicka, Ewa

    2013-01-01

    Introduction Of interest is if factors like maternal diet can influence the risk of hypospadias–affected pregnancy. Increased propionylcarnitine (C3) is regarded as a biomarker of vitamin B12 deficiency. The retrospective study was undertaken to determine whether increased propionylcarnitine and low methionine in newborns are associated with hypospadias. Material and methods 41 newborns with hypospadias and 90 control newborns without congenital anomalies were investigated. Whole blood propionylcarnitine and methionine concentrations were measured using tandem mass spectrometry. Results The mean concentration of propionylcarnitine was higher in newborns with hypospadias compared with newborns without congenital anomalies (p = 0.026). The mean methionine level in cases was insignificantly lower than in controls. Conclusion There appears to be an association between decreased vitamin B12, as indexed by an increase of propionylcarnitine, and hypospadias in the investigated group of patients. PMID:24707392

  15. Comparative genomics of transcriptional regulation of methionine metabolism in Proteobacteria.

    PubMed

    Leyn, Semen A; Suvorova, Inna A; Kholina, Tatiana D; Sherstneva, Sofia S; Novichkov, Pavel S; Gelfand, Mikhail S; Rodionov, Dmitry A

    2014-01-01

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ∼ 200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.

  16. Comparison of volatile sulphur compound production by cheese-ripening yeasts from methionine and methionine-cysteine mixtures.

    PubMed

    López Del Castillo-Lozano, M; Delile, A; Spinnler, H E; Bonnarme, P; Landaud, S

    2007-07-01

    Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with L-methionine or L-methionine/L-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with L-methionine or L-methionine/L-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when L-cysteine was supplemented, even at a low concentration (0.2 g l(-1)). This effect was due mainly to a significant decrease in L-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by L-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.

  17. Methionine, homocysteine, one carbon metabolism and fetal growth.

    PubMed

    Kalhan, Satish C; Marczewski, Susan E

    2012-06-01

    Methionine and folate are the key components of one carbon metabolism, providing the methyl groups for numerous methyl transferase reactions via the ubiquitous methyl donor, s-adenosyl methionine. Methionine metabolism is responsive to nutrient intake, is regulated by several hormones and requires a number of vitamins (B12, pyridoxine, riboflavin) as co-factors. The critical relationship between perturbations in the mother's methionine metabolism and its impact on fetal growth and development is now becoming evident. The relation of folate intake to fetal teratogenesis has been known for some time. Studies in human pregnancy show a continuous decrease in plasma homocysteine, and an increase in plasma choline concentrations with advancing gestation. A higher rate of transsulfuration of methionine in early gestation and of transmethylation in the 3rd trimester was seen in healthy pregnant women. How these processes are impacted by nutritional, hormonal and other influences in human pregnancy and their effect on fetal growth has not been examined. Isocaloric protein restriction in pregnant rats, resulted in fetal growth restriction and metabolic reprogramming. Isocaloric protein restriction in the non-pregnant rat, resulted in differential expression of a number of genes in the liver, a 50% increase in whole body serine biosynthesis and high rate of transmethylation, suggesting high methylation demands. These responses were associated with a significant decrease in intracellular taurine levels in the liver suggesting a role of cellular osmolarity in the observed metabolic responses. These unique changes in methionine and one carbon metabolism in response to physiological, nutritional and hormonal influences make these processes critical for cellular and organ function and growth.

  18. Bioavailability of methionine-hydroxy-analog free acid and S-methyl-methionine in the growing rat.

    PubMed

    Hegedüs, M; Fekete, S; Andrásofszky, E; Tamás, J; Kövári, L

    1992-01-01

    The biological utilization of DL-methionine (MET), DL-methionine-hydroxy-analog and DL-S-methyl-methionine-sulphonium-chloride (SMM) was tested in rat growth assay. Weight gain, feed efficiency, protein efficiency ratio (PER), net protein ratio (NPR) and net protein utilization (NPU) were applied as criteria. A test diet containing soybean meal as sole protein source was fortified with MET, MHA or SMM at 1.5 and 3.0 g/kg levels, respectively. All fortifications had a significant (p < 0.05) positive influence on weight gain, feed conversion and protein utilization. SMM showed the highest activity. MET and MHA were almost equally well utilized at 3.0 g/kg fortification level, however MHA proved slightly less efficient at 1.5 g/kg level than MET.

  19. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    USDA-ARS?s Scientific Manuscript database

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  20. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  1. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  2. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Methionine hydroxy analog and its calcium salts... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a) Product. Methionine hydroxy analog and its calcium salts. (b) (c) Limitations, restrictions,...

  3. Genetic and biochemical differences in populations bred for extremes in maize grain methionine content

    USDA-ARS?s Scientific Manuscript database

    Methionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Two populations selected were selected for high and low methionin...

  4. Characterisation of Potential Antimicrobial Targets in Bacillus spp. I. Aminotransferases and Methionine Regeneration in Bacillus subtilis

    DTIC Science & Technology

    2002-07-01

    targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J. Berger and Marvin H. Knodel Defence R&D...Characterisation of potential antimicrobial targets in Bacillus spp. I. Aminotransferases and methionine regeneration in Bacillus subtilis. Bradley J...examined in the gram-positive bacterium Bacillus subtilis. Homogenates of this bacterium were able to convert ketomethiobutyrate to methionine, utilising

  5. Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway.

    PubMed

    Kanai, Muneyoshi; Masuda, Mitsunori; Takaoka, Yasumichi; Ikeda, Hiroko; Masaki, Kazuo; Fujii, Tsutomu; Iefuji, Haruyuki

    2013-02-01

    To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X∆ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation between X∆ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in the X∆ado1 strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in the X∆ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the ∆ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.

  6. Genetic variants of methionine metabolism and DNA methylation.

    PubMed

    Bleich, Stefan; Semmler, Alexander; Frieling, Helge; Thumfart, L; Muschler, Marc; Hillemacher, Thomas; Kornhuber, Johannes; Kallweit, Ulf; Simon, Matthias; Linnebank, Michael

    2014-01-01

    Altered DNA methylation is associated with important and common pathologies such as cancer. The origin of altered DNA methylation is unknown. The methyl groups for DNA methylation are provided by methionine metabolism. This metabolism is characterized by a high interindividual variability, which is in part explained by genetic variants. In a cohort of 313 individuals derived from a family-based study with index cases of cerebrovascular disease, we analyzed whether global methylation of leukocyte DNA was associated with age, gender, homocysteine plasma levels or functionally relevant genetic variants. We observed an association of the G-allele of the methionine synthase variant c.2756A>G (D919G) with global methylation (% methylation ± 1 SD, AA: 41.3 ± 14.9; AG: 36.4 ± 18.2; GG: 30.8 ± 16.9; F = 4.799; p = 0.009). The methionine synthase variant c.2756A>G is associated with various types of cancer. Our data suggest that an impact on DNA methylation may contribute to the clinical relevance of the methionine synthase variant.

  7. Amino acid nutrition beyond methionine and lysine for milk protein

    USDA-ARS?s Scientific Manuscript database

    Amino acids are involved in many important physiological processes affecting the production, health, and reproduction of high-producing dairy cows. Most research and recommendations for lactating dairy cows has focused on methionine and lysine for increasing milk protein yield. This is because these...

  8. Methionine splanchnic uptake is increased in critically ill children

    USDA-ARS?s Scientific Manuscript database

    During critical illness the splanchnic area is profoundly affected. There is no information on splanchnic uptake of amino acids in vivo, in critically ill children. Methionine splanchnic uptake in critically ill children will differ from estimates in healthy adults. We studied 24 critically ill chil...

  9. Technical note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2014-11-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued, not only about their contribution to the global methane budget, but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds identified. We made use of stable isotope techniques to verify in vivo formation of methane and, in order to identify the carbon precursor, 13C-positionally labelled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labelled methionine clearly identified the sulphur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  10. Technical Note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2015-03-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued not only about their contribution to the global methane budget but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds to be identified. We made use of stable isotope techniques to verify the in vivo formation of methane, and, in order to identify the carbon precursor, 13C positionally labeled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labeled methionine clearly identified the sulfur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  11. Oxidized methionine is not a prion-specific covalent modification

    USDA-ARS?s Scientific Manuscript database

    The oxidation of methionine residues in the '-helical region of PrPC has been proposed to be important for prion formation. This proposal has been supported by structural studies, model systems and antibody-based experimental evidence. We developed a sensitive mass spectrometry-based method to stu...

  12. Whole body methionine kinetics, transmethylation, transulfuration and remethylation during pregnancy

    USDA-ARS?s Scientific Manuscript database

    There is evidence from a study of pregnant American women that methionine transmethylation (TM) and remethylation (RM) rates increase and transulfuration (TS) decreases as pregnancy progresses from trimester 1 to 3. To determine whether pregnant Indian women can make this adaptation successfully, me...

  13. Methionine sulfoxide disposition Is altered in animal models of obesity

    USDA-ARS?s Scientific Manuscript database

    Over 20% of populations in western countries are obese. Recently, two genetic studies indicate that the MSRA locus, containing the gene for the enzyme methionine sulfoxide reductase A (MsrA), is positively linked to the development of visceral adiposity. MsrA catalyzes the repair of methionyl thiol ...

  14. (11)C-Methionine uptake in secondary brain epilepsy.

    PubMed

    Lopci, E; Bello, L; Chiti, A

    2014-01-01

    Carbon-11 methionine ((11)C-Methionine) is a radio-labeled amino acid currently utilized in Positron Emission Tomography (PET) for imaging primary and metastatic brain tumors. Its clinical use relies mostly on oncologic applications, but the tracer has the potential to investigate other non-malignant conditions. So far, very limited evidence concerns the use of (11)C-Methionine in patients suffering from seizure; however, the tracer can find a proper utilization in this setting especially as a diagnostic complement to (18)F-Fluorodeoxyglucose ((18)F-FDG). Herein we report the case of a 57-year-old patient presenting with epileptic crises secondary to a brain metastasis from bladder carcinoma, who was investigated in our institution with (11)C-Methionine PET. The scan documented the disease recurrence in the left parietal lobe associated with a diffused tracer uptake in the surrounding cerebral circumvolutions, derived from the comitial status. After surgical removal of the metastatic lesion, the patient experienced a complete recovery of symptoms and no further onset of secondary seizure. Copyright © 2013 Elsevier España, S.L. and SEMNIM. All rights reserved.

  15. The role of methionine metabolism in inflammatory bowel disease

    USDA-ARS?s Scientific Manuscript database

    Methionine (Met) cycle activity is critical for normal cell functions. Met metabolites S-adenosylmethionine (SAM) and methylthioadenosine (MTA) are anti-inflammatory, yet their role in inflammatory bowel disease (IBD) is poorly understood. We hypothesize that active IBD leads to changes in Met metab...

  16. Oxidative Metabolism of Seleno-L-Methionine to L-Methionine Selenoxide by Flavin- Containing Monooxygenases

    PubMed Central

    Krause, Renee J.; Glocke, Steven C.; Sicuri, Anna Rita; Ripp, Sharon L.; Elfarra, Adnan A

    2008-01-01

    The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-L-methionine (SeMet) to L-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. Formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean ±S.D.; n=4) of 0.91 ± 0.29 mM and a Vmax value of 44 ± 8.0 nmol MetSeO/mg protein/min. Inclusion of 1-benzylimidazole, superoxide dismutase or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions. PMID:17173378

  17. Increased susceptibility to Kuru of carriers of the PRNP 129 methionine/methionine genotype.

    PubMed

    Lee, H S; Brown, P; Cervenáková, L; Garruto, R M; Alpers, M P; Gajdusek, D C; Goldfarb, L G

    2001-01-15

    Kuru reached epidemic proportions by the mid-twentieth century among the Fore people of New Guinea and disappeared after the abolition of cannibalistic rituals. To determine susceptibility to kuru and its role in the spread and elimination of the epidemic, we analyzed the PRNP gene coding sequences in 5 kuru patients; no germline mutations were found. Analysis of the PRNP 129 methionine (M)/valine (V) polymorphism in 80 patients and 95 unaffected controls demonstrated that the kuru epidemic preferentially affected individuals with the M/M genotype. A higher representation of M/M carriers was observed among the affected young Fore males entering the age of risk, whereas a lower frequency of M/M homozygotes was found among the survivors. M/V and V/V genotypes predisposed to a lower risk of disease development and longer incubation times. These findings are relevant to the current outbreak of variant Creutzfeldt-Jakob disease (vCJD) in the United Kingdom, because all vCJD patients tested thus far have been M/M carriers.

  18. Induction of caspase-dependent and -independent apoptosis in response to methionine restriction.

    PubMed

    Lu, Shan; Hoestje, Sara M; Choo, Eugene; Epner, Daniel E

    2003-02-01

    Tumor cells are more sensitive to methionine restriction than normal tissues, a phenomenon known as methionine auxotrophy. Previous studies have demonstrated that methionine restriction causes tumor cell growth arrest and eventually apoptosis. The current studies were undertaken to elucidate the molecular pathways leading to apoptosis induced by methionine restriction. We found that methionine restriction induced formation of oligonucleosomal DNA fragment and cytochrome c release from mitochondria in methionine-dependent PC3 and Hela cells. Methionine restriction also led to cleavage and activation of initiator and effector caspases in Hela cells but not PC3 cells. Furthermore, methionine restriction resulted in cleavage of BID and reduction in Bcl-2 levels in both cell lines. These data suggest that apoptosis induced by methionine restriction is mitochondria-dependent. Methionine restriction induced caspase-independent cell death in PC3 cells, whereas it stimulated caspase-dependent cell death in Hela cells. Cleavage of BID and decreased expression of Bcl-2 upon methionine deprivation may be the underlying mechanism to stimulate release of cytochrome c from mitochondria.

  19. Transgenic Mice Overexpressing Methionine Sulfoxide Reductase A: Characterization of Embryonic Fibroblasts

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Liu, Chengyu; Levine, Rodney L.

    2012-01-01

    Methionine residues in protein can be oxidized by reactive oxygen species to generate methionine sulfoxide. Aerobic organisms have methionine sulfoxide reductases capable of reducing methionine sulfoxide back to methionine. Methionine sulfoxide reductase A acts on the S-epimer of methionine sulfoxide, and it is known that altering its cellular level by genetic ablation or overexpression has notable effects on resistance to oxidative stress and on lifespan in species from microorganisms to animals. In mammals, the enzyme is present both in the cytosol and mitochondria, and this study was undertaken to assess the contribution of each subcellular compartment’s reductase activity to resistance against oxidative stresses. Non-transgenic mouse embryonic fibroblasts lack methionine sulfoxide reductase A activity, providing a convenient cell type to determine the effect of expression of the enzyme in each compartment. We created transgenic mice with methionine sulfoxide reductase A targeted to the cytosol, mitochondria, or both and studied embryonic fibroblasts derived from each line. Unexpectedly, none of the transgenic cells gained resistance to a variety of oxidative stresses even though the expressed enzymes were catalytically active when assayed in vitro. Noting that activity in vivo requires thioredoxin and thioredoxin reductase, we determined the levels of these proteins in the fibroblasts and found that they were very low in both the non-transgenic and transgenic cells. We conclude that overexpression of methionine sulfoxide reductase A did not confer resistance to oxidative stress because the cells lacked other proteins required to constitute a functional methionine sulfoxide reduction system. PMID:20510353

  20. The determination of methionine in proteins by gas-liquid chromatography.

    PubMed Central

    Ellinger, G M; Duncan, A

    1976-01-01

    Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods. PMID:949322

  1. Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.

    PubMed

    Preynat, A; Lapierre, H; Thivierge, M C; Palin, M F; Matte, J J; Desrochers, A; Girard, C L

    2009-02-01

    The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B(12), given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B(12.) To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-(13)C]glucose, [(13)C]NaHCO(3) and 3[1-(13)C,(2)H(3)] methionine. Milk and plasma concentrations of folic acid and vitamin B(12) increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 +/- 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance

  2. Efficacy of DL-methionine hydroxy analogue-free acid in comparison to DL-methionine in growing male white Pekin ducks.

    PubMed

    Kluge, H; Gessner, D K; Herzog, E; Eder, K

    2016-03-01

    The present study was performed to assess the bioefficacy of DL-methionine hydroxy analogue-free acid (MHA) in comparison to DL-methionine (DLM) as sources of methionine for growing male white Pekin ducks in the first 3 wk of life. For this aim, 580 1-day-old male ducks were allocated into 12 treatment groups and received a basal diet that contained 0.29% of methionine, 0.34% of cysteine and 0.63% of total sulphur containing amino acids or the same diet supplemented with either DLM or MHA in amounts to supply 0.05, 0.10, 0.15, 0.20, and 0.25% of methionine equivalents. Ducks fed the control diet without methionine supplement had the lowest final body weights, daily body weight gains and feed intake among all groups. Supplementation of methionine improved final body weights and daily body weight gains in a dose dependent-manner. There was, however, no significant effect of the source of methionine on all of the performance responses. Evaluation of the data of daily body weight gains with an exponential model of regression revealed a nearly identical efficacy (slope of the curves) of both compounds for growth (DLM = 100%, MHA = 101%). According to the exponential model of regression, 95% of the maximum values of daily body weight gain were reached at methionine supplementary levels of 0.080% and 0.079% for DLM and MHA, respectively. Overall, the present study indicates that MHA and DLM have a similar efficacy as sources of methionine for growing ducks. It is moreover shown that dietary methionine concentrations of 0.37% are required to reach 95% of the maximum of daily body weight gains in ducks during the first 3 wk of life. © 2015 Poultry Science Association Inc.

  3. Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew; Walter, Richard L; Mekel, Marlene; Barnett, Bobby L; Amburgey, Jack; Seibel, William L; Soper, Shari J; Djung, Jane F; Fairweather, Neil; Diven, Conrad; Rastogi, Vinit; Grinius, Leo; Klanke, Charles; Siehnel, Richard; Twinem, Tracy; Andrews, Ryan; Curnow, Alan

    2007-02-15

    In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding. 2006 Wiley-Liss, Inc.

  4. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    DOE PAGES

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; ...

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and amore » putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often stressed in the environment by reactive chlorine species (RCS) of

  5. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    SciTech Connect

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; Carlson, Hans K.; Wetmore, Kelly M.; Price, Morgan N.; Lavarone, Anthony T.; Deutschbauer, Adam M.; Arkin, Adam P.; Coates, John D.

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often

  6. Enzyme-linked immunosorbent assay for leucine and methionine enkephalins

    SciTech Connect

    Zamboni, G.; Jones, C.A.; Hughes, J.

    1983-04-01

    An enzyme-linked immunosorbent assay for enkephalins was developed by coupling the peptides to a carrier molecule (bovine serum albumin) in order to allow the antibody-antigen reaction to take place in the solid phase. The assay was shown to be highly reproducible. Its sensitivity was 14 nmol/liter for leucine enkephalin and 27 nmol/liter for methionine enkephalin, which is similar to that obtained when the same antibodies were used in radioimmunoassay.

  7. Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

    PubMed Central

    Chalova, Vesela I.; Froelich, Clifford A.; Ricke, Steven C.

    2010-01-01

    Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs. PMID:22319312

  8. Chemical modification of methionines in a cobra venom cytotoxin differentiates between lytic and binding domains.

    PubMed

    Stevens-Truss, R; Hinman, C L

    1996-08-01

    Cytotoxin-III from Naja naja atra (CTX) was chemically modified at either or both of its two methionine residues: Over 50% oxidation of methionine-26 occurred with a 1:1 molar ratio of chloramine-T:methionine; at a 5:1 molar ratio, methionine-26 was almost completely oxidized, while methionine-24 was modified only 26%; at a 10:1 molar ratio, both methionines were completely oxidized. Each oxidized derivative demonstrated a lower toxicity toward T-cells than toward heart cells. Conversely, binding to heart cells was affected more than binding to T-cells. Cyanogen bromide cleaved native CTX at both methionines, excising phenyl-alanine-25 and methionine-26 and converting methionine-24 to homoserine lactone. This treatment of CTX eliminated cytotoxicity toward both heart and T-cells, but had only a modest effect upon T-cell binding, as had 50% oxidation of methionine-26, suggesting that CTX lytic and binding regions may be distinct. A selective loss in heart cell binding following oxidation of methionine-24 further suggests that different parts of CTX may interact with the two types of target cells. Perturbation of the relatively flat hydrophobic surface of the CTX' triple-stranded beta-sheet could result from the introduction of negative charge due to methionine-24 oxidation. Alternatively, amino acid side chain participation in a CTX binding domain may be altered by the potential formation of a new hydrogen bond between tyrosine-51 and methionine-24 sulfoxide, as revealed by computer modeling of the completely oxidized CTX derivative.

  9. Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver.

    PubMed

    Cabrero, C; Puerta, J; Alemany, S

    1987-12-30

    Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.

  10. Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

    PubMed Central

    Kuhns, Lisa G.; Mahawar, Manish; Sharp, Joshua S.; Benoit, Stéphane; Maier, Robert J.

    2014-01-01

    The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions. PMID:23181726

  11. Comparative genomics of transcriptional regulation of methionine metabolism in proteobacteria

    DOE PAGES

    Leyn, Semen A.; Suvorova, Inna A.; Kholina, Tatiana D.; ...

    2014-11-20

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ~200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific andmore » genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.« less

  12. Comparative genomics of transcriptional regulation of methionine metabolism in proteobacteria

    SciTech Connect

    Leyn, Semen A.; Suvorova, Inna A.; Kholina, Tatiana D.; Sherstneva, Sofia S.; Novichkov, Pavel S.; Gelfand, Mikhail S.; Rodionov, Dmitry A.; Kuipers, Oscar P.

    2014-11-20

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ~200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.

  13. Comparative Genomics of Transcriptional Regulation of Methionine Metabolism in Proteobacteria

    PubMed Central

    Leyn, Semen A.; Suvorova, Inna A.; Kholina, Tatiana D.; Sherstneva, Sofia S.; Novichkov, Pavel S.; Gelfand, Mikhail S.; Rodionov, Dmitry A.

    2014-01-01

    Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ∼200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the metX or metZ genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria. PMID:25411846

  14. Amino acid distribution and metabolism in pituitary adenomas using positron emission tomography with D-(/sup 11/C)methionine and L-(/sup 11/C)methionine

    SciTech Connect

    Bergstroem, M.M.; Muhr, C.; Lundberg, P.O.; Bergstroem, K.L.; Lundqvist, H.; Antoni, G.; Fasth, K.J.; Langstroem B3

    1987-05-01

    Four patients with hormonally inactive pituitary adenomas were examined with positron emission tomography (PET) after injection, during different examinations, of L-(methyl-/sup 11/C)methionine and D-(methyl-/sup 11/C)methionine, respectively. After the rapid distribution phase, the enantiomer L-(/sup 11/C)methionine, which is metabolically active, showed a considerable continuous irreversible trapping attributed to amino acid metabolism. The stereoisomer D-(/sup 11/C)methionine, which does not participate in protein synthesis, showed a rapid distribution within the whole adenoma tissue, with a distribution space on the order of 100%. A minimal irreversible trapping was observed which could be explained by technical factors. It is concluded that PET using the two enantiomers allows a separation of passive distribution and metabolism, and that L-(/sup 11/C)methionine can be used for in vivo quantitative studies of amino acid metabolism of pituitary adenomas.

  15. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results.

  16. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    PubMed Central

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  17. Novel Mechanism for Scavenging of Hypochlorite Involving a Periplasmic Methionine-Rich Peptide and Methionine Sulfoxide Reductase

    PubMed Central

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; Carlson, Hans K.; Wetmore, Kelly M.; Price, Morgan N.; Iavarone, Anthony T.; Deutschbauer, Adam M.; Arkin, Adam P.

    2015-01-01

    ABSTRACT Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. PMID:25968643

  18. Polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and thymidylate synthase (TYMS) in multiple myeloma risk.

    PubMed

    Lima, Carmen S P; Ortega, Manoela M; Ozelo, Margareth C; Araujo, Renato C; De Souza, Cármino A; Lorand-Metze, Irene; Annichino-Bizzacchi, Joyce M; Costa, Fernando F

    2008-03-01

    We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

  19. Effect of Methionine Restriction on Bone Density and NK Cell Activity

    PubMed Central

    Dong, Jingming

    2016-01-01

    Methionine restriction (MR) is proven to increase the lifespan; and it also affects the bone density and the innate immune system. The aim of this study is to explore the effect of methionine restriction on bone density and natural killer (NK) cells. C57BL/6J mice were subjected to either basal diet (BD, containing 0.80% methionine) or methionine-restricted diet (containing 0.14% methionine). Mice with MR diet displayed reduced bone mass and decrease in the cytotoxicity of NK from the spleen, compared to BD animals. Also, mice with MR diet had an inferior body weight (P < 0.05) and higher plasma levels of adiponectin and FGF21 (P < 0.05) but lower concentrations of leptin and IGF-1 (P < 0.05). Overall, the investigation shows that methionine affects bone density and NK cell cytotoxicity. PMID:27882323

  20. Microbial Decomposition of Methionine and Identity of the Resulting Sulfur Products1

    PubMed Central

    Segal, William; Starkey, Robert L.

