Presence Of Methionine Research Articles

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid.

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

Derivative Spectroscopy in Conjunction with Thin Layer Chromatography Applied for Determination of Mixtures of Amino Acids in Baby Food.

The preparative thin layer chromatography using silica gel and solvent systems n. butanol : acetic acid: water (4:1:1 V/V) and 96% ethanol : water (70 : 30 v/v) were used to separate the amino acids mixture into groups that can be eluted and one or two of amino acids can be determined by derivative spectroscopy. This was done for determining histidine in presence of cystine, arginine in presence of lysine and ornithine, tryptophan in presence of tyrosine and phenylalanin, and methionine in presence of aspartic acid. The proposed method is applied for determination of these amino acids in baby food, Neslac, Dialac and Nestogen. The statistical analysis of the results were found to be good and in agreement with the ion exchange chromatographic method.

Nitrogen recycling in phenylpropanoid metabolism

Approximately 30–45% of plant organic matter is derived from l-phenylalanine, and to a lesser extent from l-tyrosine, through the cinnamate pathway. In the initial step, mediated by phenylalanine ammonia lyase, cinnamic acid and an equimolar amount of ammonium ion is generated. To determine the metabolic fate of the ammonia formed, [ 15N]- l-phenylalanine was administered to potato discs in the light under aseptic conditions. It was found, using 15N NMR spectroscopic analyses, that this nitrogen is first incorporated into the amide nitrogen of glutamine and then into glutamate. When incubations were repeated in the presence of methionine- S-sulphoximine, a known inhibitor of glutamine synthetase, only resonances corresponding to 15NH 4 + and [ 15N]- l-Phe accumulated. It is proposed that during active phenylpropanoid metabolism, the ammonia released by phenylalanine ammonia lyase/tyrosine ammonia lyase is efficiently recycled back to Phe/Tyr with glutamate serving as aminoreceptor and donor. This is the first evidence for a novel nitrogen cycle in plants.

Mechanism by which noninhibitory concentrations of glucose increase inhibitory activity ofp-coumaric acid on morning-glory seedling biomass accumulation

Noninhibitory levels of glucose-C [≤ 72 µg carbon (C)/g soil] increased the inhibitory activity ofp-coumaric acid on morning-glory seedling biomass accumulation in Cecil Bt-horizon soil. The amount ofp-coumaric acid required for a given level of inhibition of shoot and seedling biomass accumulation decreased as the concentration of glucose increased. Soil extractions with neutral EDTA (0.25 M, pH 7) after addition of combinations ofp-coumaric acid and glucose (concentrations ranging from 0 to 1.25 µmol/g soil) to the soil showed that utilization ofp-coumaric acid by microbes decreased linearly as the concentration of glucose increased. The increased inhibitory activity of a given concentration ofp-coumaric acid in the presence of glucose was not due to a reduction in soil sorption ofp-coumaric acid or effects of nitrogen-limited microbial growth. Noninhibitory levels of phenylalanine andp-hydroxybenzoic acid slowed the utilization ofp-coumaric acid by microbes in a similar manner as glucose. The presence of methionine, however, did not affect the rate ofp-coumaric acid utilization by microbes. These observations suggest that differential utilization of individual molecules in organic mixtures by soil microbes can modify, and in this case increase, the effectiveness of a given concentration of an inhibitor such asp-coumaric acid on the inhibition of seedling growth such as morning-glory.

Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli.

The enzyme encoded by Escherichia coli purU has been overproduced, purified, and characterized. The enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-FH4) to FH4 and formate. Formyl-FH4 hydrolase thus generates the formate that is used by purT-encoded 5'-phosphoribosylglycinamide transformylase for step three of de novo purine nucleotide synthesis. Formyl-FH4 hydrolase, a hexamer with 32-kDa subunits, is activated by methionine and inhibited by glycine. Heterotropic cooperativity is observed for activation by methionine in the presence of glycine and for inhibition by glycine in the presence of methionine. These results, along with previous mutant analyses, lead to the conclusion formyl-FH4 hydrolase is a regulatory enzyme whose main function is to balance the pools of FH4 and C1-FH4 in response to changing growth conditions. The enzyme uses methionine and glycine to sense the pools of C1-FH4 and FH4, respectively.

