Initiation of histone synthesis by f-met-tRNAf in an ascites cell-free system

Formyl-[³⁵S]methionine is incorporated into histones synthesized by a mouse ascites cell-free system supplemented with histone mRNA and f-[³⁵S]met-tRNA_f from yeast. Most of the [³⁵S]methionine incorporated can be shown to be at the N-terminus by Edman degradation after deformylation. This indicates that methionine can be used as the initiator amino acid for histones in this cell-free system.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Incorporation of methionine from met-tRNA-Met-F into internal positions of polypeptides by mouse liver polysomes

Two methionine accepting tRNA species corresponding to tRNA_F^Met and tRNA_M^Met from mouse ascites tumor cells were tested for their ability to donate methionine into internal positions of growing polypeptide chains on mouse liver polysomes. Both tRNA species can function in the elongation of polypeptide chains as judged by their ability to incorporate methionine into protein in the absence of chain initiation. The insertion of methionine into internal positions of polypeptide chains from Met-tRNA_F^Met was confirmed by Edman degradation and CNBr cleavage. When both tRNA^Met species were present in saturating concentrations in the cell-free system a strong preference for the incorporation of methionine from Met-tRNA_M^Met became apparent.     

Methionine or Folate and Phosphoenolpyruvate in the Biosynthesis of Threonine in Crithidia fasciculata1

SYNOPSIS. Crithidia fasciculata can synthesize threonine but it lacks an aspartokinase. The carbons of threonine may be derived from methionine when it is present in the medium. However, methionine can be synthesized by the organism provided organic sulfur is present. When both methionine and threonine are omitted from the medium, growth will occur if cysteine and high levels of folate are present. The compound common to both methionine and threonine under these conditions, α-keto-γ-hydroxy-butyrate, is derived from phosphoenolpyruvate (PEP) and the β-carbon of serine. High levels of folate are required for this coupling reaction, which is carried out by what may be called phosphoenolpyruvate-tetrahydrofolate hydroxymethyltransferase. By the use of radioactive tracers, both in growth experiments and in cell-free preparations, virtually all of the intermediates in these two series of reactions were identified.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups

Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D₂O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. ¹³C-HSQC spectra of ubiquitin produced with ¹³C/¹⁵N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-¹⁴C]glycine to ¹⁴CO₂ was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-¹⁴C]serine to ¹⁴CO₂ both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-¹⁴C]histidine to ¹⁴CO₂ is more sensitive indicator of folate deficiency than the rate of oxidation of [3-¹⁴C] serine to ¹⁴CO₂ although both are presumably tetrahydrofolate dependent.     

Oxygen utilization by Lactobacillus plantarum

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O ₂ ^– is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O ₂ ^– was markedly increased. However, the type of reaction of the O ₂ ^– decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O ₂ ^– elimination is not an enzymic reaction. Mn²⁺ phosphate(s) might be responsible for the observed elimination of O ₂ ^– . The production of O ₂ ^– is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

The Mode of Action of a Carboxypeptidase from Malt

Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.

Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.

These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH.     

A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution

During recent years, the targets of protein structure analysis using nuclear magnetic resonance spectroscopy have become larger and more complicated. As a result, a complete and precise stable isotope labeling technique has been desired. A cell-free protein synthesis system is appropriate for this purpose. In the current study, we achieved precise and complete ¹⁵N and ²H labeling using an Escherichia coli cell extract-based cell-free protein synthesis system by controlling the metabolic reactions in the system with their chemical inhibitors. The addition of aminooxyacetate, d-malate, l-methionine sulfoximine, S-methyl-l-cysteine sulfoximine, 6-diazo-5-oxo-l-norleucine, and 5-diazo-4-oxo-l-norvaline was quite effective for precise amino acid-selective ¹⁵N labeling even for aspartic acid, asparagine, glutamic acid, and glutamine, which generally suffer from severe isotope scrambling and dilution when using the conventional cell-free system. For ²H labeling, the back-protonation of the H^α and H^β positions, which commonly occurred in the conventional system, was dramatically suppressed by simply adding aminooxyacetate and d-malate to the cell-free system except for the H^α positions in methionine and cysteine.     

Amino acid requirements for growth of the rabbit blastocyst in vitro

Ten amino acids, namely, arginine, histidine, lysine, tryptophane, methionine, phenylalanine, leucine, valine, threonine and serine were indispensable for growth of rabbit blastocysts in vitro; others were nonessential. Of all the essential amino acids, arginine and lysine were required in relatively high concentrations, 10^?2 M and 10^?3 M, respectively, for optimum growth. Complete omission of the non-essential amino acids from the medium markedly reduced blastocyst growth. Interaction between serine and glycine demonstrated a partial sparing action on serine by glycine, similar to that observed between methionine and cysteine. The amino acid composition of a culture medium capable of providing continuous and consistent growth of rabbit blastocysts in vitro is described.     