    1969-01-01

    Various bacteria, actinomycetes, and filamentous fungi decomposed methionine, but only certain aerobic bacteria isolated from soil decomposed it in the absence of other organic substrates. These bacteria could grow on methionine as the only organic substrate and source of nitrogen and sulfur. Methionine was first deaminated and then demethiolated with production of methanethiol, part of which was oxidized to dimethyl disulfide. The amount of methanethiol that was oxidized varied with different cultures. A bacterial culture initially unable to grow on methionine developed capacity to do this in a medium which contained methionine and other growth substrates. The two sulfur products, methanethiol and dimethyl disulfide, are volatile and escaped from the media, resulting in a decrease in the sulfur content proportional to the amount of methionine decomposed. PMID:5788717

  1. Synthesis of specific SPECT-radiopharmaceutical for tumor imaging based on methionine: 99mTc-DTPA-bis(methionine).

    PubMed

    Hazari, Puja Panwar; Shukla, Gauri; Goel, Vijay; Chuttani, Krishna; Kumar, Nitin; Sharma, Rajnish; Mishra, Anil Kumar

    2010-02-17

    Methionine-diethylenetriaminepentaaceticacid-methionine [DTPA-bis(Met)] was synthesized by covalently conjugating two molecules of methionine (Met) to DTPA and was labeled with (99m)Tc in high radiochemical purity and specific activity (166-296 MBq/micromol). Kinetic analysis showed K(m) of 12.95 +/- 3.8 nM and a maximal transport rate velocity (V(max)) of 80.35 +/- 0.42 pmol microg protein(-1) min(-1) of (99m)Tc-DTPA-bis(Met) in U-87MG cells. DTPA-bis(Met) had dissociation constants (K(d)) of 0.067 and 0.077 nM in U-87MG and BMG, respectively. (35)S-methionine efflux was trans-stimulated by (99m)Tc-labeled DTPA conjugate demonstrating concentrative transport. The blood kinetic studies showed fast clearance with t(1/2) (F) = 36 +/- 0.5 min and t(1/2) (S) = 5 h 55 min +/- 0.85 min. U-87MG and BMG tumors saturated at approximately 2000 +/- 280 nmol/kg of (99m)Tc-DTPA-bis(Met). Initial rate of transport of (99m)Tc-DTPA-bis(Met) in U-87MG tumor was found to be 4.68 x 10(-4) micromol/kg/min. The tumor (BMG cell line, malignant glioma) grafted in athymic mice were readily identifiable in the gamma images. Semiquantitative analysis from region of interest (ROI) placed over areas counting average counts per pixel with maximum radiotracer uptake on the tumor was found to be 11.05 +/- 3.99 and compared ROI with muscle (0.55 +/- 0.13). The tumor-to-contralateral muscle tissue ratio of (99m)Tc-DTPA-bis(Met) was found to be 23 +/- 3.3. Biodistribution revealed significant tumor uptake and good contrast in the U-87MG, BMG, and EAT tumor-bearing mice. In clinical trials, the sensitivity, specificity, and positive predictive values were found to be 87.8%, 92.8%, and 96.6%, respectively. (99m)Tc-DTPA-bis(Met) showed excellent tumor targeting and has promising utility as a SPECT-radiopharmaceutical for imaging methionine-dependent human tumors and to quantify the ratio of MET(+)/HCY(-).

  2. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway.

    PubMed

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A; Ralser, Markus

    2016-04-01

    Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway.

  3. Diversity of L-methionine catabolism pathways in cheese-ripening bacteria.

    PubMed

    Bonnarme, P; Psoni, L; Spinnler, H E

    2000-12-01

    Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum. The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.

  4. Uptake of carbon-11-methionine and fluorodeoxyglucose in non-Hodgkin's lymphoma: A PET study

    SciTech Connect

    Leskinen-Kallio, S.; Ruotsalainen, U.; Nagren, K.T.; Teraes, M.J.; Joensuu, H. )

    1991-06-01

    Uptake of L-(methyl-11C)methionine (11C-methionine) and (18F)-2-fluoro-2-deoxy-D-glucose (FDG) was studied with PET in 14 patients with non-Hodgkin's lymphomas. The low molecular weight fraction of venous plasma separated by fast gel filtration was used as the input function for 11C-methionine studies, and tracer accumulation was analyzed according to Patlak and Gjedde. The average uptake rate of 11C-methionine was 0.0775 {plus minus} 0.0245 min-1 (s.d.) and of FDG 0.0355 {plus minus} 0.0293 min-1, 11C-methionine uptake rate being significantly higher than that of FDG (p less than 0.01). Carbon-11-methionine accumulated strongly in all but one of the lymphomas. FDG accumulated clearly in lymphomas of high-grade malignancy, whereas two intermediate- and three low-grade malignant lymphomas had a poor uptake rate. The tumor/plasma ratio of both 11C-methionine and FDG increased faster in high and intermediate-grade lymphomas than in low-grade lymphomas, but there was considerable overlap between the histologic grades. Carbon-11-methionine seems to be preferable in detecting tumors, while FDG was superior to 11C-methionine in distinguishing the high-grade malignant lymphomas from the other grades.

  5. Oxidation of methionine residues in polypeptide ions via gas-phase ion/ion chemistry.

    PubMed

    Pilo, Alice L; McLuckey, Scott A

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M + H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  6. Influence of protein level and supplemental methionine in practical rations for young endangered masked bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1982-01-01

    A study was conducted to examine the protein requirement of young endangered masked Bobwhite quail (Colinus virginianus ridgwayi). Five practical starting rations containing 24 to 32% protein were fed alone and supplemented with methionine for 5 weeks. Supplemental methionine significantly improved growth of quail fed diets containing 24 and 26% protein. Increasing the protein level improved growth of quail fed unsupplemented diets but did not do so when diets contained supplemental methionine. A methionine-supplemented ration containing 24% protein appeared adequate for supporting rapid growth of masked Bobwhite quail.

  7. Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

    NASA Astrophysics Data System (ADS)

    Pilo, Alice L.; McLuckey, Scott A.

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M + H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to `label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  8. Oxidation of Methionine Residues in Polypeptide Ions via Gas-Phase Ion/Ion Chemistry

    PubMed Central

    Pilo, Alice L.; McLuckey, Scott A.

    2014-01-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach to varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M+H+O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side-chain. In the case of methionine containing peptides, the [M+H+O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M+H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to ‘label’ methionine residues in polypeptides in the gas-phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications. PMID:24671696

  9. Response of growing goslings to dietary supplementation with methionine and betaine.

    PubMed

    Yang, Z; Wang, Z Y; Yang, H M; Zhao, F Z; Kong, L L

    2016-12-01

    An experiment with a 2 × 3 factorial design with two concentrations of dietary betaine (0 and 600 mg/kg) and three dietary concentrations of methionine (0, 600 and 1200 mg/kg) was conducted using goslings to estimate growth, nutrient utilisation and digestibility of amino acids from 21 to 70 d of age. Three hundred geese were randomised at 18 d of age into 6 groups with 5 replicates per treatment and 10 geese per replicate. Increasing dietary concentrations of methionine gave a linear increase in body weight and average daily gain. The coefficient of crude fat retention increased as dietary methionine increased and there was a significant non-linear response to increasing dietary methionine. Similarly, increasing supplemental methionine gave linear increases in the digestibility of methionine and cysteine. The results of this study indicated that optimal dietary supplementation of methionine could increase growth performance and methionine and cysteine utilisation in growing goslings. Betaine supplementation had no apparent sparing effect on methionine needs for growth performance, but did improve the apparent cysteine digestibility.

  10. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway

    PubMed Central

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A.

    2016-01-01

    Abstract Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway. Antioxid. Redox Signal. 24, 543–547. PMID:26596469

  11. Methionine kinetics in adult men: effects of dietary betaine on L-(2H3-methyl-1-13C)methionine

    SciTech Connect

    Storch, K.J.; Wagner, D.A.; Young, V.R. )

    1991-08-01

    The effects of a daily 3-g supplement of betaine on kinetic aspects of L-(2H3-methyl-1-13C)methionine (MET) metabolism in healthy young adult men were explored. Four groups of four subjects each were given a control diet, based on an L-amino acid mixture supplying 29.5 and 21.9 mg.kg-1.d-1 of L-methionine and L-cystine for 4 d before the tracer study, conducted on day 5 during the fed state. Two groups received the control diet and two groups received the betaine supplement. Tracer was given intravenously (iv) or orally. The transmethylation rate of MET (TM), homocysteine remethylation (RM), and oxidation of methionine were estimated from plasma methionine labeling and 13C enrichment of expired air. RM tended to increase (P = 0.14) but the TM and methionine oxidation were significantly (P less than 0.05) higher after betaine supplementation when estimated with the oral tracer. No differences were detected with the intravenous tracer. Methionine concentration in plasma obtained from blood taken from subjects in the fed state was higher (P less than 0.01) with betaine supplementation. These results suggest that excess methyl-group intake may increase the dietary requirement for methionine.

  12. Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria†

    PubMed Central

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of l-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix. PMID:9726877

  13. Insights into the reactivation of cobalamin-dependent methionine synthase

    SciTech Connect

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.

    2009-12-10

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every {approx}2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH{sup CT}) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH{sup CT} ({sub s-s}MetH{sup CT}) that offer further insight into the reactivation of MetH. The structure of {sub s-s}MetH{sup CT} with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of {sub s-s}MetH{sub CT} with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.

  14. Insights into the reactivation of cobalamin-dependent methionine synthase

    PubMed Central

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.

    2009-01-01

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every ≈2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetHCT) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetHCT (s-sMetHCT) that offer further insight into the reactivation of MetH. The structure of s-sMetHCT with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of s-sMetHCT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation. PMID:19846791

  15. Pyridinylquinazolines Selectively Inhibit Human Methionine Aminopeptidase-1 in Cells

    PubMed Central

    Zhang, Feiran; Bhat, Shridhar; Gabelli, Sandra B.; Chen, Xiaochun; Miller, Michelle S.; Nacev, Benjamin A.; Cheng, Yim Ling; Meyers, David J.; Tenney, Karen; Shim, Joong Sup; Crews, Phillip; Amzel, L. Mario; Ma, Dawei; Liu, Jun O.

    2013-01-01

    Methionine aminopeptidases (MetAPs) which remove the initiator methionine from nascent peptides are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1–4). But all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1–4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells. PMID:23634668

  16. Methionine deficiency and hepatic injury in a dietary steatohepatitis model.

    PubMed

    Oz, Helieh S; Chen, Theresa S; Neuman, Manuela

    2008-03-01

    Methionine (Meth) is an essential amino acid involved in DNA methylation and glutathione biosynthesis. We examined the effect of Meth on the development of steatohepatitis. Rats were fed (five weeks) amino acid-based Meth-choline-sufficient (A-MCS) or total deficient (MCD) diets and gavaged daily (two weeks) with vehicle (B-vehicle/MCD), or Meth replacement (C-Meth/MCD). To assess the effect of short-term deficiency, after three weeks one MCS group was fed a deficient diet (D-MCS/MCD). Animals fed the deficient diet for two weeks lost (29%) weight and after five weeks weighed one third as much as those on the sufficient diet, and also developed anemia (P < 0.01). Hepatic transaminases progressively increased from two to five weeks (P < 0.01), leading to severe hepatic pathology. Meth administration normalized hematocrit, improved weight (P < 0.05), and suppressed abnormal enzymes activities (P < 0.01). Meth administration improved blood and hepatic glutathione (GSH), S-adenosylmethionine (SAMe), and hepatic lesions (P < 0.01). The deficient diet significantly upregulated proinflammatory and fibrotic genes, which was ameliorated by Meth administration. These data support a pivotal role for methionine in the pathogenesis of the dietary model of Meth-choline-deficient (MCD) steatohepatitis (NASH).

  17. Metabolic Fate of Cysteine and Methionine in Rumen Digesta

    PubMed Central

    Nader, C. J.; Walker, D. J.

    1970-01-01

    Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35S and 14C, it was observed that a large proportion of the 35S appeared in the sulfide pool and of the 14C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14C to 35S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 35S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources. PMID:5485079

  18. Pyridinylquinazolines selectively inhibit human methionine aminopeptidase-1 in cells.

    PubMed

    Zhang, Feiran; Bhat, Shridhar; Gabelli, Sandra B; Chen, Xiaochun; Miller, Michelle S; Nacev, Benjamin A; Cheng, Yim Ling; Meyers, David J; Tenney, Karen; Shim, Joong Sup; Crews, Phillip; Amzel, L Mario; Ma, Dawei; Liu, Jun O

    2013-05-23

    Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.

  19. Ontogeny of methionine utilization and splanchnic uptake in critically ill children

    USDA-ARS?s Scientific Manuscript database

    To determine the rates of methionine splanchnic uptake and utilization in critically ill pediatric patients, we used two kinetic models: the plasma methionine enrichment,and the "intracellular" homocysteine enrichment. Twenty-four patients, eight infants, eight children, and eight adolescents, were ...

  20. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    NASA Astrophysics Data System (ADS)

    Bergès, J.; Trouillas, P.; Houée-Levin, C.

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH- elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S∴X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  1. From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span.

    PubMed

    McIsaac, R Scott; Lewis, Kaitlyn N; Gibney, Patrick A; Buffenstein, Rochelle

    2016-01-01

    Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span. © 2016 New York Academy of Sciences.

  2. Influence of dietary methionine on the metabolism of selenomethionine in rats

    SciTech Connect

    Butler, J.A.; Beilstein, M.A.; Whanger, P.D. )

    1989-07-01

    To determine the influence of methionine on selenomethionine (SeMet) metabolism, weanling male rats were fed for 8 wk a basal diet marginally deficient in sulfur amino acids, containing 2.0 micrograms selenium (Se)/g as DL-SeMet and supplemented with 0, 0.3, 0.6 or 1.2% DL-methionine. Increased dietary methionine caused decreased selenium deposition in all tissues examined but increased glutathione peroxidase activity in testes, liver and lungs. A positive correlation was found between dietary methionine and the calculated percentage of selenium associated with GSHPx. In a second experiment, {sup 75}SeMet was injected into weanling male rats which had been fed the basal diet containing 2.0 micrograms selenium as DL-SeMet with or without the addition of 1.0% methionine. The selenoamino acid content of tissues and the distribution of {sup 75}Se in erythrocyte proteins were determined. In comparison to the rats fed the basal diet without added methionine, significantly more {sup 75}Se-selenocysteine was found in liver and muscle, more {sup 75}Se was found in erythrocyte GSHPx and less {sup 75}Se was found in erythrocyte hemoglobin of rats fed 1.0% methionine. These data suggest that methionine diverts SeMet from incorporation into general proteins and enhances its conversion to selenocysteine for specific selenium-requiring proteins, such as GSHPx.

  3. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    EPA Science Inventory

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver Cytosol
    Shan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas
    S-adenosyl-L-methionine (AdoMet): ar...

  4. [Utility of 11C-methionine PET/CT in neuro-oncology].

    PubMed

    Casas Parera, Ignacio; Igirio Gamero, Jorge L; Blumenkrantz, Yamila; Bruno, Gabriel; Báez, Alejandra; Tafur Canabal, José G; Báez, Mariana; Kuchkaryan, Valeria

    2013-01-01

    Positron emission tomography (PET) with 11C-methionine (11C-methionine PET/CT) is a new technique used to evaluate primary central nervous system (CNS) tumors. We describe our experience regarding the first 4 patients with glial tumors and 11C-methionine PET/CT. This is a descriptive, observational and prospective study of 4 patients between 38-50 years of age, with different gliomas (WHO classification). MRI and 11C-methionine PET/CT were performed in all cases. Case 1, gliomatosis cerebri grade II post-radiotherapy. Case 2, oligodendroglioma grade II diagnosed and treated with radiotherapy in 1993. Case 3, glioblastoma grade IV post-radiotherapy + temozolomide. Case 4, anaplastic oligoastrocytoma grade III post-radiotherapy + temozolomide. The pattern of 11C-methionine uptake compared with MRI showed tumor progression in cases 1, 3 and 4, and in case 2 showed uptake although the final diagnosis was pseudoprogression. Unlike 18fluordeoxiglucose PET/TC, 11C-methionine uptake in normal brain tissue and pseudoprogression is low, and gliomas are displayed as metabolically active areas. The 11C-methionine PET/CT provided valuable information on the tumoral behavior and extension, although in one case presented did not differentiate tumor progression from pseudoprogression. 11C-methionine PET/CT could be a useful tool in the study and follow-up to patients with gliomas.

  5. Interactive effects of selenium, methionine, and dietary protein on survival, growth, and physiology in mallard ducklings

    USGS Publications Warehouse

    Hoffman, D.J.; Sanderson, C.J.; LeCaptain, L.J.; Cromartie, E.; Pendleton, G.W.

    1992-01-01

    Concentrations of over 100 ppm (100 mg/kg) selenium (Se) have been found in aquatic food chains associated with irrigation drainwater. Both quantity and composition of dietary protein for wild ducklings may vary in selenium-contaminated environments. Day-old mallard (Anas platyrhynchos) ducklings received one of the following diets containing 22% protein: unsupplemented (controls), 15 ppm Se (as selenomethionine), 60 ppm Se, methionine supplemented, 15 ppm Se with methionine supplement, or 60 ppm Se with methionine supplement. In a second concurrent experiment the above sequence was repeated with a protein-restricted (11%) but isocaloric diet. In a third concurrent experiment all ducklings received 44% protein with 0, 15, or 60 ppm Se added. After 4 weeks, blood and tissue samples were collected for biochemical and histological examination. With 22% protein and 60 ppm Se in the diet, duckling survival and growth was reduced and histopathological lesions of the liver occurred. Antagonistic interactive effects occurred between supplementary methionine and Se, including complete to partial alleviation of the following Se effects by methionine: mortality, hepatic lesions, and altered glutathione and thiol status. With 11% protein, growth of controls was less than that with 22% protein, Se (60 ppm) caused 100% mortality, and methionine supplementation, although protective afforded less protection than it did with 22% protein. With 44% protein, ducklings experienced physiological stress, and Se was more toxic than with methionine-supplemented 22% protein. These findings suggest the potential for antagonistic effects of Se, methionine, and protein on duckling survival and physiology.

  6. [In vitro protective effect of methionine against cisplatin's damage to the cochlear hair cell of mice].

    PubMed

    Xue, Chan; Zhou, Yong-Qing; Gao, Hai-Tao; Ma, Ying-Yu; Wang, Na; Qu, Yan

    2011-02-01

    To establish an in vitro model of mouse cochlear basilar membrane impairment using cisplatin, and observe the protective effect of methionine on the hair cells. The cochlear basilar membrane samples of thirty two Kunming mice were harvested on the 2nd day after birth and randomly divided into four groups. Each group had 16 samples. Overnight preincubation the cochlear organ followed by appropriate treatment respectively as follows: the serum-free culture medium, the serum-free culture medium with methionine and cisplatin, the cisplatinum-containing serum-free culture medium, and the methionine-containing serum-free culture medium. The protective effect of methionine for injury of cochlea hair cells induced by cisplatin was observed by myosin-VI immunofluorescence, light microscopy, laser confocal scanning microscope and hair cells counting. The outer hair cells (OHC) and inner hair cells (IHC) of control group and methionine group were not damaged. The outer and inner hair cells of cisplatin group were damaged in various degree, and had remarkable difference compared with control group and methionine group (P < 0.05). The outer hair cells and inner hair cells of cisplatin + methionine group were damaged less than the cisplatin group with remarkable difference (t(IHC) = 3.929, t(OHC) = 8.582, P < 0.05). Cisplatinum could damage the cochlear hair cells of the basal membrane in Kunming mice. Methionine might protect against cisplatin's damage on the cochlear hair cells.

  7. Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

    SciTech Connect

    Kiene, R.P.; Visscher, P.T.

    1987-10-01

    Anoxic salt marsh sediments were amended with DL-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 ..mu..M) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO/sub 2/ and only small amounts of CH/sub 4/. At higher concentrations of methionine, the proportion of the methyl-sulfur groups converted to CH/sub 4/ increased.

  8. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Methionine hydroxy analog and its calcium salts. 582.5477 Section 582.5477 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a...

  9. Methionine residues around phosphorylation sites are preferentially oxidized in vivo under stress conditions

    PubMed Central

    Veredas, Francisco J.; Cantón, Francisco R.; Aledo, J. Carlos

    2017-01-01

    Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mechanism coupling oxidative signals to changes in protein function remains unknown. An appealing hypothesis is that methionine oxidation might serve as a rheostat to control phosphorylation. To investigate this potential crosstalk between phosphorylation and methionine oxidation, we have addressed the co-occurrence of these two types of modifications within the human proteome. Here, we show that nearly all (98%) proteins containing oxidized methionine were also phosphoproteins. Furthermore, phosphorylation sites were much closer to oxidized methionines when compared to non-oxidized methionines. This proximity between modification sites cannot be accounted for by their co-localization within unstructured clusters because it was faithfully reproduced in a smaller sample of structured proteins. We also provide evidence that the oxidation of methionine located within phosphorylation motifs is a highly selective process among stress-related proteins, which supports the hypothesis of crosstalk between methionine oxidation and phosphorylation as part of the cellular defence against oxidative stress. PMID:28079140

  10. Methionine residues around phosphorylation sites are preferentially oxidized in vivo under stress conditions.

    PubMed

    Veredas, Francisco J; Cantón, Francisco R; Aledo, J Carlos

    2017-01-12

    Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mechanism coupling oxidative signals to changes in protein function remains unknown. An appealing hypothesis is that methionine oxidation might serve as a rheostat to control phosphorylation. To investigate this potential crosstalk between phosphorylation and methionine oxidation, we have addressed the co-occurrence of these two types of modifications within the human proteome. Here, we show that nearly all (98%) proteins containing oxidized methionine were also phosphoproteins. Furthermore, phosphorylation sites were much closer to oxidized methionines when compared to non-oxidized methionines. This proximity between modification sites cannot be accounted for by their co-localization within unstructured clusters because it was faithfully reproduced in a smaller sample of structured proteins. We also provide evidence that the oxidation of methionine located within phosphorylation motifs is a highly selective process among stress-related proteins, which supports the hypothesis of crosstalk between methionine oxidation and phosphorylation as part of the cellular defence against oxidative stress.

  11. A NOVEL S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE FROM RAT LIVER CYTOSOL

    EPA Science Inventory

    A Novel S-Adenosyl-L-methionine: Arsenic(III) Methyltransferase from Rat Liver Cytosol
    Shan Lin, Qing Shi, F. Brent Nix, Miroslav Styblo, Melinda A. Beck, Karen M. Herbin-Davis, Larry L. Hall, Josef B. Simeonsson, and David J. Thomas
    S-adenosyl-L-methionine (AdoMet): ar...

  12. 21 CFR 582.5477 - Methionine hydroxy analog and its calcium salts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Methionine hydroxy analog and its calcium salts. 582.5477 Section 582.5477 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Nutrients and/or Dietary Supplements 1 § 582.5477 Methionine hydroxy analog and its calcium salts. (a...

  13. Retinol status and expression of retinol-related proteins in methionine-choline deficient rats.

    PubMed

    Miyazaki, Hiroshi; Takitani, Kimitaka; Koh, Maki; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2014-01-01

    Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

  14. Genetic and biochemical differences in populations bred for extremes in maize grain methionine concentration

    PubMed Central

    2014-01-01

    Background Methionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Using divergent selection, genetically related populations with extreme differences in grain methionine content were produced. In order to better understand the molecular mechanisms controlling grain methionine content, we examined seed proteins, transcript levels of candidate genes, and genotypes of these populations. Results Two populations were selected for high or low methionine concentration for eight generations and 40 and 56% differences between the high and low populations in grain methionine concentration were observed. Mean values between the high and low methionine populations differed by greater than 1.5 standard deviations in some cycles of selection. Other amino acids and total protein concentration exhibited much smaller changes. In an effort to understand the molecular mechanisms that contribute to these differences, we compared transcript levels of candidate genes encoding high methionine seed storage proteins involved in sulfur assimilation or methionine biosynthesis. In combination, we also explored the genetic mechanisms at the SNP level through implementation of an association analysis. Significant differences in methionine-rich seed storage protein genes were observed in comparisons of high and low methionine populations, while transcripts of seed storage proteins lacking high levels of methionine were unchanged. Seed storage protein levels were consistent with transcript levels. Two genes involved in sulfur assimilation, Cys2 and CgS1 showed substantial differences in allele frequencies when two selected populations were compared to the starting populations. Major genes identified across cycles of selection by a high-stringency association analysis included dzs18, wx, dzs10, and zp27. Conclusions We

  15. Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.

    PubMed

    Borrego, Stacey L; Fahrmann, Johannes; Datta, Rupsa; Stringari, Chiara; Grapov, Dmitry; Zeller, Michael; Chen, Yumay; Wang, Ping; Baldi, Pierre; Gratton, Enrico; Fiehn, Oliver; Kaiser, Peter

    2016-01-01

    The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8. Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential. This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive

  16. Thioredoxin-dependent Redox Regulation of Cellular Signaling and Stress Response through Reversible Oxidation of Methionines

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2011-06-01

    Generation of reactive oxygen species (ROS) is a common feature of many forms of stress to which plants are exposed. Successful adaptation to changing environmental conditions requires sensitive sensors of ROS such as protein-bound methionines that are converted to their corresponding methionine sulfoxides, which in turn can influence cellular signaling pathways. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress response in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the sensitivity of individual methionines within plant and animal calmodulin to ROS, the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes.

  17. [Synthesis and spectral characteristics of selenium-chelated methionine with hexagon].

    PubMed

    Jin, Li-E; Wang, Xiao-Juan; Shen, Fan-Fan; Cao, Qing

    2013-04-01

    Using methionine and anhydrous ethanol as raw material, p-toluene sulfonic acid as catalyst, and benzene as a carrying water agent, methionine ester was first synthesized. Then, selenium-chelated methionine was prepared through the reaction of methionine ester with sodium selenite by a certain proportion, settling and crystallizing at lower temperature. The xi potential of the resultant was determined by micro-electrophoresis, through which the isoelectric point was calculated. Based on the principle of isoelectric point, it was separated and purified. The spectral properties of the resultant were analyzed by infrared spectrum, utraviolet spectrum, X ray diffraction analysis and 1H-NMR, from which we got the information of the resultant structure that has a hexagon composing of sulfur atom of methionine, nitrogen of amine and central selenium ion of four-valence.

  18. Methionine deficiency reduces autophagy and accelerates death in intestinal epithelial cells infected with enterotoxigenic Escherichia coli.

    PubMed

    Tang, Yulong; Tan, Bie; Xiong, Xia; Li, Fengna; Ren, Wenkai; Kong, Xiangfeng; Qiu, Wei; Hardwidge, Philip R; Yin, Yulong

    2015-10-01

    Infections by enterotoxigenic Escherichia coli (ETEC) result in large economic losses to the swine industry worldwide. Dietary supplementation with amino acids has been considered as a potential mechanism to improve host defenses against infection. The goal of this study was to determine whether methionine deprivation alters ETEC interactions with porcine intestinal epithelial cells. IPEC-1 cells were cultured in media with or without L-methionine. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. Methionine deprivation inhibited IPEC-1 cell autophagic responses, suggesting that the increased cytotoxicity of ETEC to methionine-deprived IPEC-1 cells might be due to defects in autophagy.

  19. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-02

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  20. The role of methionine in the intracellular accumulation and function of folates

    SciTech Connect

    Scott, J.M.; McKenna, B.; McGing, P.; Molloy, A.; Dinn, J.; Weir, D.G.