Regioselective O-Demethylation of Aporphines with Methanesulfonic Acid / Methionine: An Efficient One-Pot Transformation of Thebaine to (R)(-)-2-Methoxyapomorphine

Abstract 2-Methoxyapomorphine (4) was obtained by the rearrangement of thebaine (1) with methanesulfonic acid in the presence of methionine, a reagent system suitable for the regioselective O-demethylation of aporphine derivatives.

Physico-chemical and spectroscopic properties of the monohemic cytochrome C552 from Pseudomonas nautica 617.

A c-type monohemic ferricytochrome C552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome C552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome C552 is typical of a low-spin ferric heme.

Cytokinin-like substances and ethylene production by Azospirillum in media with different carbon sources

Production of cytokinin-like substances (CLS) from two strains and ethylene from three strains of Azospirillum in the presence of different carbo sources was studied. Ethylene production depended on the presence of methionine and different carbon sources. CLS were detected by the soybean callus test and gas chromatography. A strong stimulation of soybean callus was stated with Azospirillum No 1 and 3 in media containing malate. In culture filtrates from both strains of Azospirillum 2iP = 6(γ,γ-dimethylallylamino)purine was detected. All strains of Azospirillum produced ethylene in the presence of methionine and malate, succinate and pyruvate.

The free‐radical polymerization of acrylamide initiated with ceric sulfate in the presence of amino acids

AbstractPolymerization of acrylamide initiated with ceric (IV) ion in the presence of methionine, phenylalanine, serine and aspartic acid was investigated in sulfuric acid solution. Maximum yield was obtained at 70°C and sulfuric acid concentration of 0.16 mol/1. Both conductometric titration and FT‐IR results showed that the resulting polymers contained carboxy end groups. According to these findings and previous studies in this field, a suitable polymerization mechanism was proposed.

Low density lipoprotein oxidation by stimulated neutrophils and ferritin.

Low density lipoprotein (LDL) oxidation mediated by phorbol myristate acetate (PMA)- and formylmethionylleucylphenylalanine (FMLP) -stimulated human neutrophils was enhanced by 70% in the presence of ferritin. Iron released from ferritin by the superoxide anion generated in the respiratory burst of stimulated neutrophils is shown to be involved in lipoprotein oxidation. Ascorbate (100 microM), superoxide dismutase (10 micrograms/ml) and uric acid (430 microM) showed inhibitory effects of 30% [corrected], 70% and 50% on LDL oxidation, respectively. Ceruloplasmin (2.7 microM) potentiated LDL oxidation by stimulated neutrophils and ferritin, both alone and in the presence of methionine. Methionine (1 mM) and catalase (30 micrograms/ml) increased LDL oxidation by stimulated neutrophils and ferritin. These data suggest that LDL oxidation by stimulated neutrophils and ferritin may be relevant in inflammation when both neutrophils and ferritin are increased.

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli Over-expression of infBB.su in E. coli and purification of α- and β-forms of IF2 B.su

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, α and β, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti- E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infB B.su. In this work we have cloned infB B.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2α and -β were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2α and -β, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2β is Met 94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infB B.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.

Cytochrome b of fish mitochondria is strongly resistant to funiculosin, a powerful inhibitor of respiration

We report here some unusual properties of ubiquinol: cytochrome c reductase of eel and other fish mitochondria. The turnover rate of the reductase is clearly higher than in mammalian mitochondria and the binding constant for ubiquinone seems to be larger than in other vertebrates. Additionally, the reductase activity of fish mitochondria is resistant to some powerful inhibitors that bind to cytochrome b, in particular to funiculosin. After sequencing most of the gene of eel cytochrome b and comparing the deduced amino acid sequence with that of other fish and animals, we hypothesize that the decreased binding of funiculosin could be due to a few amino acid replacements in the third and fourth transmembrane helix of the protein. In particular, the presence of methionine instead of alanine at position 125 seems to be largely responsible for the strong resistance to funiculosin and also to the partial resistance to myxothiazol in all fish mitochondria. Correlations between some residue substitutions in cytochrome b and the different effects of funiculosin in different species are also considered.