Biosynthesis of vitamin B12: Timing of the methylation steps between uro'gen III and cobyrinic acid

The sequence of methylation between uro'gen III and cobyrinic acid has been defined by applying ¹³C pulse-labeling methods to a cell-free system from Propionibacterium shermanii. Feeding experiments using unenriched S-adenosyl methionine (¹²CH₃-SAM) followed by ¹³C-enriched SAM (¹³CH₃-SAM) (or vice versa) at various intervals caused differentiation in the ¹³C NMR signals of the SAM-derived methyl groups in cobyrinic acid (isolated as cobester). Unenriched uro'gen III and sirohydrochlorin as substrates led to cobyrinic acid containing seven and five enriched methyl groups, respectively, which on NMR analysis gave as a sequence of methylation C-2 > C-7 > C-20 > C-17 > C-12α > C-1 > C-5 C-15.     

Na+-Dependent Transport of Amino Acids and Its Significance for Growth of a Lysine-producing Bacterium

A lysine-producing mutant Brevibacterium flavum HUT 8052, a threonine plus methionine (or threonine plus homoserine) auxotroph, grew rapidly as nearly as the wild strain in a medium supplemented with NaCl (60 µg/ml), threonine (100 µg/ml), and methionine (33.3 µg/ml). With NaCl concentrations less than 20 µg/ml, the mutant grew little or very slowly, The peculiar growth behavior of the mutant including the above phenomenon could be reasonably explained by the finding of Na⁺-dependent amino acids transport and the feedback inhibition of homoserine dehydrogenase by threonine in the bacterium.

The threonine transport was stimulated by Na⁺ and Li⁺. though the latter being less effective. The transport of threonine was inhibited by serine. The uptake of serine, isoleucine, leucine and valine was also stimulated by Na⁺     

Competition of Ethionine and Methionine for Aminoacyl-tRNA Formation in an In Vitro System from Ochromonas danica*

Aminoacyl-tRNA synthetase and tRNA were isolated from the chrysomonad Ochromonas danica. The mutual effect of methionine and ethionine, and the effect of other amino acids on methionyl- and ethionyl-tRNA formation, were tested in an in vitro system. The tRNA^Met had a similar accepting capacity for either methionine or ethionine. Ethionine and methionine, but none of the other amino acids tested, competed for the same aminoacyl-tRNA synthetase. The K_m of methionine was 0.88 × 10^–5 M, and that of ethionine 5 × 10^–4 M. Ethionine inhibited methionine binding; K_i 3.4 × 10^–4 M. The respective values in a similar system isolated from E. coli were 2.2 × 10^–5, 1.95 × 10^–3, and 1.95 × 10^–3.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

15N and inhibitor studies on the photorespiratory nitrogen cycle in maize leaves

A combination of inhibitor and ¹⁵N studies were used to investigate the photorespiratory nitrogen cycle in maize, a C₄ plant. Inhibitors used included isonicotinyl hydrazide which blocks the conversion of glycine to serine, methionine sulfoximine an inhibitor of GS and azaserine an inhibitor of GOGAT. Results from levels of ammonia and amino acids and the distribution of ¹⁵N into NH₃, serine, glutamine and glutamate indicated that the photorespiratory N-cycle occurs in this C₄ plant, but the rate of flux through this pathway is low as compared with that in C₃ plants.Abbreviations Aza azasering - fw fresh weight - GOGAT glutamate synthase - GS glutamine synthetase - INH isonicotinyl hydrazide - MSO methionine sulfoximine     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Sulfate reduction in a cell-free system ofChlorella

Summary During sulfate reduction in a cell-free system ofChlorella activated sulfate of APS is transferred to a thiosulfonate reductase. The sulfate thus bound to the thiosulfonate reductase (i.e. bound sulfite) is reduced to bound sulfide in a ferredoxin dependent reaction. This bound sulfide can be used with O-acetylserine as acceptor for cysteine biosynthesis; serine and O-phosphoserine are not used. An assaysystem for thiosulfonate reductase activity using methylviologen dependent reduction of S₂O₄ ^2– to S^2– is developed and a procedure for isolating thiosulfonate reductase fromChlorella cells is presented. During isolation of thiosulfonate reductase a low weight molecular factor, needed for optimal enzyme activity was lost. The bound sulfite seems to be attached to this factor. Reduction of APS or GS-SO₃H to the level of S^2– is inhibited by cysteine. 50% inhibition of GS-SO₃H reduction was found at a molar cysteine concentration of 6.8×10^–5.Abbreviations APS adenosine-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - GSH reduced glutathion - GS-SO₃H S-sulfoglutathion - fd ferredoxin - Mv methylviologen - DTT dithiothreitol