    1983-01-01

    It is suggested that mammalian cells have evolved to respond to methionine deficiency since in such circumstances vital methylation reactions are put at risk, due to decreased levels of S-adenosyl-methionine. Decreased cellular homocysteine, as a result of decreased methionine, would also restrict cell division by decreased conversion of plasma 5-CH3-H/sub 4/PteGlu into intracellular polyglutamates. Cobalamin deficiency, either nutritional or due to exposure to the Co(I)cobalamin inactivating agent nitrous oxide, prevents the demethylation of 5-CH3-H/sub 4/PteGlu, which even in the presence of adequate amounts of homocysteine and methionine prevents rapidly proliferating cells from converting enough of the plasma 5-CH3-H/sub 4/ PteGlu into folylpolyglutamate forms to permit normal DNA biosynthesis and cell replication. This, together with the trapping of the cellular folate cofactors in the 5-CH3-H/sub 4/PteGlu form, results in megaloblastic changes occurring in tissues such as the marrow. The vital role of the methylation reactions was demonstrated by exposing monkeys to nitrous oxide which inactivated their methionine synthetase. The resultant ataxia and severe demyelination was prevented and diminished by methionine supplementation. When methionine synthetase was similarly inactivated in mice it was shown that while 5-CH3-H/sub 4/PteGlu enters mammalian cells, it is not converted into a polyglutamyl form and subsequently leaves the cell unmetabolised. In similar experiments in rats methionine was found to have only a small effect in restoring folylpolyglutamate biosynthesis. It was found that a decrease in the deoxythymidine salvage pathway by methionine has led others to the mistaken conclusion that methionine has an 'anti-folate' effect in bone marrow, i.e. that it decreases folate availability for thymidylate synthetase.

  1. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    PubMed

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

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

    PubMed

    Ferchichi, M; Hemme, D; Nardi, M

    1987-09-01

    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

  3. Effects of Dietary Methionine Levels on Choline Requirements of Starter White Pekin Ducks

    PubMed Central

    Wen, Z. G.; Tang, J.; Xie, M.; Yang, P. L.; Hou, S. S.

    2016-01-01

    A 2×5 factorial experiment, using 2 dietary methionine levels (0.28% and 0.48%) and 5 dietary choline levels (0, 394, 823, 1,239, and 1,743 mg/kg), was conducted to study the effects of dietary methionine status on choline requirements of starter white Pekin ducks from 7 to 28 days of age. Four hundred eighty 7-d-old male White Pekin ducks were randomly allotted to ten dietary treatments, each containing 6 replicate pens with 8 birds per pen. At 28 d of age, weight gain, feed intake, and feed/gain were measured and the legs of all ducks from each pen were examined for incidence of perosis. Perosis and growth depression were observed in choline-deficient ducks and supplementation of choline reduced perosis and significantly increased weight gain and feed intake regardless of dietary methionine levels (p<0.05). In addition, significant positive effects of dietary methionine supplementation on weight gain, feed intake, and feed/gain were observed at any choline level (p<0.05). Supplementation of 1,743 mg/kg choline in diets alleviated the depression of weight gain and feed intake caused by methionine deficiency at 0.28% methionine level. The interaction between choline and methionine influenced weight gain and feed intake of ducks (p<0.05). At 0.28% methionine level, 1,743 mg/kg choline group caused 4.92% and 3.23% amount of improvement in weight gain and feed intake compared with 1,239 mg/kg choline group, respectively. According to the broken-line regression, the choline requirements of starter Pekin ducks for weight gain and feed intake were 1,472 and 1,424 mg/kg at 0.28% methionine level and 946 and 907 mg/kg at 0.48% methionine level, respectively. It suggested the choline recommendations of starter Pekin ducks on a semi-purified diet were 1448 mg/kg at 0.28% methionine level and 927 mg/kg at 0.48% methionine level, respectively. Compared with the adequate methionine level, menthionine deficiency markedly increased the choline requirements of ducks. PMID

  4. 11C-Methionine PET of Myocardial Inflammation in a Rat Model of Experimental Autoimmune Myocarditis.

    PubMed

    Maya, Yoshifumi; Werner, Rudolf A; Schütz, Claudia; Wakabayashi, Hiroshi; Samnick, Samuel; Lapa, Constantin; Zechmeister, Christina; Jahns, Roland; Jahns, Valérie; Higuchi, Takahiro

    2016-12-01

    Myocarditis represents a major cause of dilated cardiomyopathy and sudden cardiac death in younger adults. Currently, definitive diagnosis of myocarditis requires endomyocardial biopsy, which is highly invasive and has the drawback of variable sensitivity due to inherent sampling error. Therefore, reliable noninvasive methods to detect and monitor cardiac inflammation are clinically relevant. In this study, we explored the potential of radiolabeled methionine to assess myocardial inflammatory activity in a rat model of experimental autoimmune myocarditis (EAM). Autoimmune myocarditis was induced by immunizing Lewis rats twice with porcine cardiac myosin and Freund complete adjuvant. Control animals were treated with adjuvant alone. Dual-tracer autoradiography was performed to assess (14)C-methionine uptake and to compare the distributions of (14)C-methionine versus (18)F-FDG. Hematoxylin and eosin staining and anti-CD68 macrophage staining were performed for histologic analysis. Additionally, cardiac (11)C-methionine PET was performed to evaluate the feasibility of in vivo imaging. (18)F-FDG PET was also conducted to compare the in vivo uptake of (11)C-methionine and (18)F-FDG. Multiple focal cardiac inflammatory lesions were histologically identified in myosin-immunized rats, whereas no cardiac lesions were observed in the controls. Autoradiographic images clearly showed a high-density accumulation of (14)C-methionine in inflammatory lesions of EAM rats, whereas no significant uptake was observed in the control animals. (14)C-methionine uptake was significantly higher in inflammatory lesions than in remote noninflammatory areas and control rat hearts. The distribution of (14)C-methionine correlated well with that of (18)F-FDG and with macrophage density. The contrast between inflammatory and noninflammatory areas was higher for (18)F-FDG than for (14)C-methionine (3.45 ± 0.68 vs. 2.07 ± 0.21, respectively; P < 0.05). In the PET imaging study, the regional (11)C-methionine

  5. Can betaine partially replace or enhance the effect of methionine by improving broiler growth and carcase characteristics?

    PubMed

    McDevitt, R M; Mack, S; Wallis, I R

    2000-09-01

    1. Growth rates and carcase characteristics were measured in male broiler chickens fed on a control diet deficient in methionine (c. 2.8 g/kg methionine) or a series of diets containing graded levels of betaine or DL-methionine or both additives. 2. We aimed to answer 2 main questions. First, can betaine replace part of the methionine in a broiler ration? Secondly is there a synergism between methionine and betaine? 3. Birds given the control diet or that supplemented only with betaine ate less, grew more slowly, had higher food convension ratio (FCR) and varied more in mass at 42 d than birds fed diets with DL-methionine. Adding 1.2 g/kg DL-methionine to the control ration produced the heaviest birds at 42 d (2500 g) with the 2nd heaviest breast muscle (366 g). 4. After correcting for treatment differences in body mass (analysis of convariance), birds fed on the control diet and the diet supplemented with betaine only, had relatively lighter breast muscles but relatively heavier abdominal fat pads than those of birds given diets supplemented with DL-methionine. However, adding betaine to diets containing added methionine further improved the relative breast muscle yield. 5. After correcting for differences in body mass between treatments, birds fed on diets containing most methionine had lighter viscera than birds fed diets deficient in methionine. This demonstrated gut plasticity, suggesting that the viscera enlarged to sequester methionine from low-methionine diets. 6. Our data refute the hypothesis that betaine can substitute for methionine in broilers fed diets that are marginally deficient in methionine plus cystine. However, betaine may improve carcase composition, especially breast meat yield.

  6. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    SciTech Connect

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

  7. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGES

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; ...

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

  8. Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences

    PubMed Central

    2015-01-01

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs. PMID:27162954

  9. Methionine restriction slows down senescence in human diploid fibroblasts.

    PubMed

    Kozieł, Rafał; Ruckenstuhl, Christoph; Albertini, Eva; Neuhaus, Michael; Netzberger, Christine; Bust, Maria; Madeo, Frank; Wiesner, Rudolf J; Jansen-Dürr, Pidder

    2014-12-01

    Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  10. Dietary methionine restriction inhibits prostatic intraepithelial neoplasia in TRAMP mice.

    PubMed

    Sinha, Raghu; Cooper, Timothy K; Rogers, Connie J; Sinha, Indu; Turbitt, William J; Calcagnotto, Ana; Perrone, Carmen E; Richie, John P

    2014-12-01

    Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P < 0.02). The reduction in PIN severity was associated with 46-64% reductions in cell proliferation rates (P < 0.02) and plasma IGF-1 levels (P < 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice. Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy. © 2014 Wiley Periodicals, Inc.

  11. Choline deprivation induces hyperhomocysteinemia in rats fed low methionine diets.

    PubMed

    Setoue, Minoru; Ohuchi, Seiya; Morita, Tatsuya; Sugiyama, Kimio

    2008-12-01

    To clarify the relationship between dietary choline level and plasma homocysteine concentration, the effects of choline deprivation on plasma homocysteine concentration and related variables were investigated in rats fed a standard (25%) casein (25C) diet or standard soybean protein (25S) diet. Using the 25S diet, the time-dependent effect of choline deprivation and the comparative effects of three kinds of lipotropes were also investigated. Feeding rats with the choline-deprived 25S diet for 10 d significantly increased plasma total homocysteine concentration to a level 2.68-times higher than that of the control group, whereas choline deprivation had no effect in rats fed the 25C diet. Increases in hepatic S-adenosylhomocysteine and homocysteine concentrations, decreases in hepatic betaine concentration and the activity of cystathionine beta-synthase, but not betaine-homocysteine S-methyltransferase, and fatty liver also occurred in rats fed the choline-deprived 25S diet. Plasma homocysteine concentration increased when rats were fed the choline-deprived 25S diet for only 3 d, and the increase persisted up to 20 d. The hyperhomocysteinemia induced by choline deprivation was effectively suppressed by betaine or methionine supplementation. Choline deprivation caused hyperhomocysteinemia also in rats fed a choline-deprived low (10%) casein diet. The results indicate that choline deprivation can easily induce prominent hyperhomocysteinemia when rats are fed relatively low methionine diets such as a standard soybean protein diet and low casein diet, possibly through the suppression of homocysteine removal by both remethylation and cystathionine formation. This hyperhomocysteinemia might be a useful model for investigating the role of betaine in the regulation of plasma homocysteine concentration.

  12. Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase

    SciTech Connect

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-09-07

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

  13. Catalysis and inhibition of Mycobacterium tuberculosis methionine aminopeptidase

    PubMed Central

    Lu, Jing-Ping; Chai, Sergio C.; Ye, Qi-Zhuang

    2010-01-01

    Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II) and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells, because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity towards the Fe(II)-form, the Mn(II)-form, or the Co(II)- and Ni(II)-forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues. PMID:20038112

  14. Effect of excessive methionine on the development of the cranial growth plate in newborn rats.

    PubMed

    Römer, Piero; Weingärtner, Jens; Desaga, Benjamin; Kubein-Meesenburg, Dietmar; Reicheneder, Claudia; Proff, Peter

    2012-09-01

    Methionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats. Twenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis. The weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group. In summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

    PubMed

    Zhu, Qingfu; El-Mergawy, Rabab G; Heinemann, Stefan H; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K E

    2013-09-01

    A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

  16. Methionine enhances the contractile activity of human colon circular smooth muscle in vitro.

    PubMed

    Choe, Eun Kyung; Moon, Jung Sun; Park, Kyu Joo

    2012-07-01

    Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 µM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 µM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and N(w)-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

  17. Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation.

    PubMed

    Aledo, Juan C; Cantón, Francisco R; Veredas, Francisco J

    2015-11-24

    Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants.

  18. Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

    PubMed Central

    Aledo, Juan C.; Cantón, Francisco R.; Veredas, Francisco J.

    2015-01-01

    Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

  19. Growth hormone signaling is necessary for lifespan extension by dietary methionine.

    PubMed

    Brown-Borg, Holly M; Rakoczy, Sharlene G; Wonderlich, Joseph A; Rojanathammanee, Lalida; Kopchick, John J; Armstrong, Vanessa; Raasakka, Debbie

    2014-12-01

    Growth hormone significantly impacts lifespan in mammals. Mouse longevity is extended when growth hormone (GH) signaling is interrupted but markedly shortened with high-plasma hormone levels. Methionine metabolism is enhanced in growth hormone deficiency, for example, in the Ames dwarf, but suppressed in GH transgenic mice. Methionine intake affects also lifespan, and thus, GH mutant mice and respective wild-type littermates were fed 0.16%, 0.43%, or 1.3% methionine to evaluate the interaction between hormone status and methionine. All wild-type and GH transgenic mice lived longer when fed 0.16% methionine but not when fed higher levels. In contrast, animals without growth hormone signaling due to hormone deficiency or resistance did not respond to altered levels of methionine in terms of lifespan, body weight, or food consumption. Taken together, our results suggest that the presence of growth hormone is necessary to sense dietary methionine changes, thus strongly linking growth and lifespan to amino acid availability.

  20. Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat.

    PubMed

    Wilson, Fiona A; Holtrop, Grietje; Calder, A Graham; Anderson, Susan E; Lobley, Gerald E; Rees, William D

    2012-06-15

    Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.

  1. Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A.

    PubMed

    Brock, Jonathan W C; Ames, Jennifer M; Thorpe, Suzanne R; Baynes, John W

    2007-01-15

    Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in

  2. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine.

    PubMed

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2016-09-23

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the

  3. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    NASA Astrophysics Data System (ADS)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2016-09-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  4. Formation of Methionine Sulfoxide during Glycoxidation and Lipoxidation of Ribonuclease A

    PubMed Central

    Brock, Jonathon WC; Ames, Jennifer M; Thorpe, Suzanne R; Baynes, John W

    2007-01-01

    Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: 1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; 2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29 > Met30 > Met13, with Met79 being resistant to oxidation; and 3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein’s overall exposure to oxidative stress from a variety of sources in

  5. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    NASA Astrophysics Data System (ADS)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2017-06-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  6. The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats.

    PubMed

    Amaral, Cátia Lira Do; Bueno, Rafaela de Barros E Lima; Burim, Regislaine Valéria; Queiroz, Regina Helena Costa; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

    2011-05-18

    Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet's effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet's effects on genomic stability and DNA methylation.

  7. Residue Phe112 of the Human-Type Corrinoid Adenosyltransferase (PduO) Enzyme of Lactobacillus reuteri Is Critical to the Formation of the Four-Coordinate Co(II) Corrinoid Substrate and to the Activity of the Enzyme

    SciTech Connect

    Mera, Paola E.; St. Maurice, Martin; Rayment, Ivan; Escalante-Semerena, Jorge C.; UW

    2009-06-08

    ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. These studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.

  8. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  9. 13C-methionine breath tests for mitochondrial liver function assessment.

    PubMed

    Candelli, M; Miele, L; Armuzzi, A; Nista, E C; Pignataro, G; Fini, L; Cazzato, I A; Zocco, M A; Bartolozzi, F; Gasbarrini, G; Grieco, A; Gasbarrini, A

    2008-01-01

    13C-methionine breath test has been proposed as a non-invasive tool for the assessment of human hepatic mithocondrial function. Two methionine breath labeled with 13C in differents point of his molecular structure have been used for breath test analisys. Aim of this study was to compare two differently 13C-labeled methionines in the evaluation of mitochondrial oxidation in basal conditions and after an acute oxidative stress. 15 healthy male subjects (mean age 30.5 +/- 3.1) received [methyl-13C]-methionine dissolved in water. Breath samples were taken at baseline and and 10, 20, 30, 45, 60, 75, 90, 105 and 120 minutes after the ingestion of the labeled substrate. Forthy-eight hours later, subjects underwent the same test 30 minutes after ethanol ingestion (0,3 g/kg of body weight). Seven-day later, subjects underwent breath test using (L-methionine-1-13COOH) as substrate, in basal condition and after ethanol ingestion. At basal condition, the cumulative percentage of 13CO2 recovered in breath during the test period (%cum-dose) was higher using L-methionine-1-13COOH than [methyl-13C]-methionine (10.25 +/- 1.0 vs 4.07 +/- 0.8; p < 0.01). After ethanol ingestion, % cum dose was significantly decreased at 60 and 120 minutes with both methionines (120 min: 10.25 +/- 1.0 vs 5.03% +/- 1.8; < 0.01 and 4.07 +/- 0.8 vs 2.16% +/- 0.9; p < 0.01, respectively). However, %cum-dose during L-methionine-1-13C-breath test was significantly lower than that observed during methyl-13C-methionine breath test (120 minutes: 5.03% +/- 1.8 vs 2.16% +/- 0.9; p < 0.01). In conclusion, breath test based on L-methionine-1-13COOH seems to show a greater reliability when compared to [methyl-13C]-methionine to assess mitochondrial function because a larger amount of labeled carbon that reaches the Krebs' cicle.

  10. The reaction of methionine with hydroxyl radical: reactive intermediates and methanethiol production.

    PubMed

    Spasojević, Ivan; Bogdanović Pristov, Jelena; Vujisić, Ljubodrag; Spasić, Mihajlo

    2012-06-01

    The mechanisms of reaction of methionine with hydroxyl radical are not fully understood. Here, we unequivocally show using electron paramagnetic resonance spin-trapping spectroscopy and GC-FID and GC-MS, the presence of specific carbon-, nitrogen- and sulfur-centered radicals as intermediates of this reaction, as well as the liberation of methanethiol as a gaseous end product. Taking into account the many roles that methionine has in eco- and biosystems, our results may elucidate redox chemistry of this amino acid and processes that methionine is involved in.

  11. Acute administration of methionine and/or methionine sulfoxide impairs redox status and induces apoptosis in rat cerebral cortex.

    PubMed

    Soares, Mayara Sandrielly Pereira; Viau, Cassiana Macagnan; Saffi, Jenifer; Costa, Marcelo Zanusso; da Silva, Tatiane Morgana; Oliveira, Pathise Souto; Azambuja, Juliana Hofstatter; Barschak, Alethéa Gatto; Braganhol, Elizandra; S Wyse, Angela T; Spanevello, Roselia Maria; Stefanello, Francieli Moro

    2017-07-04

    High plasma levels of methionine (Met) and its metabolites such as methionine sulfoxide (MetO) may occur in several genetic abnormalities. Patients with hypermethioninemia can present neurological dysfunction; however, the neurotoxicity mechanisms induced by these amino acids remain unknown. The aim of the present work was to study the effects of Met and/or MetO on oxidative stress, genotoxicity, cytotoxicity and to evaluate whether the cell death mechanism is mediated by apoptosis in the cerebral cortex of young rats. Forty-eight Wistar rats were divided into groups: saline, Met 0.4 g/Kg, MetO 0.1 g/Kg and Met 0.4 g/Kg + MetO 0.1 g/Kg, and were euthanized 1 and 3 h after subcutaneous injection. Results showed that TBARS levels were enhanced by MetO and Met+MetO 1 h and 3 h after treatment. ROS was increased at 3 h by Met, MetO and Met+MetO. SOD activity was increased in the Met group, while CAT was reduced in all experimental groups 1 h and 3 h after treatment. GPx activity was enhanced 1 h after treatment by Met, MetO and Met+MetO, however it was reduced in the same experimental groups 3 h after administration of amino acids. Caspase-3, caspase-9 and DNA damage was increased and cell viability was reduced by Met, MetO and Met+MetO at 3 h. Also, Met, MetO and Met+MetO, after 3 h, enhanced early and late apoptosis cells. Mitochondrial electrochemical potential was decreased by MetO and Met+MetO 1 h and 3 h after treatment. These findings help understand the mechanisms involved in neurotoxicity induced by hypermethioninemia.

  12. Biological efficacy and absorption of DL-methionine hydroxy analogue free acid compared to DL-methionine in chickens as affected by heat stress.

    PubMed

    Rostagno, H S; Barbosa, W A

    1995-05-01

    1. The net absorption and the biological efficacy of DL-methionine and of DL-methionine hydroxy analogue free acid (MHA-FA) were evaluated in chickens under heat stress. 2. In a growth assay, finishing broilers 21 to 42 d of age were fed on diets containing graded amounts of the two supplements; the basal diet was composed of practical ingredients. 3. From slope-ratio analysis, equimolar efficacy of MHA-FA relative to DL-methionine was determined to be 83% (confidence limits 61 to 115%) from weight gain responses, and 67% (47 to 91%) from food conversion responses. This indicates that the relative efficacy of MHA-FA is close to previous estimates of about 75% obtained under thermoneutral conditions. 4. In a balance study with caecectomised cockerels, net absorption (intake - excretion in faeces and urine) of DL-methionine and of MHA-FA, respectively, were determined to be 97.2 and 90.8%. The net absorption of MHA-FA was significantly lower than that of DL-methionine.

  13. Simultaneous determination of plasma total homocysteine and methionine by liquid chromatography-tandem mass spectrometry.

    PubMed

    Jiang, Yi; Mistretta, Brandon; Elsea, Sarah; Sun, Qin

    2017-01-01

    The sulfur-containing amino acid homocysteine is a cardiac risk factor and a biomarker for several inborn errors of metabolism in methionine synthesis. A simple LC-MS/MS method was developed and validated for determination of homocysteine and methionine in human plasma. Rapid separation was achieved using a reverse phase liquid chromatography. Mass spectrometry identification was performed in positive electrospray ionization mode for homocysteine and methionine. Accuracy, precision, linearity, recovery and sample stability were evaluated in the method validation. The test is applied in diagnosis of homocystinuria and monitoring total homocysteine levels. Moreover, simultaneous measurement of methionine helps in the differentiation of homocystinuria and some cobalamin disorders (such as cblC and cblD defects) without additional amino acid testing. Lastly, this assay is sensitive to detect reduced total homocysteine levels that are possibly seen in sulfocysteinuria and molybdenum cofactor deficiencies.

  14. Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

    PubMed

    Amarita, Felix; Yvon, Mireille; Nardi, Michele; Chambellon, Emilie; Delettre, Jerôme; Bonnarme, Pascal

    2004-12-01

    The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.

  15. Catalytic action of L-methionine gamma-lyase on selenomethionine and selenols.

    PubMed

    Esaki, N; Tanaka, H; Uemura, S; Suzuki, T; Soda, K

    1979-02-06

    We examined the catalytic action of L-methionine gamma-lyase (EC 4.4.1.11) on selenomethionine (2-amino-4-(methylseleno)butyric acid), methaneselenol, l-hexaneselenol, and benzeneselenol. The enzyme catalyzes alpha, gamma-elimination of selenomethionine to yield alpha-letobutyrate, ammonia, and methaneselenol, and also its gamma-replacement reaction with various thiols to produce S-substituted homocysteines. Selenomethionine is an even better substrate than methionine in alpha, gamma-elimination but is less effective in gamma-replacement. In addition, L-methionine gamma-lyase catalyzes gamma-replacement reaction of methionine and its derivatives with selenols to form the corresponding Se-substituted selenohomocysteines, although selenols are less efficient substituent donors than thiols. This is the first proven mechanism for the incorporation of selenium atom into amino acids.

  16. In Silico Analysis of Sequence-Structure-Function Relationship of the Escherichia coli Methionine Synthase.

    PubMed

    Kumar, Shiv; Bhagabati, Puja; Sachan, Reena; Kaushik, Aman Chandra; Dwivedi, Vivek Dhar

    2015-12-01

    The molecular evolution of various metabolic pathways in the organisms can be employed for scrutinizing the molecular aspects behind origin of life. In the present study, we chiefly concerned about the sequence-structure-function relationship between the Escherichia coli methionine synthase and their respective animal homologs by in silico approach. Using homology prediction technique, it was observed that only 79 animal species showed similarity with the E. coli methionine synthase. Also, multiple sequence alignment depicted only 25 conserved patterns between the E. coli methionine synthase and their respective animal homologs. Based on that, Pfam analysis identified the protein families of 22 conserved patterns among the attained 25 conserved patterns. Furthermore, the 3D structure was generated by HHpred and evaluated by corresponding Ramachandran plot specifying 93% of the ϕ and ψ residues angles in the most ideal regions. Hence, the designed structure was established as a good quality model for the full length of E. coli methionine synthase.

  17. Methionine and choline regulate the metabolic phenotype of a ketogenic diet.

    PubMed

    Pissios, Pavlos; Hong, Shangyu; Kennedy, Adam Richard; Prasad, Deepthi; Liu, Fen-Fen; Maratos-Flier, Eleftheria

    2013-01-01

    Low-carbohydrate ketogenic diets are commonly used as weight loss alternatives to low-fat diets, however the physiological and molecular adaptations to these diets are not completely understood. It is assumed that the metabolic phenotype of the ketogenic diet (KD) is caused by the absence of carbohydrate and high fat content, however in rodents the protein content of KD affects weight gain and ketosis. In this study we examined the role of methionine and choline in mediating the metabolic effects of KD. We have found that choline was more effective than methionine in decreasing the liver steatosis of KD-fed mice. On the other hand, methionine supplementation was more effective than choline in restoring weight gain and normalizing the expression of several fatty acid and inflammatory genes in the liver of KD-fed mice. Our results indicate that choline and methionine restriction rather than carbohydrate restriction underlies many of the metabolic effects of KD.

  18. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Karaki, Tsuyoshi; Shimizu, Akira; Kamei, Kaeko; Harada, Shigeharu; Nozaki, Tomoyoshi

    2008-08-01

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  19. Plasma methionine depletion and pharmacokinetic properties in mice of methionine γ-lyase from Citrobacter freundii, Clostridium tetani and Clostridium sporogenes.

