Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Growth Inhibition in Thiobacillus neapolitanus by Histidine, Methionine, Phenylalanine, and Threonine

Thiobacillus neapolitanus, a strict chemoautotroph, is sensitive to the addition of 10(-4)m methionine, histidine, threonine, or phenylalanine to the thiosulfate medium on which it grows. When histidine, threonine, or phenylalanine are added at the time of inoculation, spontaneous mutants tolerant to the three amino acids are selected. These mutants appear to result from a single genetic change; of 18 independently isolated histidine-tolerant mutants, all are also tolerant to phenylalanine and threonine. The uptake of (14)C-phenylalanine into exponentially growing cells of one such mutant is negligible in contrast with the uptake observed in the phenylalanine-sensitive parent. The addition of methionine to the medium slows growth, but spontaneous mutants are not selected. Inhibition of growth by these amino acids is observed only under conditions of amino acid imbalance; the addition of an equimolar mixture of 16 amino acids, in which each component is present at a concentration of 10(-3)m, causes no inhibition. Histidine and threonine inhibition may be released by equimolar amounts of any one of seven amino acids: serine, alanine, glycine, leucine, valine, tryptophan, or tyrosine; histidine inhibition is also released by isoleucine, and threonine inhibition by methionine. None of the inhibiting amino acids inhibits oxidation of thiosulfate in cell suspensions. A group of hexoses, pentoses, and Krebs cycle intermediates were tested for inhibition of growth or release of inhibition by histidine, phenylalanine, or threonine, but no effects, either inhibition or relief of inhibition, were found.     

Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [¹⁴C]threonine and [¹⁴C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.     

Amino acid supply and protein metabolism in Ehrlich ascites tumour cells. 1. Identification of limiting amino acids

The uptake and intracellular accumulation of an amino acid mixture by incubated Ehrlich ascites tumour cells was studied. The composition of the amino acid mixture simulated that of mouse intraperitoneal fluid and amino acid uptake was studied over a range of concentrations between 0.0 (no added amino acids) and 10.0-times intraperitoneal concentrations. For most amino acids uptake into cells and intracellular accumulation occurred as concentrations were increased up to 6.0-times the intraperitoneal concentrations; further increases in external amino acid concentrations did not increase concomitantly with intracellular concentrations. These data, when analysed indicated a net protein synthetic rate of 20% d-1 and that the rate of protein synthesis may be limited by the availability of the amino acids lysine, threonine and methionine.     

Growth and metabolism of the obligate photolithotroph Chlorobium thiosulfatophilum in the presence of added organic nutrients

Growth of Chlorobium vibrioforme f. thiosulfatophilum NCIB 8327 could be monitored by measurement of turbidity (E₆₀₀); absorbance at 745 and 665 nm; increase in methanol-extractable pigment (E₆₆₀); fixation of ¹⁴CO₂; and titration of thiosulphate and sulphide in the medium. Growth could be inhibited by formate, methionine, tryptophan, tyrosine, threonine, serine and glycine, but not by 14 other amino acids, shikimic acid, some alcohols, sugars or acetate. Inhibition could some-times be relieved by the presence of other amino acids. This was probably partly due to restoration of normal internal amino acid requirements by “feeding”, and partly because uptake of amino acids appeared to show some competition for two or more low specificity uptake systems. Numerous ¹⁴C-labelled amino acids, formate and glucose were shown to be photoassimilated by Chlorobium, and the labelling patterns obtained provided information on its pathways of intermediary biosynthesis. Growth inhibition by threonine could be related to the probable presence of a normal branched pathway for the synthesis of the aspartate family of amino acids, with an aspartokinase enzyme subject to strong inhibition by threonine and lysine, separately and in combination.     

Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.     

Methionine-lysine-threonine-isoleucine interrelationships in the amino acid nutrition of rice callus tissue

Growth of rice callus tissue is discouraged when methionineis excluded from CMAA medium. While determining the methionineelimination effect, the amino acid interrelationships amongmethionine, lysine, threonine and isoleucine in the nutritionof the callus tissue were found. Poor growth, found in cultureson methionine deficient media was seen only when the media containedboth threonine and lysine, simultaneously. The substitutionof homoserine for methionine was also observed. Determination of free amino acid composition in tissues revealedthat free methionine was barely detectable in tissues grownwith sufficient amounts of threonine and lysine. When the concentrationof either threonine or lysine was reduced, the free methioninecontent of the tissue increased. When the methionine deficientmedium was supplemented with homoserine, the free methioninein the tissue increased, although the tissue retained a considerableamount of free threonine and lysine. Cultivation of tissue onan isoleucine deficient medium resulted in a significant decreasein free threonine content. These experimental results suggest that the biosynthetic pathwayto methionine is cooperatively inhibited by threonine and lysine,and that threonine decomposition is inhibited by its end productisoleucine. (Received February 19, 1970; )     