Mammalian alkaloids: Synthesis and O‐methylation of (S)‐ and (R)‐3′‐hydroxycoclaurine and their N‐methylated analogues with S‐adenosyl‐L‐[methyl‐14C]methionine in presence of mammalian catechol O‐methyltransferase

AbstractO‐Methylation of the optically active 3′‐hydroxycoclaurines 3a and 3b and of the N‐methylated analogs 5a,b with S‐adenosyl‐L‐[methyl‐14C]methionine in presence of mammalian COMT was investigated in vitro. The N‐unsubstituted (1S)‐ and (1R )‐isomers 3a and 3b, respectively, afforded almost equal amounts of the corresponding N‐norreticuline 4 and N‐nororientaline 19, besides two unknown by‐products (see Fig. and Table 1). The N‐methylated (1S)‐isoquinoline 5a, on the other hand, afforded largely (S)‐orientaline ((S)‐19), while an almost equal mixture of (R)‐reticuline (6b) and (R)‐orientaline ((R)‐19) was obtained from the (1R)‐enantiomer 5b. The isoquinolines 3a,b and 5a,b were prepared by a Bischler‐Napieralski cyclization yielding O‐benzyl‐protected isoquinoline 10 (Scheme 1). The optical resolution of 10 was accomplished with 2′‐bromotartranilic acid. The N‐methylated isoquinolines were prepared by N‐formylation of 10a,b and reduction of the formamides 13a,b with diborane (→ 14a,b). Deblocking of the benzyl‐ether moieties of 10a,b and 14a,b was accomplished by catalytic hydrogenation in presence of HCl, affording directly 3a,b·HCl and 5a,b·HCl, respectively.

Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae.

The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP:adenylylsulfate-3'-phosphotransferase, EC 2.7.1.25), has been cloned. The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23,060 dalton. Translational fusions of MET14 with the beta-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca. 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium. By primer extension the MET14 transcripts were found to start between positions -25 and -45 upstream of the initiator codon. Located upstream of the MET14 gene is a perfect match (positions -222 to -229) with the previously proposed methionine-specific upstream activating sequence (UASMet). This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PHO5 gene to which the yeast protein PHO4 binds. The human oncogenic protein c-Myc also has the same recognition sequence. Furthermore, in the 270 bp upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element. The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E. coli and chromosomally integrated. We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively. We propose that both types of control are exerted on MET14.

Inhibition of Polymorphonuclear Leukocyte-Mediated Endothelial Cell Detachment by Antileukoprotease: A Comparison with Other Proteinase Inhibitors

The role of elastase and proteinase inhibitors in polymorphonuclear leukocyte(PMN)mediated injury to human umbilical cord venous endothelial cells (HUVEC) was investigated. Both purified human neutrophil elastase and PMN that were stimulated with serum-treated zymosan (STZ) induced detachment, but not lysis of HUVEC. PMN-, but not purified elastase-mediated detachment was enhanced by the presence of methionine, which indicates a role for reactive oxygen metabolites in PMN-mediated HUVEC detachment. Detachment of HUVEC could be inhibited by secretory leukocyte proteinase inhibitor or antileukoprotease (ALP), al-proteinase inhibitor (α1-PI) and N-methoxy-succinyl-ala-ala-pro-val-chloromethyl ketone (CMK). At concentrations at which elastase-mediated detachment was maximally inhibited, ALP and CMK, but not α1-PI, were also able to inhibit maximally PMN-mediated detachment. An explanation for this difference could be that the larger size of α1-PI reduces the access of α1-PI to the interface between the PMN and the HUVEC.