    PubMed

    Morozova, E A; Anufrieva, N V; Davydov, D Zh; Komarova, M V; Dyakov, I N; Rodionov, A N; Demidkina, T V; Pokrovsky, V S

    2017-04-01

    PK studies were carried out after a single i.v. administration of 500 and 1000 U/kg by measuring of MGL activity in plasma samples. L-methionine concentration was measured by mass spectrometry. After single i.v. injection of 500U/kg the circulating T1/2 of enzymes in mice varies from 73 to 123min. The AUC0-tinf values determined for MGL 500U/kg from C. freundii, C. tetani and C. sporogenes are 8.21±0.28, 9.04±0.33 and 13.88±0.39U/(ml×h), respectively. Comparison of PK parameters of three MGL sources in the dose of 500U/kg indicated the MGL C. sporogenes to have better PK parameters: clearance 0.83(95%CI: 0.779-0.871) - was lower than C. tetanii 1.27(95%CI: 1.18-1.36) and C. freundii 1.39(95%CI: 1.30-1.49). Mice plasma methionine decreased to undetectable level 10min after MGL 1000 U/kg injection. After MGL C. sporogenes 500U/kg injection plasma methionine level completely omitted after 10min till 6h, assuming the sustainability of negligible levels of methionine (<5μM) in plasma of mice for about 6h. The recovery of methionine concentration showed the advantageous efficiency of MGL from C. sporogenes: 95% 0.010-0.022 vs 0.023-0.061 for MGL C. freundii and 0.036-0.056 for MGL C. tetani. There are no significant differences between methionine cleavage after MGL C. tetani and MGL C. sporogenes i.v. injection at all doses. MGL from C. sporogenes may be considered as promising enzyme for further investigation as potential anticancer agent.

  20. Impact of methionine oxidation on calmodulin structural dynamics

    SciTech Connect

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    2015-01-09

    Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each

  1. Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa

    PubMed Central

    Berger, Louise C.; Wilson, Judith; Wood, Pamela; Berger, Bradley J.

    2001-01-01

    Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549–561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much

  2. Appropriate statistical methods to compare dose responses of methionine sources.

    PubMed

    Kratzer, D D; Littell, R C

    2006-05-01

    Two sources of methionine (Met) activity are frequently used in commercial feed formulation: DL-2-hydroxy-4-(methylthio) butanoic acid (HMTBA), most commonly available as an 88% solution with 12% water; and DL-methionine (DLM, 99% powder). Despite the fact that both compounds have been in commercial use for over 50 yr, controversy and confusion remain with respect to their relative bioefficacy (RBE). This paper presents a review of the use of a nonlinear common plateau asymptotic regression technique (NLCPAR) that has been used to compare the 2 Met sources with particular emphasis on the validity of the basic assumptions of that model. The thesis of this paper is that the controversy is due, at least in part, to the misapplication of this regression technique to estimate the RBE of HMTBA and DLM. The NLCPAR model is a bioassay with the key dependent assumptions that HMTBA is a dilution of DLM, and that each follows dose-response curves of the same form and approach a common plateau. Because both provide Met activity, it may be considered reasonable to accept these assumptions; however, specifically testing them demonstrated that the assumption of a common dose-response is not supported by data. The common plateau assumption was tested with an alternative approach of fitting nonlinear separate plateaus asymptotic regression (NLSPAR) to a set of 13 published broiler studies in which the NLCPAR model had been used to estimate RBE of HMTBA and DLM. The hypothesis of a common plateau was rejected (P < 0.01), meaning that the conclusion that HMTBA had lower bioefficacy than DLM based on the NLCPAR methodology was not valid. An example using published data demonstrated that the NLSPAR model was a significantly better fit than the NLCPAR model, and showed that HMTBA and DLM followed different dose responses. Consequently, there was no single value for RBE for the entire dose range; rather, the RBE of the 2 compounds varied with use level. The evidence presented here

  3. miR-210 expression is associated with methionine-induced differentiation of trout satellite cells.

    PubMed

    Latimer, Mary; Sabin, Nathalie; Le Cam, Aurélie; Seiliez, Iban; Biga, Peggy; Gabillard, Jean-Charles

    2017-08-15

    In fish, data on microRNAs (miRNAs) involved in myogenesis are scarce. In order to identify miRNAs involved in satellite cell differentiation, we used a methionine depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results validated that methionine removal (72 h) from the medium strongly decreased myoD1 and myogenin expression, indicating differentiation arrest. In contrast, methionine replenishment rescued expression of myoD1 and myogenin, showing a resumption of differentiation. We performed a miRNA array analysis of myogenic cells under three conditions: presence of methionine for 72 h (control), absence of methionine for 72 h (Meth-) and absence of methionine for 48 h followed by 24 h of methionine replenishment (Meth-/+). A clustering analysis identified three clusters: cluster I corresponds to miRNA upregulated only in Meth-/+ conditions; cluster II corresponds to miRNA downregulated only in Meth-/+ conditions; cluster III corresponds to miRNAs with high expression in control, low expression in Meth- conditions and intermediate expression after methionine replenishment (Meth-/+). Cluster III was very interesting because it fitted with the data obtained for myoD1 and myogenin (supporting an involvement in differentiation) and contained seven miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our previously published miRNA repertoire ( Juanchich et al., 2016), we confirmed miR-133a was expressed only in white muscle and showed that miR-210 had strong expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells, suggesting that miR-210 was potentially involved in the differentiation of satellite cells. © 2017. Published by The Company of Biologists Ltd.

  4. Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Gagey, Marie Josèphe; Frelin, Océane; Beffa, Roland; Lebrun, Marc Henri; Droux, Michel

    2015-01-01

    Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection. PMID:25856162

  5. [13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.

    PubMed Central

    He, Q Y; Mason, A B; Tam, B M; MacGillivray, R T; Woodworth, R C

    1999-01-01

    The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized. PMID:10585877

  6. New protein purification system using gold-magnetic beads and a novel peptide tag, "the methionine tag".

    PubMed

    Okada, Yoshiaki; Takano, Tomoko Y; Kobayashi, Nozomi; Hayashi, Arisa; Yonekura, Masaaki; Nishiyama, Yuji; Abe, Tomohiro; Yoshida, Takuya; Yamamoto, Takao A; Seino, Satoshi; Doi, Takefumi

    2011-05-18

    Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.

  7. Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

    PubMed Central

    Suzuki, T; Takahashi, E

    1976-01-01

    1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed. PMID:1008848

  8. Disruption of Methionine Metabolism in Drosophila melanogaster Impacts Histone Methylation and Results in Loss of Viability

    PubMed Central

    Liu, Mengying; Barnes, Valerie L.; Pile, Lori A.

    2015-01-01

    Histone methylation levels, which are determined by the action of both histone demethylases and methyltransferases, impact multiple biological processes by affecting gene expression activity. Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. The functions of methionine metabolic enzymes in regulating biological processes as well as the interaction between the methionine pathway and histone methylation, however, are still not fully understood. Here, we report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone methylation levels. Reduction of little imaginal discs (LID) histone demethylase, but not lysine-specific demethylase 2 (KDM2) demethylase, is able to counter the effects on histone methylation due to reduction of SAM synthetase (SAM-S). Taken together, these results reveal an essential role of key enzymes that control methionine metabolism and histone methylation. Additionally, these findings are an indication of a strong connection between metabolism and epigenetics. PMID:26546310

  9. Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.

    PubMed

    Richardson, David E; Regino, Celeste A S; Yao, Huirong; Johnson, Jodie V

    2003-12-15

    Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4-, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4- oxidation of free methionine have been obtained (k1 = 0.48 +/- 0.08 M(-1)s(-1) by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood ( approximately 25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of alpha1-proteinase inhibitor (alpha1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4- oxidation of alpha1-PI (0.36 +/- 0.06 M(-1)s(-1)) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the alpha1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized alpha1-PI. These results strongly suggest that HCO4- should be considered a reactive oxygen species in aerobic metabolism.

  10. Influence of dietary protein and excess methionine on choline needs for young bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1982-01-01

    Experiments were conducted with young Bobwhite quail (Colinus virginianus) to investigate the effect of differing dietary protein levels and nondetrimental amounts of excess methionine on choline needs. Growth and feed consumption of quail fed an adequate (27.3%) protein purified diet supplemented with 2000 mg/kg of choline were unaffected by increasing the level of excess methionine to 1.75%; however, greater amounts (2.0%, 2.25%) of excess methionine depressed growth (P less than .01), reduced feed consumption (P less than .01), and decreased feed utilization (P less than .05). Quail fed a purified diet containing 13.85% protein and 515 mg/kg of choline grew poorly. Growth was unaffected by additional choline in this diet. Growth was suboptimal among quail fed purified diets containing adequate or high (41.55%) levels of protein in which choline was limiting; however, a high level of protein did not in itself affect performance. Growth was improved by supplemental choline in these diets. Growth of quail fed purified diets with up to 1.35% excess methionine which were limiting (531 mg/kg) in choline was less than that of groups fed 2000 mg/kg of added dietary choline (P less than .01); however, excess methionine did not significantly influence growth of quail fed choline-deficient diets. These experiments indicate that neither high dietary protein nor excess methionine, fed at non-growth-depressing levels, increases dietary choline needs for young Bobwhite quail.

  11. d-Methionine reduces tobramycin-induced ototoxicity without antimicrobial interference in animal models.

    PubMed

    Fox, Daniel J; Cooper, Morris D; Speil, Cristian A; Roberts, Melissa H; Yanik, Susan C; Meech, Robert P; Hargrove, Tim L; Verhulst, Steven J; Rybak, Leonard P; Campbell, Kathleen C M

    2016-07-01

    Tobramycin is a critical cystic fibrosis treatment however it causes ototoxicity. This study tested d-methionine protection from tobramycin-induced ototoxicity and potential antimicrobial interference. Auditory brainstem responses (ABRs) and outer hair cell (OHC) quantifications measured protection in guinea pigs treated with tobramycin and a range of d-methionine doses. In vitro antimicrobial interference studies tested inhibition and post antibiotic effect assays. In vivo antimicrobial interference studies tested normal and neutropenic Escherichia coli murine survival and intraperitoneal lavage bacterial counts. d-Methionine conferred significant ABR threshold shift reductions. OHC protection was less robust but significant at 20kHz in the 420mg/kg/day group. In vitro studies did not detect d-methionine-induced antimicrobial interference. In vivo studies did not detect d-methionine-induced interference in normal or neutropenic mice. d-Methionine protects from tobramycin-induced ototoxicity without antimicrobial interference. The study results suggest d-met as a potential otoprotectant from clinical tobramycin use in cystic fibrosis patients. Published by Elsevier B.V.

  12. D-METHIONINE REDUCES TOBRAMYCIN-INDUCED OTOTOXICITY WITHOUT ANTIMICROBIAL INTERFERENCE IN ANIMAL MODELS

    PubMed Central

    Fox, Daniel J.; Cooper, Morris D.; Speil, Cristian A.; Roberts, Melissa H.; Yanik, Susan C.; Meech, Robert P.; Hargrove, Tim L.; Verhulst, Steven J.; Rybak, Leonard P.; Campbell, Kathleen C. M.

    2015-01-01

    Background Tobramycin is a critical cystic fibrosis treatment however it causes ototoxicity. This study tested D-methionine protection from tobramycin-induced ototoxicity and potential antimicrobial interference. Methods Auditory brainstem responses (ABR) and outer hair cell (OHC) quantifications measured protection in guinea pigs treated with tobramycin and a range of D-methionine doses. In vitro antimicrobial interference studies tested inhibition and post antibiotic effect assays. In vivo antimicrobial interference studies tested normal and neutropenic E. coli murine survival and intraperitoneal lavage bacterial counts. Results D-methionine conferred significant ABR threshold shift reductions. OHC protection was less robust but significant at 20 kHz in the 420 mg/kg/day group. In vitro studies did not detect D-methionine-induced antimicrobial interference. In vivo studies did not detect D-methionine-induced interference in normal or neutropenic mice. Conclusions D-methionine protects from tobramycin-induced ototoxicity without antimicrobial interference. The study results suggest D-met as a potential otoprotectant from clinical tobramycin use in cystic fibrosis patients. PMID:26166286

  13. Decreased rates of methionine synthesis by methylene tetrahydrofolate reductase-deficient fibroblasts and lymphoblasts.

    PubMed

    Boss, G R; Erbe, R W

    1981-06-01

    Methionine synthesis from homocysteine was measured in intact human fibroblasts and lymphoblasts using a [14C]formate label. Seven fibroblast lines and two lymphoblast lines derived from patients with 5,10-methylene tetrahydrofolate reductase deficiency had rates of methionine synthesis that were from 4 to 43% of normal. When the patients were divided by clinical status into mildly (two patients), moderately (two patients), and severely (three patients) affected, methionine biosynthesis expressed as a percent of control values was 43 and 33%, 11 and 10%, and 7, 6, and 4%, respectively, in fibroblasts. Similar data for the two lymphoblast lines were 36 and 26% for a mildly and moderately affected patient, respectively. These data are to be contrasted with the measurement of residual enzyme activity in cell extracts which agrees less precisely with the clinical status of the patients. In the presence of normal methionine synthetase activity, the rate of synthesis of methionine from homocysteine is a function of the activity of the enzyme 5,10-methylene tetrahydrofolate reductase, and measurement of the methionine biosynthetic capacity of cells deficient in this enzyme accurately reflects the clinical status of the patient from whom the cells were derived.

  14. Methionine metabolism in piglets Fed DL-methionine or its hydroxy analogue was affected by distribution of enzymes oxidizing these sources to keto-methionine.

    PubMed

    Fang, Zhengfeng; Luo, Hefeng; Wei, Hongkui; Huang, Feiruo; Qi, Zhili; Jiang, Siwen; Peng, Jian

    2010-02-10

    Previous evidence shows that the extensive catabolism of dietary essential amino acids (AA) by the intestine results in decreased availability of these AA for protein synthesis in extraintestinal tissues. This raises the possibility that extraintestinal availability of AA may be improved by supplying the animal with an AA source more of which can bypass the intestine. To test this hypothesis, six barrows (35-day-old, 8.6 +/- 1.4 kg), implanted with arterial, portal, and mesenteric catheters, were fed a DL-methionine (DL-MET) or DL-2-hydroxy-4-methylthiobutyrate (DL-HMTB) diet once hourly and infused intramesenterically with 1% p-amino hippurate. Although the directly available L-MET in DL-MET diet was about 1.2-fold that in DL-HMTB diet, the net portal appearance of L-MET was not different between the two diets. Compared with the low mRNA abundance and low activity of D-2-hydroxy acid dehydrogenase (D-HADH) and l-2-hydroxy acid oxidase (L-HAOX) in the intestine, the high mRNA abundance and high activity of D-AA oxidase (D-AAOX) indicated that the intestine had a relatively higher capacity of D-MET utilization than of dl-HMTB utilization to L-MET synthesis and its subsequent metabolism. However, in contrast to the much lower D-AAOX activity (nmol/g tissue) in the stomach than in the liver and kidney, both d-HADH and L-HAOX activity in the stomach was comparable with those in the liver and/or kidney, indicating the substantial capacity of the stomach to convert DL-HMTB to L-MET. Collectively, the difference in distribution of activity and mRNA abundance of D-AAOX, D-HADH, and L-HAOX in the piglets may offer a biological basis for the similar portal appearance of L-MET between DL-MET and DL-HMTB diets, and thus may provide new important insights into nutritional efficiency of different L-MET sources.

  15. Comparative value of L-, and D-methionine supplementation of an oat-based diet for humans.

    PubMed

    Kies, C; Fox, H; Aprahamian, S

    1975-07-01

    Total sulfur-containing amino acids have been found to be the first limiting amino acid in several foods in comparison with human amino acid requirements. Addition of methionine in appropriate amounts to these foods might be expected to improve protein value. Economically, DL-methionine would be preferable to L-methionine for this purpose. However, the comparative tuilization of L- DL-, and D-methionine is unclear. The objective of the current project was to compare the effectiveness of L-, DL-, and D-methionine supplementation of diets based on a food product known to be low in methionine value for human subjects. "Instant" oatmeal was fed to adult subjects to provide 4.0 g of nitrogen/day. In randomly arranged periods, these diets were supplemented with L-, DL-, or D-methionine at two levels (0.58 and 1.16 g of methionine/day). An unsupplemented diet was used in a control period. Diets were adequate in vitamins, minerals, and energy. Mean nitrogen balances of subjects while receiving the L-methionine supplements at the 0.58 and 1.16 g levels were minus0.10 and +0.06 g of nitrogen, respectively. At similar levels of DL-methionine supplementation, nitrogen balances were minus0.12 and minus0.15 g of nitrogen, respectively, and minus0.24 and minus0.18 g of nitrogen with D-methionine supplementation. The mean nitrogen balance when no supplement was used was minus0.22 g of nitrogen. Thus, D-methionine is seemingly poorly utilized by the human. Urinary methionine excretion data supported these results.

  16. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    PubMed Central

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  17. Methionine ligand lability of homologous monoheme cytochromes c.

    PubMed

    Levin, Benjamin D; Walsh, Kelly A; Sullivan, Kristal K; Bren, Kara L; Elliott, Sean J

    2015-01-05

    Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

  18. Methionine Uptake and Required Radiation Dose to Control Glioblastoma

    SciTech Connect

    Iuchi, Toshihiko; Hatano, Kazuo; Uchino, Yoshio; Itami, Makiko; Hasegawa, Yuzo; Kawasaki, Koichiro; Sakaida, Tsukasa; Hara, Ryusuke

    2015-09-01

    Purpose: The purpose of this study was to retrospectively assess the feasibility of radiation therapy planning for glioblastoma multiforme (GBM) based on the use of methionine (MET) positron emission tomography (PET), and the correlation among MET uptake, radiation dose, and tumor control. Methods and Materials: Twenty-two patients with GBM who underwent MET-PET prior to radiation therapy were enrolled. MET uptake in 30 regions of interest (ROIs) from 22 GBMs, biologically effective doses (BEDs) for the ROIs and their ratios (MET uptake:BED) were compared in terms of whether the ROIs were controlled for >12 months. Results: MET uptake was significantly correlated with tumor control (odds ratio [OR], 10.0; P=.005); however, there was a higher level of correlation between MET uptake:BED ratio and tumor control (OR, 40.0; P<.0001). These data indicated that the required BEDs for controlling the ROIs could be predicted in terms of MET uptake; BED could be calculated as [34.0 × MET uptake] Gy from the optimal threshold of the MET uptake:BED ratio for tumor control. Conclusions: Target delineation based on MET-PET was demonstrated to be feasible for radiation therapy treatment planning. MET-PET could not only provide precise visualization of infiltrating tumor cells but also predict the required radiation doses to control target regions.

  19. Fragmentation network of doubly charged methionine: Interpretation using graph theory.

    PubMed

    Ha, D T; Yamazaki, K; Wang, Y; Alcamí, M; Maeda, S; Kono, H; Martín, F; Kukk, E

    2016-09-07

    The fragmentation of doubly charged gas-phase methionine (HO2CCH(NH2)CH2CH2SCH3) is systematically studied using the self-consistent charge density functional tight-binding molecular dynamics (MD) simulation method. We applied graph theory to analyze the large number of the calculated MD trajectories, which appears to be a highly effective and convenient means of extracting versatile information from the large data. The present theoretical results strongly concur with the earlier studied experimental ones. Essentially, the dication dissociates into acidic group CO2H and basic group C4NSH10. The former may carry a single or no charge and stays intact in most cases, whereas the latter may hold either a single or a double charge and tends to dissociate into smaller fragments. The decay of the basic group is observed to follow the Arrhenius law. The dissociation pathways to CO2H and C4NSH10 and subsequent fragmentations are also supported by ab initio calculations.

  20. Meta-analysis of lactation performance in dairy cows receiving supplemental dietary methionine sources or postruminal infusion of methionine.

    PubMed

    Zanton, G I; Bowman, G R; Vázquez-Añón, M; Rode, L M

    2014-11-01

    The objectives of our study were to evaluate the productive response to methionine supplementation in lactating dairy cows and to define a relationship between metabolizable Met (MP Met) intake and production. A database of 64 papers meeting the selection criteria was developed evaluating postruminally infused dl-methionine (9 papers with 18 control diets and 35 treatment comparisons), 2-hydroxy-4-methylthio butanoic acid (HMTBa) provided as either a liquid or Ca salt form (17 papers with 34 control diets and 46 treatment comparisons), Mepron (Evonik Industries, Essen, Germany; 18 papers with 35 control diets and 42 treatment comparisons), and Smartamine (Adisseo Inc., Antony, France; 20 papers with 30 control diets and 39 treatment comparisons). Dietary ingredients and their accompanying nutritional compositions as described in the reports were entered into the Cornell-Penn-Miner software to model the diets and to predict nutrients that were not reported in the original publication. Data were analyzed using a weighted analysis of response to supplementation compared with the intraexperiment control, as well as through a regression analysis to changing dietary MP Met. Data included in the analysis were from experiments published between 1970 and 2011 with cows supplemented with between 3.5 and 67.9 g of Met or its equivalent from HMTBa. Cows supplemented with Smartamine consumed more, whereas cows supplemented with Mepron consumed less DM compared with controls. Milk yield did not significantly respond to Met supplementation, although it tended to increase for cows supplemented with HMTBa and Mepron. Milk protein yield was increased due to supplementation from all sources or from infusion, and protein concentration was greater for all supplements or infusion of dl-Met, except for cows supplemented with HMTBa. Irrespective of Met source, milk protein yield increased 2.23 g of protein/g of MP Met until reaching the breakpoint. Milk fat yield was increased for Mepron

  1. Selenium-methionine and chromium-methionine supplementation of sheep around parturition: impacts on dam and offspring performance.

    PubMed

    Mousaie, Amir; Valizadeh, Reza; Chamsaz, Mahmoud

    2017-04-01

    To examine the effects of maternal energy restriction along with selenium-methionine (Se-Met) and chromium-methionine (Cr-Met) supplementation on performance of pregnant sheep and their offspring, the following treatments were allotted randomly to 40 multiparous Baluchi ewes (53.9 ± 1.15 kg of body weight [BW]) from 5 weeks prior to 5 weeks after parturition: (1) Control diet (60% and 100% of NRC energy requirements in pre- and post-partum, respectively); (2) Control diet plus 5 mg Se-Met/kg dry matter (DM); (3) Control diet plus 3 mg Cr-Met/kg DM and (4) Control diet plus 5 mg Se-Met and 3 mg Cr-Met/kg DM (Se-Cr-Met) of concentrate diet. The results indicated that Cr-Met alone or in combination with Se-Met increased average DM intake of ewes. In addition, Group Cr-Met had higher average BW than the Control (p < 0.05). Se-Met and/or Cr-Met supplementation led to decreased average serum cholesterol of the ewes (p < 0.05). Groups Cr-Met and Se-Cr-Met displayed decreased average serum malondialdehyde compared to the Control (p < 0.05). At 24 h post-partum, Group Se-Cr-Met had a greater serum Se content than the Control (p = 0.006). Compared with the Control, the Se concentration in milk was significantly increased from 30 to 138 µg/l and 197 µg/l in Groups Se-Met and Se-Cr-Met, respectively (p < 0.01), which proved that Se-Met supplementation can increase the Se concentration of ewe milk. Furthermore, feeding Cr-Met may attenuate BW loss post-partum and Se-Met and/or Cr-Met supplements may ameliorate oxidative stress condition in ewes around parturition.

  2. Divergent modulation of swine ileal microbiota by formic acid and methionine hydroxy analogue-free acid.

    PubMed

    Apajalahti, J; Rademacher, M; Htoo, J K; Redshaw, M; Kettunen, A

    2009-06-01

    Management of intestinal microbiota of monogastric animals has increased in importance since the ban of growth promoting antibiotics in many countries. Organic acids have been used as alternatives to antibiotics by many feed manufacturers. Regardless of the wide usage, the effect, dose response and mode of action of acids on intestinal microbes is poorly understood. In this study, we investigated the effects of dietary supplementation of three commonly used products, namely formic acid (FA) (90%), dl-methionine (DLM) (99%) and liquid methionine hydroxy analogue-free acid (88%), on ileal microbiota of pigs. Laboratory simulation system, mimicking swine ileum, was used to study the products at various concentrations and combinations. Furthermore, selected combinations were tested in a piglet trial to confirm the findings made in in vitro studies. FA turned out to have a dual effect on ileal microbiota. At concentrations below 0.5%, it significantly stimulated bacteria, but at higher inclusion rates it was highly inhibitory. This finding, which was consistent in in vitro and in vivo studies, implies that reducing the dose of FA does not lead to a diluted inhibitory effect, but in fact, an opposite, stimulatory effect on intestinal microbiota. It is highly important that feed compounders acknowledge this finding. Unlike FA, the inhibitory effect of methionine hydroxy analogue on ileal bacteria was linearly dose dependent and significant at inclusion levels above 0.2%, in vitro. Partial replacement of methionine hydroxy analogue by FA, or FA by methionine hydroxy analogue, led to an unpredictable outcome due to the dual effects of FA; e.g., a minor inclusion of added FA changed the inhibitory effect of methionine hydroxy analogue into microbial stimulation by FA. Inhibition of ileal microbiota by methionine hydroxy analogue was detected only in in vitro studies, suggesting that intact methionine hydroxy analogue may not have reached the ileum, in live animals. Therefore

  3. Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense.

    PubMed

    Goldberg, B; Rattendi, D; Lloyd, D; Yarlett, N; Bacchi, C J

    2000-05-01

    Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense

  4. The Cus efflux system removes toxic ions via a methionine shuttle.

    PubMed

    Su, Chih-Chia; Long, Feng; Yu, Edward W

    2011-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell. These efflux systems span the entire cell envelope to mediate the phenomenon of bacterial multidrug resistance. The three parts of the efflux complexes are: (1) a membrane fusion protein (MFP) connecting (2) a substrate-binding inner membrane transporter to (3) an outer membrane-anchored channel in the periplasmic space. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. We recently determined the crystal structures of both the inner membrane transporter CusA and MFP CusB of the CusCBA tripartite efflux system from E. coli. These are the first structures of the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here, we summarize the structural information of these two efflux proteins and present the accumulated evidence that this efflux system utilizes methionine residues to bind and export Cu(I)/Ag(I). Genetic and structural analyses suggest that the CusA pump is capable of picking up the metal ions from both the periplasm and cytoplasm. We propose a stepwise shuttle mechanism for this pump to extrude metal ions from the cell.