The influence of peptides on the uptake of amino acids inFusobacterium; predicted interactions withPorphyromonas gingivalis

A study of the uptake of amino acids and its influence by a peptide source was carried out withFusobacterium varium as a convenient representative of the genus. Reference strains and a clinical isolate had similar amino acid uptake profiles, but most amino acids were incorporated at lower concentrations by the latter. In general, high levels of serine, asparagine, glutamate, cysteine, and arginine were incorporated by all species. Histidine, lysine, threonine, and aspartate were taken up at lower levels, whereas the nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, glycine, proline, phenylalanine, and methionine were poorly metabolized. Yeast extract, as a source of peptides, stimulated the uptake of several amino acids such as histidine and glutamate, whereas others such as methionine, threonine, and asparagine were repressed. The incorporation of some amino acids such as aspartate, ornithine, lysine, and arginine was unaffected by the presence of peptides. Equimolar nitrogen concentrations of amino acids or ammonia could not replace the peptide requirement, emphasizing the importance of peptides as an energy source. The limited capacity ofFusobacterium spp. to hydrolyze proteins increased approximately 30% in the presence of the proteolytic species,Porphyromonas gingivalis, and may represent one bacterial interaction in which peptides may become available toFusobacterium species in vivo.     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

Summary Diploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 10⁷ suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.Abbreviation LT lysine + threonine in equimolar concentration     

Regulation of the synthesis of aspartokinase 3 of Escherichia coli by amino acids other than lysine

Summary When Escherichia coli B is grown in the presence of methionine, leucine and some other amino acids, lysine-sensitive aspartokinase (aspartokinase III) and aspartic semialdehyde dehydrogenase syntheses are derepressed. This can be explained by a synergistic inhibition between lysine and these amino acids on the lysine-sensitive aspartokinase, which leads to a decrease of the lysine intracellular pool.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Pre-proparathyroid hormone: analysis of radioactive tryptic peptides and amino acid sequence.

The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.     

General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

THE INFLUENCE OF EXCESS METHIONINE ON THE FREE AMINO ACIDS OF BRAIN AND LIVER OF THE WEANLING RAT*

Abstract— —High circulating levels of l -methionine produced by inclusion in the diet or parenteral injection of the amino acid caused alterations in the free amino acid pattern of liver and brain tissues. Acute effects following l -methionine injection were more pronounced than those following long term feeding where adaptation played a role. The net effect following parenteral injection was to increase the total free amino acids of liver while decreasing those of brain. Individually, hepatic levels of aspartic acid, threonine, serine, glutamine, glutamic acid, glycine, and alanine were depressed while levels of taurine, cystathionine, methionine, lysine, and ornithine were markedly elevated. Brain levels of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, and γ-aminobutyric acid were markedly depressed and increased levels of cystathionine, methionine, lysine, and glutamine were observed. A generalized aminoaciduria occurred shortly after excessive methionine intake. Disruption of the free amino acid pools was of two kinds. The first depended on the continued presence of excess l -methionine, the second did not.     

Nutrition of Myxococcus xanthus FBa and Some of Its Auxotrophic Mutants

A defined medium containing 15 amino acids plus salts was used to study the nutrition of Myxococcus xanthus FBa. The amino acids phenylalanine, leucine, isoleucine, valine, and methionine were essential for growth, whereas glycine, proline, asparagine, alanine, lysine, and threonine stimulated growth. An unusual pattern of requirement was found in the aromatic amino acids. Phenylalanine was essential and served as the precursor of tyrosine. Growth in the absence of tryptophan was adaptive, with cells reaching a growth rate equal to that of controls after a lag of about a week. (14)C-labeled ribose and glucose were not appreciably metabolized. Auxotrophs requiring purines and pyrimidines were isolated and were used to study the fate of externally supplied nucleic acid derivatives. Appropriate mutants could satisfy their requirements with free bases, nucleosides, and nucleotides, and could hydrolyze nucleic acids and use the products. However, studies using (14)C-ribose-labeled uridine (isolated from a Salmonella typhimurium pyrimidine auxotroph) showed that externally supplied nucleic acid derivatives were incorporated almost solely into the nucleic acids of the myxobacters, with little used either for energy-yielding oxidations or other cell anabolism.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Amino-acid synthesis in adult Dacus oleae (Gmelin) (Diptera Tephritidae) determined with [U-14C] glucose

The ability of adult Dacus oleae for amino-acid synthesis from [U-14C] glucose was investigated. The relatively high specific activity radiometric measurements indicated that both sexes were able to synthesize the amino acids: alanine, aspartic acid, cystine, glutamic acid, glycine, hydroxyproline, proline and tyrosine; therefore, these amino acids are considered as nutritionally dispensable for D. oleae. On the other hand, the amino acids: arginine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, serine, threonine, and valine, showed a very low specific activity and therefore are considered as nutritionally indispensable. It was not possible to conclude about tryptophane, since the acid hydrolysis destroyed this amino acid.