Serum has a differential effect on DNA replication in a human melanoma cell line cultured in methionine or 5′-deoxy-5′-methylthioadenosine

A human melanoma cell line called MeWo-LC1 exhibits a reduced ability to synthesize DNA when cultured in serum-supplemented medium containing 5′-deoxy-5′-methylthioadenosine (MeSAdo) in place of methionine. However, DNA replication in these cells occurs normally if the cells are cultured in serum-free medium containing transferin, and MeSAdo in place of methionine. Although the presence of serum alters the cells' ability to respond to MeSAdo, it is not likely a consequence of any increased extracellular metabolism by MeSAdo-phosphorylase or adenosine deaminase activity, or due to the diminished uptake of the nucleoside. In the presence of methionine, MeSAdo appears to have a stronger cytostatic effect in medium containibg serum than in serum-free medium supplemented with transferrin. MeWo-LC1 cells contain MeSAdo-phosphorylase activity as measured both in vivo and in vitro. The diminished replication of DNA in medium containing serum and MeSAdo is likely not due to the inhibition of polyamine synthesis by the nucleoside. These results indicated that serum (factors) can have an important influence upon the ability of MeSAdo to act as a methio-source for cells cultured in the absence of methionine.

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E. coli which are conditionally defective in AdoMet synthesis. Temperature-sensitive mutants in metK, the structural gene for the S-adenosylmethionine synthetase (AdoMet synthetase) expressed in minimal medium, were constructed by in vitro mutagenesis of a plasmid-borne copy of metK. By homologous recombination, the chromosomal copy was replaced with the mutated metK gene. Both heat- and cold-sensitive mutants were examined. At the nonpermissive temperature, two such mutants had 200-fold-reduced intracellular AdoMet levels and required either methionine or vitamin B12 for growth. In the presence of methionine or vitamin B12, the mutants grew at normal rates even though the AdoMet levels remained 0.5% of wild type. A third mutant when placed at nonpermissive temperature had less than 0.2% of the normal AdoMet level and did not grow on minimal medium even in the presence of methionine or vitamin B12. All of these mutants grew normally on yeast-extract-based medium in which an alternate form of S-adenosylmethionine synthetase was expressed.

The presence of methionine or threonine at position 381 in vitronectin is correlated with proteolytic cleavage at arginine 379.

Vitronectin (serum spreading factor, complement S protein, epibolin) is a glycoprotein that mediates cell adhesion and interacts with components of the complement, coagulation, and fibrinolytic systems. It circulates in plasma as a 75-kDa single chain polypeptide and as a two-chain form consisting of 65- and 10-kDa polypeptides linked by a disulfide bond. An individual may have a predominance of the single chain or the two-chain form inherited as a Mendelian trait or have approximately equal amounts of both forms. Inspection of published cDNA sequences suggests that either methionine or threonine can occur at position 381, which is adjacent to the presumed site of proteolytic cleavage (Arg379-Ala380) that gives rise to the two-chain form. We have determined the presence of the Met381 and/or Thr381 alleles of the vitronectin gene in 42 individuals by oligonucleotide hybridization to genomic DNA. To determine whether this polymorphism is correlated with the susceptibility to cleavage of the Arg379-Ala380 peptide bond in vivo, we have prepared immunoblots of plasma from the same group of individuals. Nineteen individuals homozygous for the Thr381 allele had 17 +/- 9% (mean +/- S.D.) single chain vitronectin in their plasma samples. Nine individuals homozygous for the Met381 allele had 66 +/- 4% single chain vitronectin. Fourteen heterozygous individuals had 38 +/- 13% single chain vitronectin. The differences in mean values were statistically significant (p less than 0.001). These results suggest that the presence of threonine rather than methionine at position 381 of vitronectin increases the susceptibility of the protein to cleavage of the Arg379-Ala380 peptide bond in vivo.