  5. Control of protein life-span by N-terminal methionine excision

    PubMed Central

    Giglione, Carmela; Vallon, Olivier; Meinnel, Thierry

    2003-01-01

    Peptide deformylases (PDFs) have been discovered recently in eukaryotic genomes, and it appears that N-terminal methionine excision (NME) is a conserved pathway in all compartments where protein synthesis occurs. This work aimed at uncovering the function(s) of NME in a whole proteome, using the chloroplast-encoded proteins of both Arabidopsis thaliana and Chlamydomonas reinhardtii as model systems. Dis ruption of PDF1B in A.thaliana led to an albino phenotype, and an extreme sensitivity to the PDF- specific inhibitor actinonin. In contrast, a knockout line for PDF1A exhibited no apparent phenotype. Photosystem II activity in C.reinhardtii cells was substantially reduced by the presence of actinonin. Pulse–chase experiments revealed that PDF inhibi tion leads to destabilization of a crucial subset of chloroplast-encoded photosystem II components in C.reinhardtii. The same proteins were destabilized in pdf1b. Site-directed substitutions altering NME of the most sensitive target, subunit D2, resulted in similar effects. Thus, plastid NME is a critical mechanism specifically influencing the life-span of photosystem II polypeptides. A general role of NME in modulating the half-life of key subsets of proteins is suggested. PMID:12505980

  6. Cysteine dietary supplementation reverses the decrease in mitochondrial ROS production at complex I induced by methionine restriction.

    PubMed

    Gomez, A; Gomez, J; Lopez Torres, M; Naudi, A; Mota-Martorell, N; Pamplona, R; Barja, G

    2015-06-01

    It has been described that dietary cysteine reverses many of the beneficial changes induced by methionine restriction in aging rodents. In this investigation male Wistar rats were subjected to diets low in methionine, supplemented with cysteine, or simultaneously low in methionine and supplemented with cysteine. The results obtained in liver showed that cysteine supplementation reverses the decrease in mitochondrial ROS generation induced by methionine restriction at complex I. Methionine restriction also decreased various markers of oxidative and non-oxidative stress on mitochondrial proteins which were not reversed by cysteine. Instead, cysteine supplementation also lowered protein damage in association with decreases in mTOR activation. The results of the present study add the decrease in mitochondrial ROS production to the various beneficial changes induced by methionine restriction that are reversed by cysteine dietary supplementation.

  7. Recycling of 5'-methylthioadenosine-ribose carbon atoms into methionine in tomato tissue in relation to ethylene production.

    PubMed

    Wang, S Y; Adams, D O; Lieberman, M

    1982-07-01

    The ribose moiety of 5'-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-(14)C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C(2)H(4) and other unidentified compounds. However, when [U-(14)C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C(2)H(4), but not MTA. This suggests that C(2)H(4) formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O(2) dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.

  8. Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato.

    PubMed

    Tu, H M; Godfrey, L W; Sun, S S

    1998-07-01

    A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation. To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato. Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides. The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein. The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves. In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5-11 kDa) polypeptide was detected. These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

  9. 1- sup 13 C; methyl-2H3 methionine kinetics in humans: Methionine conservation and cystine sparing

    SciTech Connect

    Storch, K.J.; Wagner, D.A.; Burke, J.F.; Young, V.R. )

    1990-05-01

    Methionine (Met) conservation in healthy young adult men (4/diet group) was explored by supplying one of the following three L-amino acid based diets: (1) adequate Met but no cystine; (2) neither Met nor cystine; or (3) no Met but cystine supplementation. After 5 days, subjects received a continuous intravenous infusion of L-(1-13C; methyl-2H3)Met for 5 h while the diet was given as small isocaloric isonitrogenous meals. Estimates were made of rates of Met incorporation into protein synthesis (S) and release from body proteins (B), transmethylation (TM), remethylation of homocysteine (RM), and transsulfuration (TS). For the adequate Met diet, the rates were S = 24 +/- 2, B = 18 +/- 1, TM = 12.4 +/- 1.7, RM = 4.7 +/- 1.1, and TS = 7.6 +/- 0.6 (SE) mumol.kg-1.h-1. The sulfur amino acid-devoid diet significantly (P less than 0.05) reduced S, TM, RM, and TS. Supplementation of this diet with cystine reduced Met oxidation (P = 0.05). Therefore, two loci are quantitatively important regulatory points in Met conservation in vivo: (1) the distribution of Met between the pathways of protein anabolism and TM (Met locus) and (2) the distribution of homocysteine between RM and TS (homocysteine locus).

  10. Spectroscopic characterization of active-site variants of the PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri: insights into the mechanism of four-coordinate Co(II)corrinoid formation.

    PubMed

    Park, Kiyoung; Mera, Paola E; Escalante-Semerena, Jorge C; Brunold, Thomas C

    2012-04-16

    The PduO-type adenosine 5'-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co(1+)cobalamin (Cbl) and Co(1+)cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi(+)), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co(2+) → Co(1+) reduction, the enzyme drastically weakens the axial ligand-Co(2+) bond so as to generate effectively four-coordinate (4c) Co(2+)corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co(2+)corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co(2+)corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi(+). Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi(+). Finally, when ATP analogues were used as cosubstrate, only "unactivated" five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by

  11. Functional Consequences of Methionine Oxidation of hERG Potassium Channels

    PubMed Central

    Su, Zhi; Limberis, James; Martin, Ruth L.; Xu, Rong; Kolbe, Katrin; Heinemann, Stefan H.; Hoshi, Toshinori; Cox, Bryan F.; Gintant, Gary A.

    2010-01-01

    Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in hERG1 (human ether à go-go related gene 1), which encodes cardiac IKr channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 µM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to −60 or −100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or −10 mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel. PMID:17624316

  12. Metabolic engineering of Escherichia coli for microbial production of L-methionine.

    PubMed

    Huang, Jian-Feng; Liu, Zhi-Qiang; Jin, Li-Qun; Tang, Xiao-Ling; Shen, Zhen-Yang; Yin, Huan-Huan; Zheng, Yu-Guo

    2017-04-01

    L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli ΔmetJ ΔmetI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 ΔmetJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H. Finally, addition of Na2 S2 O3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H was able to produce 9.75 g/L L-methionine with productivity of 0.20 g/L/h in a 5 L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. © 2016 Wiley Periodicals, Inc.

  13. [Association of serum homocysteine and methionine levels with cognition and functioning in bipolar disorder].

    PubMed

    Doğanavşargil Baysal, G Özge; Gökmen, Zehra; Akbaş, Halide; Cinemre, Buket; Metin, Özmen; Karaman, Taha

    2013-01-01

    The relationship between homocysteine (HCY) levels and cognitive impairments, particularly executive functions in bipolar disorder (BD), has recently been investigated. However, conflicting results were reported. The aim of the present study is to investigate changes in serum HCY, methionine, vitamine B12 and levels in BD patients are relative to controls and to investigate the relationship between HCY, methionine, vitamin B12, and folate levels and clinical features, cognitive functions and psychosocial functioning in euthymic BD patients and controls. Sixty BD type I euthymic patients and twenty controls were assessed with Global Assessment of Functioning and a battery of neuropsychological tests including the Wisconsin card sorting test, the Rey's auditory verbal learning test, the Cancellation test, Trail making test A, Trail making test B, and the Stroop test. HCY, vitamin B12, methionine and folate levels were measured together after collecting blood samples from both patient and controls. Mean serum methionine concentration was different between groups. Low serum methionine was found to be a predictor of BD. However, a statistically significant difference was not detected between groups for mean serum values of HCY, folate, or vitamin B12. HCY levels showed a positive correlation with illness duration, the number of total episodes, and the number of manic episodes. A significant correlation was not found between HCY, methionine, folate, B12 levels with cognitive functions and functioning in the BD group. Low serum methionine was found to be a predictor of BD, a condition which can lead to a decrease in SAM synthesis and thus to a variety of complications in methylation reactions. Additional studies are needed to clarify the impact of single carbon metabolism on BD.

  14. Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

    PubMed

    Bolten, Christoph J; Schröder, Hartwig; Dickschat, Jeroen; Wittmann, Christoph

    2010-08-01

    In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

  15. Hepatic effects of a methionine-choline-deficient diet in hepatocyte RXR{alpha}-null mice

    SciTech Connect

    Gyamfi, Maxwell Afari; Tanaka, Yuji; He Lin; Klaassen, Curtis D.; Wan, Y.-J.Y.

    2009-01-15

    Retinoid X receptor-{alpha} (RXR{alpha}) is an obligate partner for several nuclear hormone receptors that regulate important physiological processes in the liver. In this study the impact of hepatocyte RXR{alpha} deficiency on methionine and choline deficient (MCD) diet-induced steatosis, oxidative stress, inflammation, and hepatic transporters gene expression were examined. The mRNA of sterol regulatory element-binding protein (SREBP)-regulated genes, important for lipid synthesis, were not altered in wild type (WT) mice, but were increased 2.0- to 5.4-fold in hepatocyte RXR{alpha}-null (H-RXR{alpha}-null) mice fed a MCD diet for 14 days. Furthermore, hepatic mRNAs and proteins essential for fatty acid {beta}-oxidation were not altered in WT mice, but were decreased in the MCD diet-fed H-RXR{alpha}-null mice, resulting in increased hepatic free fatty acid levels. Cyp2e1 enzyme activity and lipid peroxide levels were induced only in MCD-fed WT mice. In contrast, hepatic mRNA levels of pro-inflammatory factors were increased only in H-RXR{alpha}-null mice fed the MCD diet. Hepatic uptake transporters Oatp1a1 and Oatp1b2 mRNA levels were decreased in WT mice fed the MCD diet, whereas the efflux transporter Mrp4 was increased. However, in the H-RXR{alpha}-null mice, the MCD diet only moderately decreased Oatp1a1 and induced both Oatp1a4 and Mrp4 gene expression. Whereas the MCD diet increased serum bile acid levels and alkaline phosphatase activity in both WT and H-RXR{alpha}-null mice, serum ALT levels were induced (2.9-fold) only in the H-RXR{alpha}-null mice. In conclusion, these data suggest a critical role for RXR{alpha} in hepatic fatty acid homeostasis and protection against MCD-induced hepatocyte injury.

  16. In vivo metabolism of L-methionine in mice: evidence for stereoselective formation of methionine-d-sulfoxide and quantitation of other major metabolites.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2006-12-01

    Flavin-containing monooxygenases (FMOs) 1-4 oxidize methionine (Met) to methionine sulfoxide (MetO). FMO3, the primary isoform expressed in adult human liver, has the lowest Km and favors methionine-d-sulfoxide (Met-d-O) formation over methionine-l-sulfoxide. Because female mice, but not males, also express FMO3 in liver, levels of Met and its major metabolites were determined in male or female mice dosed with 400 mg/kg Met i.p. The results show that Met levels in male and female mouse liver or plasma increased significantly at both 15 and 30 min after the Met treatment; Met plasma and liver levels at 30 min were similar to or lower than the corresponding levels at 15 min. Liver and plasma MetO levels increased significantly in both sexes at 30 min, and Met-d-O was the major MetO diastereomer detected. Interestingly, less than 0.1% of the Met dose was excreted in the urine (0-24 h) as Met and Met-d-O. S-Adenosylmethionine (SAM) was the major metabolite detected in liver at 15 min. Liver SAM levels at 30 min were lower than the levels at 15 min, and the plasma SAM levels at both 15 and 30 min were much lower than the corresponding levels in the liver. Increases in liver and/or plasma S-adenosyl-l-homocysteine, 5'-deoxy-5'-(methylthio)adenosine, and N-acetyl-l-methionine were also detected. Taken together, these results suggest that mice extensively and rapidly used the Met dose. Although mice exhibited increases in tissue MetO levels, a major role for FMO3 in Met-d-O formation is not certain since the MetO increases were mostly similar in both males and females.

  17. Tissue glutathione and cysteine levels in methionine-restricted rats.

    PubMed

    Richie, John P; Komninou, Despina; Leutzinger, Yvonne; Kleinman, Wayne; Orentreich, Norman; Malloy, Virginia; Zimmerman, Jay A

    2004-09-01

    Previously, we demonstrated that lifelong methionine (Met) restriction (MR) increases lifespan, decreases the incidence of aging-related diseases, increases blood glutathione (GSH) levels, and prevents loss of GSH during aging in rats. Our present objective was to elucidate the effects of MR on GSH metabolism and transport by determining the time course and nature of GSH and cysteine changes in blood and other tissues in young and mature rats. Male F-344 rats were placed on control (0.86% Met) or MR (0.17% Met) defined amino acid diets at age 7 wk and killed at different times thereafter. MR was also initiated in adult (12-mo-old) rats. Throughout the first 2 mo of MR, blood GSH levels increased 84% and liver GSH decreased 66% in relation to controls. After this period, liver GSH levels remained constant through at least 6 mo. GSH levels also decreased in the pancreas (80%) and kidney (22%) but remained unchanged in other tissues examined after 11 wk of MR. The increase in blood GSH was evident as soon as 1 wk after initiating MR and reached a plateau by 6 wk. A similar increase in erythrocyte GSH levels was observed when MR was administered to mature adult rats. Fasting decreased liver GSH in controls but had no further effect in MR animals. By 1 mo, cysteine levels had decreased in all tissues except brain. These results suggest that adaptive changes occur in the metabolism of Met, cysteine, and/or GSH as a result of MR in young and adult rats. These early metabolic changes lead to conservation of GSH levels in most extrahepatic tissues and increased GSH in erythrocytes by depleting liver GSH to a critical level.

  18. Methionine increases BDNF DNA methylation and improves memory in epilepsy

    PubMed Central

    Parrish, R Ryley; Buckingham, Susan C; Mascia, Katherine L; Johnson, Jarvis J; Matyjasik, Michal M; Lockhart, Roxanne M; Lubin, Farah D

    2015-01-01

    Objective Temporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus. Methods Using the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model. Results We found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade. Interpretation Our findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE. PMID:25909085

  19. Impact of food supplementation and methionine on high densities of cotton rats: Support of the amino-acid-quality hypothesis?

    USGS Publications Warehouse

    Webb, R.E.; Leslie, David M.; Lochmiller, R.L.; Masters, R.E.

    2005-01-01

    Considerable research supports the tenet that quantity and quality of food limit vertebrate populations. We evaluated predictions that increased availabilities of food and the essential amino acid methionine were related to population limitation of the hispid cotton rat (Sigmodon hispidus). Effects of supplemental food and methionine on density, survival, and reproductive parameters of wild cotton rats were assessed in north-central Oklahoma in 1998-1999. Twelve enclosed groups of 16 adult cotton rats each (8 male, 8 female) were randomly assigned to either no supplementation (control), supplementation with a mixed ration that had methionine at slightly below maintenance levels (0.20%), or a methionine-enhanced mixed ration (1.20%). In general, densities of cotton rats were twice as high and were sustained longer with dietary supplementation, and methionine-supplemented populations maintained the highest densities. Treatment effects on survival depended on time of year, with higher survival in supplemented enclosures in October and November. Per capita recruitment was highest with methionine-enhanced food. Treatment effects on proportions of overall and female cotton rats in reproductive condition depended on sampling date, but males were most reproductively active with methionine supplementation. Methionine supplementation resulted in an earlier and longer reproductive season. Density-dependent and density-independent factors no doubt interplay to determine population dynamics of cotton rats, but our results suggest that methionine plays a role in the population dynamics of wild cotton rats, apparently by enhancing overall density, recruitment, and reproductive activity of males.

  20. The relative contribution of genes operating in the S-methylmethionine cycle to methionine metabolism in Arabidopsis seeds.

    PubMed

    Cohen, Hagai; Salmon, Asaf; Tietel, Zipora; Hacham, Yael; Amir, Rachel

    2017-05-01

    Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine synthesis in seeds. In addition, mutual effects exist between the S -methylmethionine cycle and the aspartate family pathway in seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. The previous lines of evidence proposed that the S-methylmethionine (SMM) cycle contributes to methionine synthesis in seeds where methionine that is produced in non-seed tissues is converted to SMM and then transported via the phloem into the seeds. However, the relative regulatory roles of the S-methyltransferases operating within this cycle in seeds are yet to be fully understood. In the current study, we generated transgenic Arabidopsis seeds with altered expression of three HOMOCYSTEINE S-METHYLTRANSFERASEs (HMTs) and METHIONINE S-METHYLTRANSFERASE (MMT), and profiled them for transcript and metabolic changes. The results revealed that AtHMT1 and AtHMT3, but not AtHMT2 and AtMMT, are the predominant enzymes operating in seeds as altered expression of these two genes affected the levels of methionine and SMM in transgenic seeds. Their manipulations resulted in adapted expression level of genes participating in methionine synthesis through the SMM and aspartate family pathways. Taken together, our findings provide new insights into the regulatory roles of the SMM cycle and the mutual effects existing between the two methionine biosynthesis pathways, highlighting the complexity of the metabolism of methionine and SMM in seeds.

  1. Effects of methionine and betaine supplementation on growth performance, carcase composition and metabolism of lipids in male broilers.

    PubMed

    Zhan, X A; Li, J X; Xu, Z R; Zhao, R Q

    2006-10-01

    1. This study was conducted to investigate the effects of methionine and betaine supplementation on growth performance, carcase composition and lipid metabolism in growing broilers. 2. A total of 450 commercial broilers, 22 d of age, were randomly allocated to three groups, each of which included three replicates (50 birds per replicate). The groups received the same methionine-deficient diet supplemented with 0 or 1 g/kg methionine, or 0.5 g/kg betaine, respectively. 3. Methionine and betaine supplementation significantly improved weight gain and feed conversion. Supplemental methionine and betaine also significantly increased breast muscle yield and decreased abdominal fat content. Meanwhile, addition of methionine and betaine significantly increased the contents of creatine and free carnitine in liver, the activity of hormone-sensitive lipase in abdominal fat and the concentration of free fatty acid in serum, whereas uric acid concentration in serum was significantly decreased. 4. The results of this study suggest that betaine can spare methionine in its function as an essential amino acid and is as effective as methionine in improving performance and carcase quality of growing broilers if the diet is moderately deficient in methionine. The decrease in abdominal fat may be due to the increased carnitine synthesis in liver and hormone-sensitive lipase activity in abdominal fat.

  2. Definitive Endoderm Differentiation of Human Embryonic Stem Cells Combined with Selective Elimination of Undifferentiated Cells by Methionine Deprivation.

    PubMed

    Tsuyama, Tomonori; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    Human embryonic stem cells (ESCs) show a characteristic feature in that they are highly dependent on methionine metabolism. Undifferentiated human ESCs cannot survive under condition that methionine is deprived from culture medium. We describe here a procedure for definitive endoderm differentiation from human ESCs, in which human ESCs are subject to 10 days' (d) differentiation combined with methionine deprivation between differentiation days (d) 8 to (d) 10. Methionine deprivation results in elimination of undifferentiated cells from the culture with no significant loss of definitive endoderm cells, as compared to those cultured under complete condition throughout the whole culture period.

  3. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity

    PubMed Central

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M.; Bortolato, Marco

    2015-01-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous SNP that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood posttranslational mechanisms. One posttranslational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, while brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  4. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase.

    PubMed

    Elshorbagy, Amany K; Valdivia-Garcia, Maria; Mattocks, Dwight A L; Plummer, Jason D; Smith, A David; Drevon, Christian A; Refsum, Helga; Perrone, Carmen E

    2011-01-01

    Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation.

  5. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase

    PubMed Central

    Elshorbagy, Amany K.; Valdivia-Garcia, Maria; Mattocks, Dwight A. L.; Plummer, Jason D.; Smith, A. David; Drevon, Christian A.; Refsum, Helga; Perrone, Carmen E.

    2011-01-01

    Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation. PMID:20871132

  6. Methionine deprivation suppresses triple-negative breast cancer metastasis in vitro and in vivo

    PubMed Central

    Jang, Young Jin; Son, Joe Eun; Kwon, Jung Yeon; Lim, Tae-gyu; Kim, Sunghoon; Park, Jung Han Yoon; Kim, Jong-Eun; Lee, Ki Won

    2016-01-01

    Nutrient deprivation strategies have been proposed as an adjuvant therapy for cancer cells due to their increased metabolic demand. We examined the specific inhibitory effects of amino acid deprivation on the metastatic phenotypes of the human triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and Hs 578T, as well as the orthotopic 4T1 mouse TNBC tumor model. Among the 10 essential amino acids tested, methionine deprivation elicited the strongest inhibitory effects on the migration and invasion of these cancer cells. Methionine deprivation reduced the phosphorylation of focal adhesion kinase, as well as the activity and mRNA expression of matrix metalloproteinases MMP-2 and MMP-9, two major markers of metastasis, while increasing the mRNA expression of tissue inhibitor of metalloproteinase 1 in MDA-MB-231 cells. Furthermore, methionine restriction downregulated the metastasis-related factor urokinase plasminogen activatior and upregulated plasminogen activator inhibitor 1 mRNA expression. Animals on the methionine-deprived diet showed lower lung metastasis rates compared to mice on the control diet. Taken together, these results suggest that methionine restriction could provide a potential nutritional strategy for more effective cancer therapy. PMID:27579534

  7. The N-Terminal Methionine of Cellular Proteins as a Degradation Signal

    PubMed Central

    Kim, Heon-Ki; Kim, Ryu-Ryun; Oh, Jang-Hyun; Cho, Hanna; Varshavsky, Alexander; Hwang, Cheol-Sang

    2014-01-01

    SUMMARY The Arg/N-end rule pathway targets for degradation proteins that bear specific unacetylated N-terminal residues while the Ac/N-end rule pathway targets proteins through their Nα-terminally acetylated (Nt-acetylated) residues. Here we show that Ubr1, the ubiquitin ligase of the Arg/N-end rule pathway, recognizes unacetylated N-terminal methionine if it is followed by a hydrophobic residue. This capability of Ubr1 expands the range of substrates that can be targeted for degradation by the Arg/N-end rule pathway, because virtually all nascent cellular proteins bear N-terminal methionine. We identified Msn4, Sry1, Arl3, and Pre5 as examples of normal or misfolded proteins that can be destroyed through the recognition of their unacetylated N-terminal methionine. Inasmuch as proteins bearing the Nt-acetylated N-terminal methionine residue are substrates of the Ac/N-end rule pathway, the resulting complementarity of the Arg/N-end rule and Ac/N-end rule pathways enables the elimination of protein substrates regardless of acetylation state of N-terminal methionine in these substrates. PMID:24361105

  8. Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

    PubMed Central

    Bozzi, Aaron T.; Bane, Lukas B.; Weihofen, Wilhelm A.; McCabe, Anne L.; Singharoy, Abhishek; Chipot, Christophe J.; Schulten, Klaus

    2016-01-01

    Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it—more effectively than any other amino acid—increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium. PMID:27573840

  9. Methionine supplementation influences melanin-based plumage colouration in Eurasian kestrel, Falco tinnunculus, nestlings.

    PubMed

    Parejo, Deseada; Silva, Nadia

    2009-11-01

    The extent to which the expression of melanin-based plumage colouration in birds is genetically or environmentally determined is controversial. Here, we performed a between-nest design supplementation with either the sulphur amino acid dl-methionine or with water to investigate the importance of the non-genetic component of melanin-based plumage colouration in the Eurasian kestrel, Falco tinnunculus. Methionine affects growth and immunity, thus we aimed to modify nestling growth and immunity before feather development. Then, we measured the effect of the experiment on colouration of two melanin-based plumage patches of nestling kestrels. We found that methionine slowed down nestling growth through treatment administration and that nestlings compensated by speeding up their growth later. We did not find any effects of methionine on nestling immunity (i.e. lymphocyte counts, natural antibody levels or complement-mediated immunity). Effects on growth seemed to be mirrored by changes in nestling colouration in the two sexes: methionine-nestlings showed less intense brown plumage on their backs compared with control nestlings. These results provide support for a non-genetic determination of a melanin-based plumage patch in the two sexes of nestling kestrels.

  10. Intermediates in the Recycling of 5-Methylthioribose to Methionine in Fruits 1

    PubMed Central

    Kushad, Mosbah M.; Richardson, Daryl G.; Ferro, Adolph J.

    1983-01-01

    The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [14CH3]MTR was not metabolized in cell free extract from avocado fruit. Either [14CH3]MTR plus ATP or [14CH3]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [35S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-14C]α-KMB and [U-14C]methionine, but not [U-14C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene. PMID:16663204

  11. [Determination of methionine and tryptophan level in veterinary infusions containing amino acids].

    PubMed

    Kovácsné Hadady, K

    1989-09-01

    Method has been developed for determination of methionine in veterinary infusions containing amino acid. Methionine was measured on the basis of color reaction with sodium pentacyano-nitrosyl-ferrate in alkaline solution. Reproducibility of the method was good (recovery: 95.9-101.9%, r.s.d. = 2.3%, p = 0.95, n = 6). Tryptophane was determined on the basis of its UV absorbency at 280 nm, methionine did not interfere on this wavelength. Reproducibility of tryptophane determination was with an order better than that of methionine determination (recovery: 97.7-98.3%, r.s.d. = 0.15%, p = 0.95, n = 9). Methionine could be regained from infusions that had been heated at 120 degrees C for sixty minutes in the same quantity as from infusions which had not been heated. On the other hand tryptophane could be regained with 10% positive error from the heated infusions. The error can mainly be attributed to absorbency increase of glucose which is present in the infusion.

  12. Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress

    PubMed Central

    Suzuki, Satoko; Kodera, Yoshio; Saito, Tatsuya; Fujimoto, Kazumi; Momozono, Akari; Hayashi, Akinori; Kamata, Yuji; Shichiri, Masayoshi

    2016-01-01

    Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases. PMID:27929071

  13. Functional characterisation of the methionine sulfoxide reductase repertoire in Trypanosoma brucei.

    PubMed

    Guerrero, Sergio A; Arias, Diego G; Cabeza, Matias S; Law, Michelle C Y; D'Amico, Maria; Kumar, Ambika; Wilkinson, Shane R

    2017-09-01

    To combat the deleterious effects that oxidation of the sulfur atom in methionine to sulfoxide may bring, aerobic cells express repair pathways involving methionine sulfoxide reductases (MSRs) to reverse the above reaction. Here, we show that Trypanosoma brucei, the causative agent of African trypanosomiasis, expresses two distinct trypanothione-dependent MSRs that can be distinguished from each other based on sequence, sub-cellular localisation and substrate preference. One enzyme found in the parasite's cytosol, shows homology to the MSRA family of repair proteins and preferentially metabolises the S epimer of methionine sulfoxide. The second, which contains sequence motifs present in MSRBs, is restricted to the mitochondrion and can only catalyse reduction of the R form of peptide-bound methionine sulfoxide. The importance of these proteins to the parasite was demonstrated using functional genomic-based approaches to produce cells with reduced or elevated expression levels of MSRA, which exhibited altered susceptibility to exogenous H2O2. These findings identify new reparative pathways that function to fix oxidatively damaged methionine within this medically important parasite. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters.

    PubMed

    Bozzi, Aaron T; Bane, Lukas B; Weihofen, Wilhelm A; McCabe, Anne L; Singharoy, Abhishek; Chipot, Christophe J; Schulten, Klaus; Gaudet, Rachelle

    2016-09-13

    Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

  15. Parasites suppress immune-enhancing effect of methionine in nestling great tits.

    PubMed

    Wegmann, Michèle; Voegeli, Beatrice; Richner, Heinz

    2015-01-01

    After birth, an organism needs to invest both in somatic growth and in the development of efficient immune functions to counter the effects of pathogens, and hence an investment trade-off is predicted. To explore this trade-off, we simultaneously exposed nestling great tits (Parus major) to a common ectoparasite, while stimulating immune function. Using a 2 × 2 experimental design, we first infested half of the nests with hen fleas (Ceratophyllus gallinae) on day 3 post-hatch and later, on day 9-13 post-hatch, and then supplemented half of the nestlings within each nest with an immuno-enhancing amino acid (methionine). We then assessed the non-specific immune response by measuring both the inflammatory response to a lipopolysaccharide (LPS) and assessing the levels of acute phase proteins (APP). In parasite-infested nestlings, methionine had a negative effect on body mass close to fledging. Methionine had an immune-enhancing effect in the absence of ectoparasites only. The inflammatory response to LPS was significantly lower in nestlings infested with fleas and was also lower in nestlings supplemented with methionine. These patterns of immune responses suggest an immunosuppressive effect of ectoparasites that could neutralise the immune-enhancing effect of methionine. Our study thus suggests that the trade-off between investment in life history traits and immune function is only partly dependent on available resources, but shows that parasites may influence this trade-off in a more complex way, by also inhibiting important physiological functions.

  16. Methionine and S-Adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis

    PubMed Central

    Zubiete-Franco, Imanol; García-Rodríguez, Juan Luis; Martínez-Uña, Maite; Martínez-Lopez, Nuria; Woodhoo, Ashwin; Juan, Virginia Gutiérrez-De; Beraza, Naiara; Lage-Medina, Sergio; Andrade, Fernando; Fernandez, Marta Llarena; Aldámiz-Echevarría, Luis; Fernández-Ramos, David; Falcon-Perez, Juan Manuel; Lopitz-Otsoa, Fernando; Fernandez-Tussy, Pablo; Barbier-Torres, Lucía; Luka, Zigmund; Wagner, Conrad; García-Monzón, Carmelo; Lu, Shelly C.; Aspichueta, Patricia; Mato, José María; Martínez-Chantar, María Luz; Varela-Rey, Marta

    2015-01-01

    Background & Aims Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe NAFLD. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. Methods We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. Results We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and Methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameloriated liver steatosis. Conclusions These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis. PMID:26394163

  17. Dietary crude protein has minimal effect on the activity of selected enzymes of methionine catabolism in kittens fed diets near-limiting in methionine.

    PubMed

    Strieker, M J; Morris, J G; Avery, E H; Freedland, R A; Rogers, Q R

    2008-04-01

    Previous experiments have shown that increasing the dietary crude protein (CP) of cats does not increase urea cycle enzymes or alanine amino transferase as occurs in rats. Also when an essential amino acid (EAA) is limiting in a diet for growing kittens, the kittens do not exhibit an amino acid imbalance when other EAAs are added to the diet. To study the metabolic basis for these observations which are different from that found in omnivores and herbivores, the hypothesis that increased dietary CP decreases methionine catabolism, so more is spared for growth, was tested. Fifteen male kittens were randomly assigned to one of three dietary treatments. Each diet contained 2.5 g l-methionine/kg diet and 200, 300 or 500 g CP/kg diet. The livers and kidneys were removed and assayed for methionine transaminase (MTA), cystathionase (CASE) and cystathionine synthase (CS). Free amino acid concentrations were determined in liver, kidney and plasma. The 300 and 500 g CP/kg groups had significantly greater kidney weights and body weight gains than the 200 g CP/kg group. Hepatic MTA activity was lower in the 300 than the 200 or 500 g CP/kg groups (p < 0.05). Renal MTA and CASE activities were 35% and 50% greater, respectively, for the 500 g CP/kg group than for the 200 g CP/kg diet group (p < 0.05). Renal CS activities for the 300 and 500 g CP/kg groups were 29% (p > 0.05) and 38% (p < 0.05) greater, respectively, than the 200 g CP/kg group. Cyst(e)ine concentrations were lower in the livers of the 500 g CP/kg group than the 200 g CP/kg group (p < 0.05). Cystathionine was lower in plasma and kidney from the 500 g CP/kg diet group than from the 200 g CP/kg diet group (p < 0.05). It was concluded that the metabolic basis for the increased growth of kittens fed diets marginally limiting in methionine, with increasing concentrations of dietary CP, was not mediated through decreased enzyme activity associated with the catabolism of methionine, but was the result of an increase in

  18. Impact of Methionine Oxidation on Calmodulin Structural Dynamics

    PubMed Central

    McCarthy, Megan R.; Thompson, Andrew R.; Nitu, Florentin; Moen, Rebecca J.; Olenek, Michael J.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of either M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron-electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous x-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: In both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ~4 nm (closed) and another at ~6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1.5 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift. PMID:25478640

  19. Gastrointestinal methionine shuttle: Priority handling of precious goods.

    PubMed

    Mastrototaro, Lucia; Sponder, Gerhard; Saremi, Behnam; Aschenbach, Jörg R

    2016-12-01

    Methionine (Met) is a neutral, sulfur-containing, essential amino acid with biological functions in the initiation and prolongation step of protein synthesis, transmethylation reactions, the synthesis of cysteine and cystine, and as a component of antioxidant systems. Its key importance is reflected by the fact that it is usually absorbed from the diet with highest efficiency among all proteinogenic amino acids but may yet not optimally support metabolism and health. As such, crystalline Met supplements are partly used in man and heavily used in production of animal species (poultry, fish, shrimps, pigs and cattle) to provide improved health and performance. The main intention of this review is to analyze the current knowledge on transport proteins with proven or hypothetical relevance for Met absorption in the gastrointestinal tract, especially the small intestine. These transporters include Na(+) -dependent B(0) AT1 and ATB(0,+) and the Na(+) -independent exchanger b(0,+) /rBAT in the apical membrane, which may be supported by the Na(+) -dependent systems ASCT2 and IMINO. The basolateral exit of Met appears to be largely limited to a single uniporter protein, LAT4. Insufficient or overtaxed efflux via LAT4 may lead to significant intracellular accumulation and metabolism of Met in the absorptive state. The latter can release large amounts of homocysteine into the blood, which favors atherosclerosis and other cardiovascular, as well as neurological, diseases. When LAT4 is defective, basolateral Met exit may be compensated to a certain degree by the Met exchange proteins 4F2hc/LAT2 or 4F2hc/LAT1; while carriers 4F2hc/y(+) LAT1, 4F2hc/y(+) LAT2, SNAT1 and SNAT2, may serve primarily for basolateral Met import. Expression of SNAT2 is increased when amino acid supply from the lumen ceases, suggesting a key role for Met supply of enterocytes in interdigestive periods. Enterocytes themselves have a huge requirement for Met to synthesize mucins and glutathione. © 2016

  20. Conversion of methionine into homocysteic acid in heavily oxidized proteomics samples.

    PubMed

    Bern, Marshall; Saladino, Jessica; Sharp, Joshua S

    2010-03-01

    Analysis of protein oxidation is necessary in numerous areas of biochemistry, including hydroxyl radical surface mapping, oxidative stress assays, and pharmaceutical stability testing. Mass spectrometry is one of the tools most often used to identify protein oxidation products, and previous studies have attempted to identify and characterize all of the major oxidation products detected by mass spectrometry for each amino acid residue. In this note, we present evidence that in heavily oxidized protein samples, such as those produced by hydroxyl radical surface mapping, a major oxidation product of methionine is homocysteic acid. The formation of homocysteic acid from methionine was previously unrecognized in other mass spectrometric analyses, and has important implications for the analysis of oxidized samples, as well as potential implications as to the functional consequences of methionine oxidation. Copyright (c) 2010 John Wiley & Sons, Ltd.

  1. Conversion of Methionine to Homocysteic Acid in Heavily Oxidized Proteomics Samples

    PubMed Central

    Bern, Marshall; Saladino, Jessica; Sharp, Joshua S.

    2010-01-01

    Analysis of protein oxidation is necessary in numerous areas of biochemistry, including hydroxyl radical surface mapping, oxidative stress assays, and pharmaceutical stability testing. Mass spectrometry is one of the tools most often used to identify protein oxidation products, and previous studies have attempted to identify and characterize all of the major oxidation products detected by mass spectrometry for each amino acid residue. In this note, we present evidence that in heavily oxidized protein samples, such as those produced by hydroxyl radical surface mapping, a major oxidation product of methionine is homocysteic acid. The formation of homocysteic acid from methionine was previously unrecognized in other mass spectrometric analyses, and has important implications for the analysis of oxidized samples, as well as potential implications as to the functional consequences of methionine oxidation. PMID:20169556

  2. Quantitative requirements of the hatchling green sea turtle for lysine, tryptophan, and methionine.

    PubMed

    Wood, J R; Wood, F E

    1977-02-01

    The quantitative requirement for the amino acids lysine, tryptophan, and methionine was determined for the hatchling green sea turtle (Chelonia mydas). Hatchling green sea turtles were fed synthetic diets of purified substances with the composition of the diet varying in the amount of lysine, tryptophan or methionine present. The lysine requirement was found to be 4.8% of the crude protein (N X 6.25) or 1.7% of the dry diet. The tryptophan requirement was found to be 0.63% of the crude protein or 0.22% of the dry diet. The methionine requirement, in the presence of adequate cystine (3.1% of the crude protein), was found to be 1.5% of the crude protein or 0.49% of the dry diet.

  3. Flavour sulphides are produced from methionine by two different pathways by Geotrichum candidum.

    PubMed

    Demarigny, Y; Berger, C; Desmasures, N; Gueguen, M; Spinnler, H E

    2000-08-01

    We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5'-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography-mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography-mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.

  4. Methionine-mediated gene expression and characterization of the CmhR regulon in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Shafeeq, Sulman

    2016-01-01

    This study investigated the transcriptomic response of Streptococcus pneumoniae D39 to methionine. Transcriptome comparison of the S. pneumoniae D39 wild-type grown in chemically defined medium with 0–10 mM methionine revealed the elevated expression of various genes/operons involved in methionine synthesis and transport (fhs, folD, gshT, metA, metB-csd, metEF, metQ, tcyB, spd-0150, spd-0431 and spd-0618). Furthermore, β-galactosidase assays and quantitative RT-PCR studies demonstrated that the transcriptional regulator, CmhR (SPD-0588), acts as a transcriptional activator of the fhs, folD, metB-csd, metEF, metQ and spd-0431 genes. A putative regulatory site of CmhR was identified in the promoter region of CmhR-regulated genes and this CmhR site was further confirmed by promoter mutational experiments. PMID:28348831

  5. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  6. Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters.

    PubMed

    Marie, Benjamin; Joubert, Caroline; Belliard, Corinne; Tayale, Alexandre; Zanella-Cléon, Isabelle; Marin, Frédéric; Gueguen, Yannick; Montagnani, Caroline

    2012-05-01

    Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO3 and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.

  7. Fluoresence quenching of riboflavin in aqueous solution by methionin and cystein

    NASA Astrophysics Data System (ADS)

    Drössler, P.; Holzer, W.; Penzkofer, A.; Hegemann, P.

    2003-01-01

    The fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes of riboflavin in methanol, DMSO, water, and aqueous solutions of the sulphur atom containing amino acids methionin and cystein have been determined. In methanol, DMSO, and water (pH=4-8) only dynamic fluorescence reduction due to intersystem crossing and internal conversion is observed. In aqueous methionin solutions of pH=5.25-9 a pH independent static and dynamic fluorescence quenching occurs probably due to riboflavin anion-methionin cation pair formation. In aqueous cystein solutions (pH range from 4.15 to 9) the fluorescence quenching increases with rising pH due to cystein thiolate formation. The cystein thiol form present at low pH does not react with neutral riboflavin. Cystein thiolate present at high pH seems to react with neutral riboflavin causing riboflavin deprotonation (anion formation) by cystein thiolate reduction to the cystein thiol form.

  8. Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase*

    PubMed Central

    Samson, Andre L.; Knaupp, Anja S.; Kass, Itamar; Kleifeld, Oded; Marijanovic, Emilia M.; Hughes, Victoria A.; Lupton, Chris J.; Buckle, Ashley M.; Bottomley, Stephen P.; Medcalf, Robert L.

    2014-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression. PMID:25086035

  9. Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice

    PubMed Central

    Gupta, Sapna; Wang, Liqun; Kruger, Warren D.

    2015-01-01

    Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the TgI278T Cbs−/− mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. TgI278T Cbs−/− mice on betaine, from the time of weaning until for 240 days of age, had a 40% decrease in mean tHcy level and a 137% increase in serum methionine levels. Betaine-treated Tg-I278T Cbs−/− mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs−/− animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs+/− mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice. PMID:26231230

  10. Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice.

    PubMed

    Gupta, Sapna; Wang, Liqun; Kruger, Warren D

    2016-01-01

    Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the Tg-I278T Cbs (-/-) mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. Tg-I278T Cbs (-/-) mice on betaine, from the time of weaning until for 240 days of age, had a 40 % decrease in mean tHcy level and a 137 % increase in serum methionine levels. Betaine-treated Tg-I278T Cbs (-/-) mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs (-/-) animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs (+/-) mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice.

  11. Isotopic Dilution GC/MS Method for Methionine Determination in Biological Media

    NASA Astrophysics Data System (ADS)

    Horj, Elena; Iordache, Andreea; Culea, Monica

    2011-10-01

    The isotopic dilution mass spectrometry technique is the method of choice for sensitive and accurate determination of analytes in biological samples. The aim of this work was to establish a sensitive analytical method for the determination of methionine in different biological media. Quantitation of methionine from the resultant tracer spectrum requires deconvolution of the enrichment of the isotopomers. Deconvolution of the ion abundance ratios to yield tracer-to-tracee ratio for the isotopomer was done using Brauman's least squares approach. Comparison with regression curve calculation method is presented. The method was applied for amino-acids determination in beef, pork and fish meat.

  12. Metabolic adaptations to methionine restriction that benefit health and lifespan in rodents.

    PubMed

    Perrone, Carmen E; Malloy, Virginia L; Orentreich, David S; Orentreich, Norman

    2013-07-01

    Restriction of dietary methionine by 80% slows the progression of aged-related diseases and prolongs lifespan in rodents. A salient feature of the methionine restriction phenotype is the significant reduction of adipose tissue mass, which is associated with improvement of insulin sensitivity. These beneficial effects of MR involve a host of metabolic adaptations leading to increased mitochondrial biogenesis and function, elevated energy expenditure, changes of lipid and carbohydrate homeostasis, and decreased oxidative damage and inflammation. This review summarizes observations from MR studies and provides insight about potential mediators of tissue-specific responses associated with MR's favorable metabolic effects that contribute to health and lifespan extension.

  13. Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration

    SciTech Connect

    Wiederhold, M.D.; Ou, D.W.

    1986-03-05

    Regulation of eicosanoid biosynthesis by neuropeptides was investigated in human peripheral blood monocytes from normal donors. Metabolites of /sup 3/H-arachidonic acid (/sup 3/H-AA) were analyzed by thin layer and high pressure liquid chromatography following exposure to 0.2 ..mu..gm/ml and 2.0 ..mu..gm/ml of leucine (L-ENK) and methionine (M-ENK) enkephalin. Supernatants of cultured cells were analyzed. The data indicate that both leucine and methionine enkephalin can stimulate eicosanoid biosynthesis in human monocytes, and may indicate a possible regulatory mechanism between the central nervous system and the reticuloendothelial system.

  14. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  15. Metabolism of excess methionine in the liver of intact rat: an in vivo /sup 2/H NMR study

    SciTech Connect

    London, R.E.; Gabel, S.A.; Funk, A.

    1987-11-03

    L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo /sup 2/H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-(methyl-/sup 2/H/sub 3/)methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small /sup 2/H resonances assigned to N/sup 1/-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.

  16. Modified bean seed protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations

    USDA-ARS?s Scientific Manuscript database

    The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three dimensional structure...

  17. The reliability of methionine challenge test is not affected by a deficiency in pyridoxal phosphate, folic acid, or cobalamin.

    PubMed

    Nooij, Linda S; van Eyck, Jim; Klip, Helen; van de Leur, Josephus J C M; Bosma, Joost P F

    2011-01-01

    Abnormal methionine challenge test results cannot be explained by a deficiency in pyridoxal phosphate, folic acid, or cobalamin. The methionine challenge test is reliable for demonstrating hyperhomocysteinemia. To research the status of pyridoxal phosphate, folic acid, and cobalamin as part of an examination for hyperhomocysteinemia and to establish their relationship to the results of methionine challenge tests in women, who were tested at least 3 months after they had ended a pregnancy which was complicated by preeclampsia or other vascular-related pregnancy complications. In the Isala clinics in Zwolle, women with vascular-related complications of pregnancy were tested at least 3 months postpartum for hyperhomocysteinemia, by performing a methionine challenge test, as well as tests to measure their vitamin status. The diagnosis of hyperhomocysteinemia was made after two abnormal test results. The χ(2) test was used to compare the vitamin status of the group with normal results to those with an abnormal result of methionine challenge tests. No statistically significant differences in the vitamin status were found between the group of women with an abnormal and the group with normal results of the methionine challenge tests. Abnormal methionine challenge test results cannot be explained by a deficiency in pyridoxal phosphate, folic acid, or cobalamin. We demonstrate that, when women are tested 3 months postpartum, the methionine challenge test is reliable for demonstrating hyperhomocysteinemia as a risk factor for vascular-related complications of pregnancy.

  18. Influence of methionine administration during chelation of cadmium by CaNa(3)DTPA and DMPS in the rat.

    PubMed

    Tandon, S K; Singh, S; Prasad, S

    1997-07-01

    Influence of methionine administration was investigated in rats on the efficacy of calcium trisodium diethylenetriamine pentaacetate (CaNa(3)DTPA) and 2,3-dimercaptopropane-1 sulfonate (DMPS) in the treatment of cadmium intoxication. CaNa(3)DTPA, DMPS or methionine were quite effective in mobilizing cadmium from blood and all the tissues examined in cadmium pre-exposed animals. The combination of CaNa(3)DTPA and methionine was more efficient in reducing hepatic, renal and heart cadmium levels while that of DMPS and methionine was more efficient in lowering liver, kidney and brain cadmium levels than either of them alone. The combinations were also highly effective in enhancing the urinary and the fecal excretion of cadmium. The treatment with CaNa(3)DTPA, DMPS or methionine was quite effective in reversing cadmium inhibited tissue enzymes and alterations in blood and serum biochemical levels. The combined treatment with a chelator and methionine was more effective than the chelators alone in restoring cadmium induced changes in hepatic and renal transaminases. The treatment with CaNa(3)DTPA, DMPS or methionine appreciably decreased the depletion of endogenous zinc, copper and iron due to cadmium but the combined treatments were more efficient than the individuals in restoring the kidney and the brain copper levels only. The results show that the administration of methionine during chelation therapy may be beneficial in the treatment of cadmium intoxication.

  19. [Phenotypic and technological influences of the Lupinus mutabilis (Tarwi) seed on its methionine availability and sulfur content].

    PubMed

    Oliveros, M; Schoeneberger, H; Gross, R; Reynoso, Z

    1983-09-01

    The present study was carried out to determine the content of available methionine and sulphur in seed cultivars of Lupinus mutabilis from different Andean regions, and to study the influence of processing on methionine and sulphur contents. An additional objective was to evaluate interrelationships among these chemical characteristics and protein quality, as measured by the protein efficiency ratio (PER) method. Results revealed a high variability in the content of available methionine and sulphur between the different ecotypes and varieties of Lupinus mutabilis. Fertilization with CaSO4 (200 kg/ha) did alter the content of available methionine and sulphur in Lupinus albus seeds. Traditional water-debittering of lupines did not affect the methionine content of the seeds, whereas oil-extraction and alcohol-debittering led to a decrease in available methionine (14 and 23% reduction, respectively). Production of a protein isolate further reduced the methionine content (54%). Regression analysis revealed a high correlation between available methionine and sulphur (r = 0.83), between sulphur and PER (r = 0.98) in the processed lupine samples, and lupine mixtures with other protein sources.

  20. The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity.

    PubMed

    Labrenz, Steven R; Calmann, Melissa A; Heavner, George A; Tolman, Glen

    2008-01-01

    Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.

  1. Bioavailability of D-methionine relative to L-methionine for nursery pigs using the slope-ratio assay

    PubMed Central

    Kong, Changsu; Ahn, Jong Young

    2016-01-01

    This experiment was conducted to determine the bioavailability of D-methionine (Met) relative to L-Met for nursery pigs using the slope-ratio assay. A total of 50 crossbred barrows with an initial BW of 13.5 kg (SD = 1.0) were used in an N balance study. A Met-deficient basal diet (BD) was formulated to contain an adequate amount of all amino acids (AA) for 10–20 kg pigs except for Met. The two reference diets were prepared by supplementing the BD with 0.4 or 0.8 g L-Met/kg at the expense of corn starch, and an equivalent concentration of D-Met was added to the BD for the two test diets. The pigs were adapted to the experimental diets for 5 d and then total but separated collection of feces and urine was conducted for 4 d according to the marker-to-marker procedure. Nitrogen intakes were similar across the treatments. Fecal N output was not affected by Met supplementation regardless of source and consequently apparent N digestibility did not change. Conversely, there was a negative linear response (P < 0.01) to Met supplementation with both Met isomers in urinary N output, which resulted in increased retained N (g/4 d) and N retention (% of intake). No quadratic response was observed in any of the N balance criteria. The estimated bioavailability of D-Met relative to L-Met from urinary N output (g/4 d) and N retention (% of intake) as dependent variables using supplemental Met intake (g/4 d) as an independent variable were 87.6% and 89.6%, respectively; however, approximately 95% of the fiducial limits for the relative bioavailability estimates included 100%. In conclusion, with an absence of statistical significance, the present study indicated that the mean relative bioequivalence of D- to L-Met was 87.6% based on urinary N output or 89.6% based on N retention. PMID:27651987

  2. Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.

    PubMed

    Leahy, D J; Erickson, H P; Aukhil, I; Joshi, P; Hendrickson, W A

    1994-05-01

    Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.

  3. Methionine-enriched diet decreases hippocampal antioxidant defences and impairs spontaneous behaviour and long-term potentiation in rats.

    PubMed

    Viggiano, Alessandro; Viggiano, Emanuela; Monda, Marcellino; Ingrosso, Diego; Perna, Alessandra F; De Luca, Bruno

    2012-08-30

    Diets high in methionine lead to elevation of plasma homocysteine levels which are possibly linked to neurodegenerative diseases and oxidative stress. In the present study, we investigated the effects of methionine-enriched diet on antioxidant defences, on rat spontaneous behaviour and on the ability to sustain long-term potentiation in the dentate gyrus (DG). Sprague-Dawley rats were fed either a standard laboratory diet or a methionine enriched-diet (1% or 5% methionine in drinking water) for 8 weeks. After the 8 weeks, the animals were tested for spontaneous motor activity and habituation in an open field maze, for anxiety-like behaviour in an elevated plus maze and for the ability to sustain long-term potentiation (LTP) induced in the dentate gyrus under urethane anaesthesia. The brains were then removed and histochemically stained for superoxide dismutase (SOD) activity. Rats fed on 5% methionine significantly reduced total distance travelled during the open field test and exhibited no habituation with respect to the other two groups. Rats fed on 5% methionine also showed a significant increase of the anxiety level. Moreover, in this group, the ability to induce LTP in DG was impaired. SOD activity was significantly increased in the cerebral cortex of the rats fed on 1% and 5% methionine with respect to the control group. In conclusion, 5% methionine in drinking water led to evident impairment of locomotor skills and of synaptic plasticity. SOD activity in the cortex was increased in both the groups fed on 1% and 5% methionine, thus suggesting that metabolic adjustments, triggered by the methionine-enriched diet, are likely mediated by reactive oxygen species. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

    PubMed

    Gonda, Itay; Lev, Shery; Bar, Einat; Sikron, Noga; Portnoy, Vitaly; Davidovich-Rikanati, Rachel; Burger, Joseph; Schaffer, Arthur A; Tadmor, Ya'akov; Giovannonni, James J; Huang, Mingyun; Fei, Zhangjun; Katzir, Nurit; Fait, Aaron; Lewinsohn, Efraim

    2013-05-01

    Sulfur-containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur-containing and other volatiles. L-methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C- and ²H-labeled L-methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an L-methionine aminotransferase and preserves the main carbon skeleton of L-methionine. The second route apparently involves the action of an L-methionine-γ-lyase activity, releasing methanethiol, a backbone for formation of thiol-derived aroma volatiles. Exogenous L-methionine also generated non-sulfur volatiles by further metabolism of α-ketobutyrate, a product of L-methionine-γ-lyase activity. α-Ketobutyrate was further metabolized into L-isoleucine and other important melon volatiles, including non-sulfur branched and straight-chain esters. Cell-free extracts derived from ripe melon fruit exhibited L-methionine-γ-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-γ-lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co-segregated with the levels of sulfur-containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that L-methionine is a precursor of both sulfur and non-sulfur aroma volatiles in melon fruit.

  5. The Efficiency of Methionine as a Radioprotectant of Bacillus anthracis for Cell Viability and Outgrowth Time after UVC and Gamma Irradiation

    DTIC Science & Technology

    2015-03-01

    helped to prevent spores from clumping or aggregating to the sides of the reactor. Models were created using SolidWorks software...left) and a profile (right) of the carousel. The model was created using SolidWorks software...aminobutyric acid, methionine sulfoxide and methionine sulfone [45]. Figure 5 shows the formation of the higher oxidation states of methionine

  6. Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

    USDA-ARS?s Scientific Manuscript database

    We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pi...

  7. Prediction of (L)-methionine VCD spectra in the gas phase and water solution.

    PubMed

    Rode, Joanna E; Dobrowolski, Jan Cz; Sadlej, Joanna

    2013-11-21

    In this paper we provide a computational study of the l-methionine conformational landscape and VCD spectra in the gas phase and a water environment simulated by implicit PCM and the hybrid model, i.e., a combination of explicit "microsolvation" and implicit models. In the gas phase, two groups of conformers differing in H-bonding, i.e., OH···NH2 and NH···O═C, could be distinguished based solely on the IR ν(OH) and ν(NH) stretching vibrations range. On the other hand, VCD better reflected chain differences. The most stable OH···NH2 conformer was predicted to be easily detected, and the presence of two out of four NH···O═C conformers could be confirmed. Three zwitterionic methionine conformers were shown to dominate in water. Their VCD spectra, simulated within the hybrid model at the B3LYP-IEF-PCM/aug-cc-pVDZ level of theory, indicated that they could be recognized in the mixture. Use of the hybrid model is crucial for good reproduction of the hydrogen bonding pattern in the VCD spectra of methionine in water solution. However, the 1300-800 cm(-1) region of the skeleton vibrations of methionine appeared to be relatively insensitive to the model of the solvent.

  8. Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2†

    PubMed Central

    Dias, Benjamin; Weimer, Bart

    1998-01-01

    l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases. PMID:9726878

  9. [Biomass production enriched in intracellular methionine by a mutant of Saccharomyces cerevisiae].

    PubMed

    Albornoz, I J; Sánchez Crispin, J A; Moreno, R

    1993-01-01

    According with FAO reported data the methionine intracellular content in Saccharomyces cerevisiae is higher than another yeast. For increasing the yeast methionine internal concentration three S. cerevisiae mutant strains were chosen (M2, M4 y M9), obtained by ethionine (0.1 mg/ml) and norleucine (0.33 mg/ml) resistance by González Miliani (personal communication). The resistance levels in culture were modified until selection of a mutant LF-M9 etr norr, which shows resistance to ethionine (6 mg/ml). Optimal conditions for growth were fixed on shaked flasks and later mutationaly experiments were conducted with nitrosoguanidine (0.5 mg/ml) and U.V. light (240 nm in 9 minutes d = 32 cm). Mutants obtained were selected on plate replicates and microbiological test, using Escherichia coli 303 (Wollman, met- b1- strr) as the indicating strain. The mutant LF-M9 treo- etr norr, shows an intracellular methionine content 3 times higher than the control strain DSM D273-10B and 1.8 times higher than parental strain M9. The mutant was cultivated on different agroindustrial wastes and the optimal growth was reached in acid hydrolysates of cassava foliage. Fermentations in 1 litre stirred fermentor were accomplished using these medium and the biomass obtained was 6.3 g of the yeast (dry weight) enriched in methionine per litre of extract.

  10. Characteristics of viable Brevibacterium linens cells, methionine and cysteine in milkfat-coated microcapsules.

    PubMed

    Kim, S C; Olson, N F

    1985-01-01

    The potential for microencapsulation of viable Brevibacterium linens with methionine or cysteine in milkfat to produce sulphur compounds was examined in this study. More than 80 per cent of B. linens cells were encapsulated and then numbers inside capsules increased about three-fold during 48 h at 26 degrees C under anaerobic conditions before a slow decline. Most of micro-organisms (7 x 10(7)/ml) were still viable in the capsules after 15 days. More than 90 per cent of cysteine and methionine were encapsulated and 90, 80 and 60 per cent of the encapsulated amino acids were maintained in the capsules at 4, 12 and 26 degrees C, respectively, after 24 h. Most of the cysteine was oxidized to cystine during microencapsulation but still available to micro-organisms whereas methionine remained in the reduced form. Partition coefficients of methionine and cysteine to milkfat were 0.117 and 0.275, respectively, indicating that most of these substrates would be available to cells of B. linens in the capsules.

  11. Purification and characterization of L-methionine gamma-lyase from brevibacterium linens BL2

    PubMed

    Dias; Weimer

    1998-09-01

    L-Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the alpha,gamma elimination of methionine to produce methanethiol, alpha-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25 degreesC but was active between 5 and 50 degreesC. Activity was inhibited by carbonyl reagents, completely inactivated by DL-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of L-methionine gamma-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 &mgr;mol min-1 mg-1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

  12. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    EPA Science Inventory

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
    S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

    Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  13. Dietary methionine restriction increases fat oxidation in obese adults with metabolic syndrome.

    PubMed

    Plaisance, Eric P; Greenway, Frank L; Boudreau, Anik; Hill, Kasey L; Johnson, William D; Krajcik, Rozlyn A; Perrone, Carmen E; Orentreich, Norman; Cefalu, William T; Gettys, Thomas W

    2011-05-01

    In preclinical reports, restriction of dietary methionine intake was shown to enhance metabolic flexibility, improve lipid profiles, and reduce fat deposition. The present report is the outcome of a "proof of concept" study to evaluate the efficacy of dietary methionine restriction (MR) in humans with metabolic syndrome. Twenty-six obese subjects (six male and 20 female) meeting criteria for metabolic syndrome were randomized to a diet restricted to 2 mg methionine/kg body weight per day and were provided capsules containing either placebo (n = 12) or 33 mg methionine/kg body weight per day (n = 14). Energy expenditure, body composition, insulin sensitivity, and biomarkers of metabolic syndrome were measured before and after 16 wk on the respective diets. Insulin sensitivity and biomarkers of metabolic syndrome improved comparably in both dietary groups. Rates of energy expenditure were unaffected by the diets, but dietary MR produced a significant increase in fat oxidation (MR, 12.1 ± 6.0% increase; control, 8.1 ± 3.3% decrease) and reduction in intrahepatic lipid content (MR liver/spleen attenuation ratio, 8.1 ± 3.3% increase; control ratio, 2.2 ± 2.1% increase) that was independent of the comparable reduction in weight and adiposity that occurred in both groups. Sixteen weeks of dietary MR in subjects with metabolic syndrome produced a shift in fuel oxidation that was independent of the weight loss, decreased adiposity, and improved insulin sensitivity that was common to both diets.

  14. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    EPA Science Inventory

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)

    Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  15. Structure-function relationship in an archaebacterial methionine sulphoxide reductase B.

    PubMed

    Carella, Michela; Becher, Juliane; Ohlenschläger, Oliver; Ramachandran, Ramadurai; Gührs, Karl-Heinz; Wellenreuther, Gerd; Meyer-Klaucke, Wolfram; Heinemann, Stefan H; Görlach, Matthias

    2011-01-01

    Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs. © 2010 Blackwell Publishing Ltd.

  16. Zwitterionic self-assembly of L-methionine nanogratings on the Ag(111) surface.

    PubMed

    Schiffrin, Agustin; Riemann, Andreas; Auwärter, Willi; Pennec, Yan; Weber-Bargioni, Alex; Cvetko, Dean; Cossaro, Albano; Morgante, Alberto; Barth, Johannes V

    2007-03-27

    The engineering of complex architectures from functional molecules on surfaces provides new pathways to control matter at the nanoscale. In this article, we present a combined study addressing the self-assembly of the amino acid L-methionine on Ag(111). Scanning tunneling microscopy data reveal spontaneous ordering in extended molecular chains oriented along high-symmetry substrate directions. At intermediate coverages, regular biomolecular gratings evolve whose periodicity can be tuned at the nanometer scale by varying the methionine surface concentration. Their characteristics and stability were confirmed by helium atomic scattering. X-ray photoemission spectroscopy and high-resolution scanning tunneling microscopy data reveal that the L-methionine chaining is mediated by zwitterionic coupling, accounting for both lateral links and molecular dimerization. This methionine molecular recognition scheme is reminiscent of sheet structures in amino acid crystals and was corroborated by molecular mechanics calculations. Our findings suggest that zwitterionic assembly of amino acids represents a general construction motif to achieve biomolecular nanoarchitectures on surfaces.

  17. Independent and Additive Effects of Glutamic Acid and Methionine on Yeast Longevity

    PubMed Central

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2013-01-01

    It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan. PMID:24244480

  18. Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

    USDA-ARS?s Scientific Manuscript database

    Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...

  19. Levels of Key Enzymes of Methionine-Homocysteine Metabolism in Preeclampsia

    PubMed Central

    Pérez-Sepúlveda, Alejandra; España-Perrot, Pedro P.; Fernández B, Ximena; Ahumada, Verónica; Bustos, Vicente; Arraztoa, José Antonio; Dobierzewska, Aneta; Figueroa-Diesel, Horacio; Rice, Gregory E.; Illanes, Sebastián E.

    2013-01-01

    Objective. To evaluate the role of key enzymes in the methionine-homocysteine metabolism (MHM) in the physiopathology of preeclampsia (PE). Methods. Plasma and placenta from pregnant women (32 controls and 16 PE patients) were analyzed after informed consent. Protein was quantified by western blot. RNA was obtained with RNA purification kit and was quantified by reverse transcritase followed by real-time PCR (RT-qPCR). Identification of the C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) and A2756G methionine synthase (MTR) SNP was performed using PCR followed by a high-resolution melting (HRM) analysis. S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) were measured in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). The SNP association analysis was carried out using Fisher's exact test. Statistical analysis was performed using a Mann-Whitney test. Results. RNA expression of MTHFR and MTR was significantly higher in patients with PE as compared with controls. Protein, SAM, and SAH levels showed no significant difference between preeclamptic patients and controls. No statistical differences between controls and PE patients were observed with the different SNPs studied. Conclusion. The RNA expression of MTHFR and MTR is elevated in placentas of PE patients, highlighting a potential compensation mechanism of the methionine-homocysteine metabolism in the physiopathology of this disease. PMID:24024209

  20. Independent and additive effects of glutamic acid and methionine on yeast longevity.

    PubMed

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2013-01-01

    It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan.

  1. 1H, 15N and 13C NMR Assignments of Mouse Methionine Sulfoxide Reductase B2

    PubMed Central

    Breivik, Åshild S.; Aachmann, Finn L.; Sal, Lena S.; Kim, Hwa-Young; Del Conte, Rebecca; Gladyshev, Vadim N.; Dikiy, Alexander

    2011-01-01

    A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with 15N and 15N/13C was generated. We report here the 1H, 15N and 13C NMR assignments of the reduced form of this protein. PMID:19636904

  2. Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.

    PubMed

    Narayanan, Sai Shyam; Ramanujan, Ajeena; Krishna, Shyam; Nampoothiri, Kesavan Madhavan

    2008-12-01

    The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.

  3. Interrelations between Glycine Betaine Catabolism and Methionine Biosynthesis in Sinorhizobium meliloti Strain 102F34

    PubMed Central

    Barra, Lise; Fontenelle, Catherine; Ermel, Gwennola; Trautwetter, Annie; Walker, Graham C.; Blanco, Carlos

    2006-01-01

    Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B12-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment. PMID:17015658

  4. Corynebacterium diphtheriae Methionine Sulfoxide Reductase A Exploits a Unique Mycothiol Redox Relay Mechanism*

    PubMed Central

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-01-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen. PMID:25752606

  5. Evaluating a quantitative methionine requirement for juvenile Pacific white shrimp Litopenaeus vannamei

    USDA-ARS?s Scientific Manuscript database

    A 10-wk feeding trial was conducted as a third study (all conducted in our laboratory) to determine a quantitative requirement of juvenile Litopenaeus vannamei for sulfur amino acid methionine. Juvenile shrimp (mean weight 0.61 +/- 0.13 g) were reared in 110-L aquaria in a seawater recirculating sy...

  6. Overexpression of methionine-R-sulfoxide reductases has no influence on fruit fly aging

    PubMed Central

    Shchedrina, Valentina A.; Vorbrüggen, Gerd; Cheon Lee, Byung; Kim, Hwa-Young; Kabil, Hadise; Harshman, Lawrence G.; Gladyshev, Vadim N.

    2009-01-01

    Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine residues in proteins. This function implicated Msrs in antioxidant defense and the regulation of aging. There are two known Msr types in animals: MsrA specific for the reduction of methionine-S-sulfoxide, and MsrB that catalyzes the reduction of methionine-R-sulfoxide. In a previous study, overexpression of MsrA in the nervous system of Drosophila was found to extend lifespan by 70%. Overexpression of MsrA in yeast also extended lifespan, whereas MsrB overexpression did so only under calorie restriction conditions. The effect of MsrB overexpression on lifespan has not yet been characterized in any animal model systems. Here, the GAL4-UAS binary system was used to drive overexpression of cytosolic Drosophila MsrB and mitochondrial mouse MsrB2 in whole body, fatbody, and the nervous system of flies. In contrast to MsrA, MsrB overexpression had no consistent effect on the lifespan of fruit flies on both corn meal and sugar yeast diets. Physical activity, fecundity, and stress resistance were also similar in MsrB-overexpressing and control flies. Thus, MsrA and MsrB, the two proteins with identical function in antioxidant protein repair, have different effects on aging in fruit flies. PMID:19409408

  7. Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

    PubMed Central

    Walasek, Paula; Honek, John F

    2005-01-01

    Background The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region. Results To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme. Conclusion Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease. PMID:16221305

  8. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  9. Carbon-11-methionine and PET in evaluation of treatment response of breast cancer.

    PubMed Central

    Huovinen, R.; Leskinen-Kallio, S.; Någren, K.; Lehikoinen, P.; Ruotsalainen, U.; Teräs, M.

    1993-01-01

    Uptake of L-methyl-11C-methionine (11C-methionine) in breast cancer metastases was studied with positron emission tomography (PET). Eight patients with soft tissue metastases were studied twice: before the onset of chemotherapy (4), hormonal therapy (3) or radiotherapy (1) and 3-14 weeks later. The radioactivity concentration of the low molecular weight fraction of venous plasma samples separated by fast gel filtration was used as input function. The input corrected uptake rate of 11C-methionine (Ki) in breast cancer metastases before the treatment ranged between 0.035 and 0.186 1 min-1 and the standardised uptake value (SUV) between 2.0 and 11.4. The uptake of 11C-methionine into the metastases decreased when clinical objective stability or regression of the metastases was later obtained and increased in cases where progressive disease was seen during treatment. We conclude that metabolic changes in the amino acid metabolism detected by PET precede the clinical response, and may be of clinical value in predicting the treatment response. Images Figure 1 PMID:8471437

  10. PET evaluation of spinal cord tumor using sup 11 C-methionine

    SciTech Connect

    Higano, S.; Shishido, F.; Nagashima, M.; Tomura, N.; Murakami, M.; Inugami, A.; Fujita, H.; Tabata, K.; Yasui, N.; Uemura, K. )

    1990-03-01

    A cervical cord tumor was examined with positron emission tomography using L-methyl-({sup 11}C)methionine. The radioactive tracer accumulated in the solid parts (but not in the associated cysts) of the neoplasm, which at histology was found to be an ependymoma.

  11. Zwitterionic self-assembly of l-methionine nanogratings on the Ag(111) surface

    PubMed Central

    Schiffrin, Agustin; Riemann, Andreas; Auwärter, Willi; Pennec, Yan; Weber-Bargioni, Alex; Cvetko, Dean; Cossaro, Albano; Morgante, Alberto; Barth, Johannes V.

    2007-01-01

    The engineering of complex architectures from functional molecules on surfaces provides new pathways to control matter at the nanoscale. In this article, we present a combined study addressing the self-assembly of the amino acid l-methionine on Ag(111). Scanning tunneling microscopy data reveal spontaneous ordering in extended molecular chains oriented along high-symmetry substrate directions. At intermediate coverages, regular biomolecular gratings evolve whose periodicity can be tuned at the nanometer scale by varying the methionine surface concentration. Their characteristics and stability were confirmed by helium atomic scattering. X-ray photoemission spectroscopy and high-resolution scanning tunneling microscopy data reveal that the l-methionine chaining is mediated by zwitterionic coupling, accounting for both lateral links and molecular dimerization. This methionine molecular recognition scheme is reminiscent of sheet structures in amino acid crystals and was corroborated by molecular mechanics calculations. Our findings suggest that zwitterionic assembly of amino acids represents a general construction motif to achieve biomolecular nanoarchitectures on surfaces. PMID:17372212

  12. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE

    EPA Science Inventory

    CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
    S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE

    Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental...

  13. CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC (III) METHYLTRANSFERASE (CYT19)

    EPA Science Inventory

    CLONING, EXPRESSION, AND CHARACTERIZATION OF RAT S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE (cyt19)

    Stephen B. Waters1 , Felicia Walton1 , Miroslav Styblo1 , Karen Herbin-Davis2, and David J. Thomas2 1 School of Medicine, University of North Carolina at Chape...

  14. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    PubMed

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  15. Coupling Oxidative Signals to Protein Phosphorylation via Methionine Oxidation in Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    The mechanisms involved in sensing oxidative signaling molecules such as H2O2 in plant and animal cells are not completely understood. In the present study, we tested the postulate that oxidation of methionine (Met) to Met sulfoxide (MetSO) can couple oxidative signals to changes in protein phosphor...

  16. Methionine kinetics and balance at the 1985 FAO/WHO/UNU intake requirement in adult men studied with L-(2H3-methyl-1-13C)methionine as a tracer

    SciTech Connect

    Young, V.R.; Wagner, D.A.; Burini, R.; Storch, K.J. )

    1991-08-01

    The upper range of the requirement for methionine plus cystine in healthy adults was proposed in 1985 by FAO/WHO/UNU to be 13 mg.kg body wt-1.d-1. To explore the validity of this estimate, five healthy, young adult men were given for 7 d a diet based on an L-amino acid mixture supplying 13 mg methionine.kg-1.d-1 (87 mumol.kg-1.d-1) without cystine. Constant intravenous infusions of L-(2H3-methyl-1-13C)methionine were given on days 5 and 7 while subjects were in the fed and postabsorptive states, respectively. Estimates were made of methionine oxidation, and daily methionine balance was derived from the intake-oxidation data. For the five subjects, methionine balances were -0.9, +0.7, +3.5, -3.1, and -3.8 mg kg-1.d-1, or -6, +5, +23, -21, and -26 mumol.kg-1.d-1. These findings lead to the conclusion that the upper range of the requirement for methionine plus cystine probably exceeds 13 mg.kg-1.d-1 in healthy young adults. The implications of this conclusion for establishing an appropriate amount of sulfur amino acids in an amino acid requirement pattern for adults is discussed.

  17. Methionine oxidation as a major cause of the functional impairment of oxidized actin.

    PubMed

    Dalle-Donne, I; Rossi, R; Giustarini, D; Gagliano, N; Di Simplicio, P; Colombo, R; Milzani, A

    2002-05-01

    A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl

  18. Methionine as a potential precursor for halogenated compounds by the reaction with iron minerals

    NASA Astrophysics Data System (ADS)

    Tubbesing, C.; Krause, T.; Mulder, I.; Kotte, K.; Schöler, H. F.

    2012-04-01

    Volatile halogenated compounds (VOX) play an important role in different photochemical reactions within the troposphere and the stratosphere. Soils and sediments seem to act as a major natural source for VOX, but investigations of the reaction mechanisms are rather scarce. To get further information on potential intermediates the reaction of the amino acid methionine with the ferrous and ferric iron minerals pyrite and ferrihydrite as well as solute ferrous sulfate was studied using a gas chromatography-flame ionization detector (GC-FID). Methionine is an important amino acid in the biosynthesis of plants used as a starting compound for the messenger ethene with aminocyclopropane carboxylic acid as an intermediate product. This pathway may also occur under abiotic conditions. Ethene is assumed as precursor for various halogenated C2-compounds like vinyl chloride and dichloroethene. Due to its ubiquity by an average concentration of 10 to 290 ng/g soil and its potential to regenerate in soils and organic litter by microorganisms, methionine may be an important educt for both abiotic and biotic terrestrial halogenation processes. In laboratory tests methionine was exposed to different iron species like pyrite, iron sulfate or ferrihydrite. The oxidant H2O2 was used to start the reaction. Production values of methyl chloride and other halogenated compounds are discussed in the context of methionine as their potential precursor and several Fe-minerals as soil-borne catalysers. Several possible intermediates for the production of VOX have been detected e.g. methane, ethene or propane. A formation of isobutylene is noteworthy for some cases. In addition to VOC the production of methyl chloride and dimethyl sulfide (DMS) was observed. Only the DMS bears upon a specific mineral. The samples containing pyrite reveal the highest concentrations. To get a better assessment of methionine, respectively VOC released from methionine as precursors for halogenated compounds

  19. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  20. Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig.

    PubMed

    Halsted, Charles H; Villanueva, Jesus A; Devlin, Angela M; Niemelä, Onni; Parkkila, Seppo; Garrow, Timothy A; Wallock, Lynn M; Shigenaga, Mark K; Melnyk, Stepan; James, S Jill

    2002-07-23

    Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2'-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.

  1. Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts.

    PubMed

    Tan, Amelia W J; Lee, Pin-Rou; Seow, Yi-Xin; Ong, Peter K C; Liu, Shao-Quan

    2012-08-01

    Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.

  2. Influence of methionine oxidation on the aggregation of recombinant human growth hormone.

    PubMed

    Mulinacci, Filippo; Poirier, Emilie; Capelle, Martinus A H; Gurny, Robert; Arvinte, Tudor

    2013-09-01

    Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group. In previous papers, we showed that oxidation of the two most accessible methionine residues of recombinant human growth hormone (r-hGH), Met¹⁴ and Met¹²⁵, has no influence on the conformation of the protein [1]. However, the oxidized r-hGH is less thermally stable than the native protein [2]. In the current work, the consequences of the oxidation of these two methionine residues on the aggregation of r-hGH were investigated. The aggregation properties and kinetics of the native and oxidized r-hGH were measured in different buffers with both spectroscopic and chromatographic methods. Stabilities of oxidized and non-oxidized r-hGH were studied after storage at 37°C and freeze/thawing cycles. Methionine oxidation influenced the aggregation properties of r-hGH. In accelerated stability studies at 37°C, oxidized hormone aggregated more and faster than non-oxidized hormone. In freezing/thawing stability studies, it was found that oxidized r-hGH was less stable than its non-oxidized counterpart. In case of hGH, we have shown that chemical degradations such as oxidation can affect its physical stability and can induce aggregation.

  3. Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

    PubMed Central

    Halsted, Charles H.; Villanueva, Jesus A.; Devlin, Angela M.; Niemelä, Onni; Parkkila, Seppo; Garrow, Timothy A.; Wallock, Lynn M.; Shigenaga, Mark K.; Melnyk, Stepan; James, S. Jill

    2002-01-01

    Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2′-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury. PMID:12122204

  4. THE DISTRIBUTION OF METHIONINE-ENKEPHALIN AND LEUCINE-ENKEPHALIN IN THE BRAIN AND PERIPHERAL TISSUES

    PubMed Central

    Hughes, J; Kosterlitz, HW; Smith, TW

    1997-01-01

    A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD-2 resin. Elution with methanol separates the enkephalins and α-endorphin from β-endorphin. Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine- and leucine-enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins. An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed ∈-endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts. A differential assay of methionine- and leucine-enkephalin was made either by destroying methionine-enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography. The ratio of methionine-enkephalin to leucine-enkephalin varied greatly in different brain regions. The highest proportions of leucine-enkephalin were found in the cerebral cortex and hippocampus. Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides. It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine- and leucine-enkephalin may subserve separate neuronal functions. PMID:9142421

  5. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: potential roles in toxicity.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2009-05-01

    L-methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 degrees C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-d-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-dl-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  6. Kinetic evidence for separate systems in transport of D- and L-methionine by rat small intestine

    SciTech Connect

    Brachet, P.; Alvarado, F.; Puigserver, A.

    1987-03-01

    The kinetics of D- and L-methionine uptake by rings of everted intestine in vitro are consistent with a saturable Michaelis-Menten component plus a linear, diffusional one. All the data could be fit with a diffusion constant, which was essentially the same, independent of whether it was estimated by iteration or by using the extracellular marker, (/sup 3/H), inulin. Similar results were obtained from in vivo perfusion experiments, except that the diffusional term was negligible. D-(3,4-/sup 14/C)Methionine was found to inhibit L-methionine uptake by intestinal rings according to fully noncompetitive kinetics. Another set of experiments with jejunal brush-border membrane vesicles showed that D-methionine uptake is dependent on a Na/sup +/ gradient and is significantly inhibited by L-(/sup 35/S) methionine and L-prolie, but not by ..beta..-alanine and ..cap alpha..-methylaminoisobutyric acid. The results indicate that, in rat jejunum, D-methionine is taken up through a Na/sup +/-dependent pathway distinct from the neutral amino acid (L-methionine) carrier and from the amino acid (L-proline,..cap alpha..-methylaminoisobutyric acid, ..beta..-alanine) carrier.

  7. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    PubMed

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. S-adenosyl methionine (SAMe) for depression in adults.

    PubMed

    Galizia, Ilaria; Oldani, Lucio; Macritchie, Karine; Amari, Erica; Dougall, Dominic; Jones, Tessa N; Lam, Raymond W; Massei, Guido Jacopo; Yatham, Lakshmi N; Young, Allan H

    2016-10-10

    Depression is a recurrent illness with high rates of chronicity, treatment-resistance and significant economic impact. There is evidence in the literature that S-adenosyl methionine (SAMe), a naturally occurring compound in the human body, has antidepressant efficacy. This product may be an important addition to the armamentarium of antidepressant agents. To assess the effects of SAMe in comparison with placebo or antidepressants for the treatment of depression in adults. We searched the Cochrane Common Mental Disorders Group's Specialised Register (CCMDCTR Studies and Reference Register), MEDLINE, EMBASE, PsycINFO, international trial registers ClinicalTrials.gov and the World Health Organization trials portal (ICTRP). We checked reference lists, performed handsearching and contacted experts in the field. The CCMDCTR literature search was last updated on 5 February 2016. Randomised controlled trials comparing SAMe with placebo or antidepressants in adults with a diagnosis of major depression. Two authors independently performed extraction of data and assessment of risk of bias. We contacted trialists of included studies for additional information. This systematic review included eight trials comparing SAMe with either placebo, imipramine, desipramine or escitalopram. We accepted trials that used SAMe as monotherapy or as add-on therapy to selective serotonin reuptake inhibitors (SSRIs), and we accepted both oral and parenteral administration. The review involved 934 adults, of both sexes, from inpatient and outpatient settings.The trials were at low risk of reporting bias. We judged the risk of selection, performance, detection and attrition bias as unclear or low, and one study was at high risk of attrition bias.There was no strong evidence of a difference in terms of change in depressive symptoms from baseline to end of treatment between SAMe and placebo as monotherapy (standardised mean difference (SMD) -0.54, 95% confidence interval (CI) -1.54 to 0.46; P = 0

  9. Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

    PubMed

    Tang, Xiaohu; Keenan, Melissa M; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J Will; Freedland, Stephen J; Murphy, Susan K; Chi, Jen-Tsan

    2015-04-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  10. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

    PubMed Central

    Tang, Xiaohu; Keenan, Melissa M.; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J. Will; Freedland, Stephen J.; Murphy, Susan K.; Chi, Jen-Tsan

    2015-01-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  11. The importance of transmethylation reactions to methionine metabolism in sheep: effects of supplementation with creatine and choline.

    PubMed

    Lobley, G E; Connell, A; Revell, D

    1996-01-01

    The influence of administering the methylated products choline and creatine on methionine irreversible-loss rate (ILR) and recycling from homocysteine has been investigated in sheep fed close to energy and N equilibrium. Two methods to estimate methionine recycling were compared. The first involved [U-13C]methionine infused as part of a labelled amino acid mixture obtained from hydrolysed algal protein. In this approach the isotope dilution of methionine with all five C atoms labelled (m + 5) will represent the ILR which does not recycle through homocysteine, while that which includes molecules with C-1-C-4 labelled will allow for loss of the labelled methyl (5)-C atom and replacement by an unlabelled moiety in the remethylation of homocysteine. The second method involved a combined infusion of [1-13C]- and [S-methyl-2H3]methionine. These two approaches gave similar data for methionine ILR which does not include label recycled to the amino acid from homocysteine but differed for recycled methionine fluxes. Consequently the two procedures differed in the calculated extent of homocysteine methylation under control conditions (6 v. 28%). These extents of remethylation are within the range observed for the fed human subject, despite the fact that fewer dietary methyl groups are available for the ruminant. Using combined data from the infusions, significant depression of methionine recycling occurred in blood (P < 0.05), with a similar trend for plasma (P = 0.077), when choline plus creatine were infused. Wool growth, assessed by intradermal injection of [35S]cysteine, was not altered by supplementation with the methylated products. From changes in the label pattern of free methionine in aortal, hepatic portal and hepatic venous blood during U-13C-labelled algal hydrolysate infusion, the major sites of homocysteine remethylation appear to be the portal-drained viscera and the liver. This was confirmed by analysis of free methionine enrichments in various tissues

  12. Quantitative study in vivo of methionine cycle in humans using (methyl-/sup 2/H/sub 3/)- and (1-/sup 13/C)methionine

    SciTech Connect

    Storch, K.J.; Wagner, D.A.; Burke, J.F.; Young, V.R.

    1988-09-01

    Kinetic aspects of body methionine (MET) metabolism were examined in healthy young men during the fed and postabsorptive (PA) states. Rates of MET incorporation (S) into and release (B) from body proteins; transmethylation (TM); and remethylation (RM) and transsulfuration (TS) of homocysteine (HCY) were estimated with the aid of a 5-h constant intravenous infusion of (methyl-2H3)- and (1-13C)methionine. The isotopic data (plasma methionine labeling and 13C enrichment of expired air) were submitted to a stochastic model of amino acid metabolism. During the fed state, the subjects (n = 4) received, at 20-min intervals, small isonitrogenous isocaloric meals containing a complete L-amino acid mixture supplying MET at a rate equivalent to 198 mumol.kg body wt-1.day-1. The PA subjects (n = 4) received the isotope after a 10-h overnight fast. For the PA group, the components of MET metabolism were as follows: S, 20 +/- 0.5; B, 24 +/- 0.5; TM, 5.8 +/- 0.6; RM, 1.8 +/- 0.4; and TS, 4.0 +/- 0.4 (+/-SE) mumol.kg-1.h-1. During the fed state the values were S, 26 +/- 2.5; B, 18 +/- 2; TM, 14 +/- 1.3; RM, 5.7 +/- 0.9; and TS 8.3 +/- 0.6 mumol.kg-1.h-1. The meal-induced changes in B, TM, RM, and TS were significant (P less than 0.05). Comparison of the partitioning of MET between S and TM (these two pathways of MET disposal constitute the ''methionine locus'') in the PA and in the fed states indicates that the MET locus is of regulatory importance in MET homeostasis. A twofold increase in the partitioning of MET to TM was observed in the fed state. The increase in HCY recycling, relative to TS (these two pathways of HCY disposal constitute the ''HCY locus''), in the fed state did not reach statistical significance when compared with the PA state. Total daily TM are estimated to be 238 +/- 22 mumol/kg. This is similar to the estimate generated by the methyl balance model of Mudd and Poole which approximated 241 mumol/kg.

  13. Effect of dietary chromium-L-methionine on glucose metabolism of beef steers.

    PubMed

    Kegley, E B; Galloway, D L; Fakler, T M

    2000-12-01

    Thirty-six crossbred steers (288 +/- 3.7 kg initial BW) were used to determine the effect of Cr, as chromium-L-methionine, on glucose tolerance and insulin sensitivity in beef calves. Calves were fed a control diet or the diet supplemented with 400 or 800 microg Cr/kg of diet as chromium-L-methionine. Calves were kept in drylots (six calves/pen; two pens/dietary treatment). Steers were caught twice a day in locking headgates and individually fed their respective diets for a period of 22, 23, or 24 d prior to the metabolic challenges. Calves received a totally mixed diet containing 54% corn, 38% cottonseed hulls, and 5% soybean meal. On d 21, 22, and 23, four calves/dietary treatment were fitted with an indwelling jugular catheter. Approximately 24 h after catheterization, an intravenous glucose tolerance test (500 mg glucose/kg of BW), followed 5 h later by an intravenous insulin challenge test (0.1 IU insulin/kg of BW), was conducted. There was no effect (P > 0.10) of dietary treatment on ADG or ADFI. During the intravenous glucose tolerance test, serum insulin concentrations were increased by supplemental chromium-L-methionine (linear effect of Cr, P < 0.05). There was a time x treatment interaction (P < 0.05) on plasma glucose concentrations after the glucose infusion. Plasma glucose concentrations of calves fed 400 microg Cr/kg of diet were lower than those of controls and calves supplemented with 800 microg Cr/kg of diet (quadratic effect of Cr, P < 0.05) 5 and 10 min after the glucose infusion. Supplemental chromium-L-methionine increased the glucose clearance rate from 5 to 10 min after the insulin challenge test (linear effect of Cr, P < 0.05). Glucose half-life from 5 to 10 min after the insulin infusion was also decreased by supplemental chromium-L-methionine (linear effect of Cr, P < 0.10). These data indicate that supplemental Cr, as chromium-L-methionine, increased glucose clearance rate after an insulin infusion and increased the insulin response to

  14. Effects of methionine on the cytoplasmic distribution of actin and tubulin during neural tube closure in rat embryos

    PubMed Central

    Moephuli, Shadrack R.; Klein, Norman W.; Baldwin, Michael T.; Krider, Hallie M.

    1997-01-01

    Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [methyl-14C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-14C-labeled proteins were identified as actin, αβ-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and αβ-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology. PMID:9012820

  15. Identification and characterization of a methionine γ-lyase in the calicheamicin biosynthetic cluster of Micromonospora echinospora.

    PubMed

    Song, Haigang; Xu, Ri; Guo, Zhihong

    2015-01-02

    CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal-5'-phosphate-dependent enzyme and exhibits high sequence homology to cystathionine γ-lyases and cystathionine γ-synthases. However, it was found to be active towards methionine and to convert this amino acid into α-ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor-bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ-lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ-lyase producing methanethiol as a building block in biosynthesis of calicheamicins.

  16. Structural Transitions Induced by a Recombinant Methionine-Trigger in Silk Spidroin

    NASA Astrophysics Data System (ADS)

    Wilson, Donna; Winkler, Stefan; Valluzzi, Regina; Kaplan, David

    2000-03-01

    Control of beta sheet formation is an important factor in the understanding and prediction of structural transitions and protein folding. In genetically engineered silk proteins this control has been achieved using oxidative triggers. A genetically engineered variant of a spider silk protein, and a peptide analog, based on the consensus sequence of Nephila clavipes dragline silk, were modified to include methionines flanking the beta sheet forming polyalanine regions. These methionines could be selectively reduced and oxidized, altering the bulkiness and charge of the sulfhydryl group to control beta sheet formation by steric hindrance. Biophysical characterization and monitoring of structural transitions and intermediates were accomplished through attenuated total reflectance infrared spectroscopy (ATR-IR) for solution state structures in both oxidized and reduced forms. For solid state structural characterization, IR microscopy and reflectance IR experiments were performed. Electron diffraction data as well as circular dichroism studies provide structural corroboration for all experiments in which reproducible sample preparation was achieved.

  17. Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions.

    PubMed

    Chugha, Preeti; Sage, Harvey J; Oas, Terrence G

    2006-03-01

    Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.

  18. One-pot modification of 5'-capped RNA based on methionine analogs.

    PubMed

    Muttach, Fabian; Rentmeister, Andrea

    2016-09-01

    This paper outlines chemically and enzymatically synthesized S-adenosylmethionine (AdoMet) analogs and their use in the site-specific modification of RNA by methyltransferases, enabling the facile attachment of clickable moieties to the nucleic acid. We then focus on methodological aspects of setting up a methyltransferase-based enzymatic cascade reaction starting from methionine analogs. This strategy is applied to the one-pot modification of the mRNA cap which is subsequently derivatized in copper-free and copper-catalyzed click reactions. We show that high transfer efficiencies to the cap are obtained using Se-propargyl-, hexenynyl- and azido-bearing methionine analogs. By switching to other methyltransferases our one-pot modification approach should be directly applicable to the regiospecific modification of other target molecules including nucleic acids, proteins and small molecules.

  19. Fibrin Clot Structure and Mechanics Associated with Specific Oxidation of Methionine Residues in Fibrinogen

    PubMed Central

    Weigandt, Katie M.; White, Nathan; Chung, Dominic; Ellingson, Erica; Wang, Yi; Fu, Xiaoyun; Pozzo, Danilo C.

    2012-01-01

    Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels. PMID:23283239

  20. Isolation and primary structure of a methionine- and cystine-rich seed protein of Cannabis sativa.

    PubMed

    Odani, S; Odani, S

    1998-04-01

    A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

  1. Stabilization of methionine-rich protein in Saccharomyces cerevisiae: targeting of BZN protein into the peroxisome.

    PubMed

    Nicaud, J M; Raynal, A; Beyou, A; Merkamm, M; Ito, H; Labat, N

    1994-01-01

    We have constructed a gene coding for the 12-kDa intermediate form of the 2s methionine-rich protein from Bertholletia excelsa seeds. This protein, expressed intracellularly in yeast, is characterised by a 20-min half-life. By adding 11 amino acids corresponding to the peroxisome-targeting sequence (PTSc) of luciferase, we have significantly increased its half-life. This stabilization allowed accumulation of the BZN protein into the peroxisome as judged by cell fractionation. Accumulation of the 12-kDa protein results in a significant increase of the total methionine content in yeast cells (30%) indicating that such a microorganism could represent a practicable protected shuttle for an animal-feed additive.

  2. Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

    NASA Astrophysics Data System (ADS)

    Yermolaieva, Olena; Xu, Rong; Schinstock, Carrie; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2004-02-01

    Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

  3. Protective effect of S-adenosyl-L-methionine against CCl4-induced hepatotoxicity in cultured hepatocytes.

    PubMed

    Tsuji, M; Kodama, K; Oguchi, K

    1990-02-01

    Effect of S-adenosyl-L-methionine disulfate tosylate salt (SAMe-ST) and L-methionine (L-Met) on primary cultured rat hepatocytes were studied. In cultured hepatocytes treated with CCl4, SAMe-ST and L-Met suppressed the decrease in urea-nitrogen secretion as well as the leakages of GOT and GPT. The membrane-protective action of these two compounds was verified by the histological data. Failure of SAMe-ST to counteract CCl4-induced reduction of radioactive leucine incorporation into the trichloroacetic acid-insoluble materials in hepatocytes indicates that the observed effects of SAMe-ST or L-Met do not involve acceleration of protein synthesis. The present results indicate that SAMe-ST remarkably protects hepatocytes from CCl4-induced hepatotoxicity, probably by either changing the structure or compositions of membrane phospholipids or by modifying the interaction of CCl4 with the intracellular drug-metabolizing enzyme systems.

  4. New Enzyme Prodrug and Methionine-Depletion Combination Therapy of Breast Cancer Designed for Effective Delivery to the Tumor

    DTIC Science & Technology

    2009-10-01

    methylselenol , α-ketobutyrate, and ammonia. Methylselenol has been shown to be cytotoxic to various cancer cells. To accomplish the specific aims...is planned to repeat this test for MDA-MB-231 cells with 750 µM L-methionine. 10 Figure 4: Effect of SeMet conversion to methylselenol ...to methylselenol on MCF-7 breast cancer cells. Cells were grown in medium adjusted to 2000 µM of L-methionine. Cell viability was assessed using

  5. A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs

    PubMed Central

    Castellano, Rosa; Perruchot, Marie-Hélène; Conde-Aguilera, José Alberto; van Milgen, Jaap; Collin, Anne; Tesseraud, Sophie; Mercier, Yves; Gondret, Florence

    2015-01-01

    Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue

  6. Effects of methionine supplementation on the expression of oxidative stress-related genes in acute heat stress-exposed broilers.

    PubMed

    Del Vesco, Ana Paula; Gasparino, Eliane; Grieser, Daiane de Oliveira; Zancanela, Vittor; Soares, Maria Amélia Menck; Neto, Adhemar Rodrigues de Oliveira

    2015-02-28

    The aim of the present study was to evaluate the effects of heat stress (HS) and methionine supplementation on the markers of stress and on the gene expression levels of uncoupling proteins (UCP), betaine-homocysteine methyltransferase (BHMT), cystathionine β-synthase (CBS), glutathione synthetase (GSS) and glutathione peroxidase 7 (GPx7). Broilers from 1 to 21 d and from 22 to 42 d of age were divided into three treatment groups related to methionine supplementation: without methionine supplementation (MD); recommended level of methionine supplementation (DL1); excess methionine supplementation (DL2). The broilers were either kept at a comfortable thermal temperature or exposed to HS (38°C for 24 h). During the starter period, we observed the effects of the interaction between diet and environment on the gene expression levels of UCP, BHMT and GSS. Higher gene expression levels of UCP and BHMT were observed in broilers that were maintained at thermal comfort conditions and received the MD diet. HS broilers fed the DL1 and DL2 diets had the highest expression level of GSS. The expression levels of the CBS and GPx7 genes were influenced by both the environment and methionine supplementation. During the grower period, the gene expression levels of BHMT, CBS, GSS and GPx7 were affected by the diet × environment interaction. A higher expression level of BHMT was observed in broilers maintained at thermal comfort conditions and on the MD diet. HS induced higher expression levels of CBS, GSS and GPx7 in broilers that received the DL1 and DL2 diets. The present results suggest that under HS conditions, methionine supplementation could mitigate the effects of stress, since methionine contributed to the increased expression levels of genes related to antioxidant activity.

  7. [The feed value in growing pigs of a new cultivar of field beans (Vicia faba L.) supplemented with DL-methionine or DL-methionine-hydroxyanalog].

    PubMed

    Abel, H j; Burghard, G

    2002-02-01

    A basal control mixture of barley, soy bean meal and soy bean oil was replaced by 25% of the new field bean-cultivar 'Divine' and the resulting two mixtures were supplemented with minerals, trace elements, vitamins and amino acids according to the ideal protein concept. The control diet was adjusted with DL-methionine (DL-Met), the field bean mixture either with DL-Met or DL-methionine-hydroxyanalogue (DL-MHA) assuming biological equivalence on a molar basis for both supplements. The three experimental diets were fed to growing pigs (35-40 kg bwt.). Spontaneous urine samples were analysed separately for determining parameters that characterize the acid-base status of the pigs. There were no significant differences between experimental groups in nutrient digestibilities. The level of bacterially fermentable substances was increased in the diets containing field beans. The field beans contained 14 mg ME/kg DM. There were no significant (p < 0,05) differences in N- and mineral-retentions (Ca, P, Na, K) between the treatments. The stronger alkalinity found in urine after feeding the field bean mixtures resulted from a higher electrolyte balance of the diet.

  8. Genetic Studies of Sulfadiazine-resistant and Methionine-requiring Neisseria Isolated From Clinical Material

    PubMed Central

    Catlin, B. Wesley

    1967-01-01

    Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species. Images PMID:4962305

  9. Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

    PubMed

    Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

    2017-04-01

    l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  10. Biochemical and histologic presentations of female Wistar rats administered with different doses of paracetamol/methionine.

    PubMed

    Iyanda, A A; Adeniyi, F A A

    2011-12-20

    This study was carried out to compare the hepatoprotective effect of methionine on paracetamol treated rats at both the peaks of toxicity and absorption. Female Wistar rats were divided into 17 groups consisting of eight rats per group and treated with different doses of paracetamol/methionine (5:1). Each control rat received 5 ml of physiologic saline. The study was terminated at two different end points -the 4th and 16th hours. Results show that rats administered with toxic doses (1000 mg/kg, 3000 mg/kg, 5000 mg/kg BW) of paracetamol exhibited significant increases in the levels of ALT, AST, γ- GT compared with controls. These increases were much higher at the 16th than 4th hour but serum total protein, albumin and globulin were significantly decreased by the end of the 16th hour. Histology results of rats in the 3000 and 5000 mg/kg (by the end of the 16th hour) confirmed hepatic damage, light microscopic evaluation of liver showed remarkable centrilobular necrosis. Moreover, the presence of mononuclear cells in liver section of rats intoxicated with APAP (5000 mg/kg) suggests a possible involvement of inflammatory process which resulted in regurgitation of bilirubin leading to its elevated level as well as increase activity of ALP. The hepatoprotective effect of methionine, on the other hand, was demonstrated in these rats at the 4th and 16th hours, and both results were comparable and therefore not significantly different but elevation in GGT level still persisted. In conclusion, data obtained from this study suggest that these agents may be capable of inducing GGT, although further study is required to establish a possible relationship between methionine and this enzyme in some other animal species.

  11. Effect of methionine and lactic acid bacteria as aflatoxin binder on broiler performance

    NASA Astrophysics Data System (ADS)

    Istiqomah, Lusty; Damayanti, Ema; Julendra, Hardi; Suryani, Ade Erma; Sakti, Awistaros Angger; Anggraeni, Ayu Septi

    2017-06-01

    The use of aflatoxin binder product based amino acids, lacic acid bacteria, and natural product gived the opportunity to be an alternative biological decontamination of aflatoxins. A study was conducted to determine the efficacy of aflatoxin binder administration (amino acid methionine and lactic acid bacteria (Lactobacillus plantarum G7)) as feed additive on broiler performance. In this study, 75 Lohmann unsexed day old chicks were distributed randomly into 5 units of cages, each filled with 15 broilers. Five cages were assigned into 5 treatments groups and fed with feed contained aflatoxin. The treatments as follow: P1 (aflatoxin feed without aflatoxin binder), P3 (aflatoxin feed + 0.8% of methionine + 1% of LAB), P4 (aflatoxin feed + 1.2% of methionine + 1% of LAB), P5 (aflatoxin feed + 1% of LAB), and K0 (commercial feed). The measurement of aflatoxin content in feed was performed by Enzyme Linked Immunosorbent Assay method using AgraQuant® Total Aflatoxin Assay Romer Labs procedure. The experimental period was 35 days with feeding and drinking ad libitum. LAB was administered into drinking water, while methionine into feed. Vaccination program of Newcastle Disease (ND) was using active vaccine at 4 and 18 day old, while Infectious Bursal Disease (IBD) was given at 8 day old. Parameter of body weight was observed weekly, while feed consumption noted daily. The result showed that aflatoxin in feed for 35 days period did not significantly affect the body weight gain and feed conversion. The lowest percentage of organ damage at 21 day old was found in P5 treatment (55%), while at 35day old was found in P4 treatment (64%). It could be concluded that technological process of detoxifying aflatoxin could be applied in an attempt to reduce the effect on the toxicity of aflatoxin in poultry feed.

  12. Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

    PubMed

    Kulikova, Vitalia V; Morozova, Elena A; Revtovich, Svetlana V; Kotlov, Mikhail I; Anufrieva, Natalya V; Bazhulina, Natalya P; Raboni, Samanta; Faggiano, Serena; Gabellieri, Edi; Cioni, Patrizia; Belyi, Yury F; Mozzarelli, Andrea; Demidkina, Tatyana V

    2017-09-01

    The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL(-1) , 4.4 U mL(-1) , 1.2 U mL(-1) , and 3.4 U mL(-1) , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  13. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    PubMed Central

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    2010-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel diverse toxic compounds from the cell.1,2 The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions.3,4 No prior structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide important new structural information about the HME sub-family of RND efflux pumps. The structures suggest that the metal binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. Intriguingly, this cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, utilizing these methionine pairs/clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites. PMID:20865003

  14. NdgR, a Common Transcriptional Activator for Methionine and Leucine Biosynthesis in Streptomyces coelicolor

    PubMed Central

    Kim, Songhee H.; Lee, Bo-Rahm; Kim, Ji-Nu

    2012-01-01

    We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts. PMID:23065973

  15. Dietary B vitamin and methionine intakes and lung cancer risk among female never smokers in China

    PubMed Central

    Takata, Yumie; Cai, Qiuyin; Beeghly-Fadiel, Alicia; Li, Honglan; Shrubsole, Martha J.; Ji, Bu-Tian; Yang, Gong; Chow, Wong-Ho; Gao, Yu-Tang; Zheng, Wei; Shu, Xiao-Ou

    2012-01-01

    Purpose B vitamins and methionine have been postulated to have potential effects on carcinogenesis; however, findings from previous epidemiologic studies on B vitamins, methionine, and lung cancer risk are inconsistent. We investigated associations of dietary intakes of B vitamins (i.e., riboflavin, niacin, vitamin B6, folate, and vitamin B12) and methionine with lung cancer risk among female never smokers. Methods The Shanghai Women’s Health Study, a population-based, prospective cohort study, included 74,941 women. During a median follow-up of 11.2 years, 428 incident lung cancer cases accrued among 71,267 women with no history of smoking or cancer at baseline. Baseline dietary intakes were derived from a validated, interviewer-administered food frequency questionnaire. Cancer incidence and vital status were ascertained through annual linkage to the Shanghai Cancer Registry and Shanghai Vital Statistics Registry databases and through biennial in-person follow-ups with participants. Adjusted hazard ratios (HR) and 95% confidence intervals (CI) were calculated using Cox regression. Results Dietary riboflavin intake was inversely associated with lung cancer risk (HR = 0.62; 95% CI = 0.43–0.89; P-trend = 0.03 for the highest quartile compared with the lowest). A higher than median intake of methionine was associated with lower risk of lung cancer (HR = 0.78; 95% CI = 0.60–0.99), however, there was no dose-response relation. Intakes of other B vitamins were not associated with lung cancer risk. Conclusions Our study suggests that dietary riboflavin intake may be inversely associated with lung cancer risk among female never smokers, which warrants further investigation. PMID:23065072

  16. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

    SciTech Connect

    Long, Feng; Su, Chih-Chia; Zimmermann, Michael T.; Boyken, Scott E.; Rajashankar, Kanagalaghatta R.; Jernigan, Robert L.; Yu, Edward W.

    2010-09-23

    Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs - three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

  17. Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens.

    PubMed

    Arfi, Kenza; Landaud, Sophie; Bonnarme, Pascal

    2006-03-01

    Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

  18. Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet.

    PubMed

    Tanaka, Naoki; Takahashi, Shogo; Fang, Zhong-Ze; Matsubara, Tsutomu; Krausz, Kristopher W; Qu, Aijuan; Gonzalez, Frank J

    2014-11-01

    Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1 week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH. Published by Elsevier B.V.

  19. Enhancing stress tolerance by overexpression of a methionine sulfoxide reductase A (MsrA) gene in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-04-01

    Proteins are subjected to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological functions. Methionine as a sulfur-containing amino acid is easily oxidized to methionine sulfoxide (MetSO). The modified methionine can be repaired by methionine sulfoxide reductase (Msr), an enzyme that reverses oxidation of methionine in proteins. In this study, a methionine sulfoxide reductase A (PoMsrA) gene from Pleurotus ostreatus was cloned and characterized. Furthermore, the function of PoMsrA gene was analyzed by overexpression in P. ostreatus via Agrobacterium-mediated transformation. Stable integration of the target gene into the genome of P. ostreatus was confirmed by PCR, fluorescence observation, and Southern blot hybridization. qRT-PCR analysis showed that PoMsrA was highly expressed in the stage of mature and young fruiting bodies as well as the osmotic stress condition of 0.3 M NaCl. Additionally, the transgenic strains with PoMsrA overexpression exhibited an enhanced tolerance to high temperature, high osmotic stress, and oxidative stress. This suggests that PoMsrA is an active player in the protection of the cellular proteins from oxidative stress damage.

  20. The allosteric regulatory mechanism of the Escherichia coli MetNI methionine ATP binding cassette (ABC) transporter.

    PubMed

    Yang, Janet G; Rees, Douglas C

    2015-04-03

    The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of D- and L-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by L-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

    PubMed Central

    Pan, Hai; Chen, Kenneth; Chu, Liping; Kinderman, Francis; Apostol, Izydor; Huang, Gang

    2009-01-01

    Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements. PMID:19165723

  2. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn.

    PubMed

    Pan, Hai; Chen, Kenneth; Chu, Liping; Kinderman, Francis; Apostol, Izydor; Huang, Gang

    2009-02-01

    Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.

  3. Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

    PubMed Central

    Berney, Michael; Berney-Meyer, Linda; Wong, Ka-Wing; Chen, Bing; Chen, Mei; Kim, John; Wang, Jingxin; Harris, David; Parkhill, Julian; Chan, John; Wang, Feng; Jacobs, William R.

    2015-01-01

    Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ΔmetA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ΔmetA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ΔmetA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb—the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action. PMID:26221021