In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.     

No photoperiodoc control of the formation of turions in eight clones of Spirodela polyrhiza

The influence of daily photoperiod (8, 16, 24 h) on eight clones of Spirodela polyrhiza was tested in two different nutrient media. The number of vegetative fronds and resting turions formed after 50 days of cultivation were scored. The specific turion yield (STY; number of turions formed per vegetative frond) was used to evaluate the effectiveness of turion formation of the tested clones. All clones formed turions in both nutrient media. The STY varied substantially between the different clones, ranging from 0.22 +/- 0.03 (clone SC from Cuba) to 3.9 +/- 0.3 (clone 9256 from Finland) in continuous light. The STY increased with increasing duration of the photoperiod. This increase may have been due to the extended period of photosynthesis rather than that of a photoperiodic long-day response. Shorter photoperiods did not stimulate turion formation in any of the clones. S. polyrhiza is a day-neutral plant with respect to turion formation, as noted previously (Appenroth et al. 1990. Annals of Botany 66: 163-168). In accordance with this conclusion, no correlation was detected between the STY and the latitude at which the clones occur naturally. Environmental factors other than shortening of photoperiods seem to be effective in signalling seasonal changes of growth conditions in advance to S. polyrhiza.     

Oxic and anoxic growth of a new Citrobacter species on amino acids

A facultatively anaerobic bacterium, strain P-88, was enriched selectively under dual limitation by glutamate and oxygen in a chemostat. The new strain is a gram-negative motile rod. The mol% guanine plus cytosine of the DNA is 51.4±0.6 mol%. The organism grows on citrate as a sole source of carbon and energy, does not form acetoin, does not induce lysine decarboxylase and was thus classified as a species of the genus Citrobacter. A remarkable characteristic of the new isolate is its ability to grow on several amino acids with either a respiratory or a fermentative type of metabolism. Under strictly anoxic conditions glutamate was fermented to acetate, H₂, CO₂ and ammonia. Asparagine, aspartate and serine could also be fermented. Furthermore, all type strains of the genus Citrobacter were shown to have the same fermentative abilities. Based on enzyme activities determined in cell-free extracts a combination of the methylaspartate pathway and the mixed acid fermentation of Enterobacteriaceae is proposed to explain the glutamate fermentation pattern observed in cultures of strain P-88. Analysis of the growth of strain P-88 in continuous culture with various degrees of oxygen supply, demonstrated that the bacterium can rapidly switch between oxic and anoxic metabolism. Cultures of strain P-88 grown under oxygen limitation simultaneously respire and ferment glutamate, suggesting that the organism is particularly well adapted to growth in microoxic environments.     

Unusual regulation of the uptake system for branched-chain amino acids in Corynebacterium glutamicum

The uptake of branched-chain amino acids in threonine-dehydratase deficient mutants of Corynebacterium glutamicum is dependent on the presence of relatively high (>1 mM) intracellular concentrations of isoleucine, valine or leucine. This indicates that the respective uptake-system is induced by its substrate, i.e. branched-chain amino acids, at the internal side. This unusual regulation presumably is the reason for the failure to obtain mutants deficient in isoleucine uptake by use of a selection scheme which starts from isoleucine auxotroph mutants. The physiological meaning of this regulation is discussed with respect to isoleucine efflux and the cyclic retention hypothesis.Abbreviations amp ampicillin - dw dry weight - Km kanamycin - kb kilobase(s) - NMG N-methyl-N-nitro-N-nitrosoguanidine - ®, resistant resistance     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Effects of amino acids on methionine-sulfoximine-induced heterocyst formation in Anabaena

Heterocyst development in ammonia-grown cultures of Anabaena variabilis and Anabaena 7120 was fully induced by the amino-acid analog methionine sulfoximine (MSO) at concentrations of 0.5–1.0 M. Glutamine, glutamate, aspartate, and alanine at 0.5 mM blocked the induction of heterocysts by MSO in A. variabilis. With Anabaena 7120, glutamine and glutamate were fully effective and alanine partially effective in preventing MSO-induced heterocyst formation. In MSO-treated algae, glutamine synthetase activity was reduced to less than 15% of control values within 4–6 h. Inactivation of the enzyme was prevented by all four amino acids tested.     

Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during prolonged cycling

Brain serotonin (5-hydroxytryptamine, 5-HT) has been suggested to be involved in central fatigue during prolonged exercise. Changes in the ratio of plasma free tryptophan (free Trp) to branched-chain amino acids (BCAA) are associated with altered brain 5-HT synthesis. The purposes of this study were to describe systematically the effects of prolonged exercise on changes in plasma free Trp and BCAA and to examine the effects of carbohydrate (CHO) feedings on these same variables. Eight well-trained men [VO2max = 57.8 (SE 4.1) ml kg-1 min-1] cycled for up to 255 min at a power output corresponding to VO2 at lactate threshold (approximately 68% VO2max) on three occasions separated by at least 1 week. Subjects drank 5 ml kg-1 body wt-1 of either a water placebo, or a liquid beverage containing a moderate (6% CHO) or high (12% CHO) concentration of carbohydrate beginning at min 14 of exercise and every 30 min thereafter. Exercise time to fatigue was shorter in subjects receiving placebo [190 (SE 4) min] as compared to 6% CHO [235 (SE 10) min] and 12% CHO [234 (SE 9) min] (P < 0.05). Glucose and insulin decreased in the placebo group, and free Trp, free-Trp/BCAA, and free fatty acids increased approximately five- to sevenfold (P < 0.05). These changes were attenuated in a dose-related manner by the carbohydrate drinks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL--phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme production.     

Preference of cabbage white butterflies and honey bees for nectar that contains amino acids

Summary Amino acids occur in most floral nectars but their role in pollinator attraction is relatively unstudied. Nectars of butterfly-pollinated flower tend to have higher concentrations of amino acids than do flowers pollinated by bees and many other animals, suggesting that amino acids are important attractants of butterflies to flowers. In order to determine whether amino acids are important in attracting butterflies and bees, we tested the preference of cabbage white butterflies (Pieris rapae) and honey bees (Apis mellifera) by allowing them to feed from artificial flowers containing sugar-only or sugar-amino acid mimics ofLantana camara nectar. Honey bees and female cabbage white butterflies consumed more sugar-amino acid nectar than sugar-only nectar. In addition, female cabbage white butterflies visited artificial flowers containing sugar-amino acid nectars more frequently than flowers containing sugar-only nectars; honey bees spent more time consuming the sugar-amino acid nectar. Male cabbage white butterflies did not discriminate between the two nectars. These results support the hypothesis that the amino acids of nectar contribute to pollinator attraction and/or feeding.     

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed.     

Isolation and quantitation of isotopically labeled amino acids from biological samples

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phtaaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Becuase the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken byb gas-phase acid hydrolysis (103% recovery after 5 h at 150°C: S.D = 3.5%, n = 14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 μl could be injcted, representing approximately 200 μl of deproteinized plasma. The methods were linear up to injection of 0.5 μmol of all amino acids (OPA: r²=0.995−0.999; FMOC: r²=0.992−0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50–500 nmol), was less than 3% above 100 mmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.     

Side-chain conformation angles of amino acids: effect of temperature factor cut-off

The paper presents the analysis of the side-chain conformation angles of amino acids in 90% non-identical protein structures. The analysis has been carried out using 113,699 residues, which is higher compared to the previous studies. In the present study, one more quality check, namely, temperature factor cut-off, has been introduced in addition to resolution and R-factor cut-offs. Due to this, the present calculation reveals the approximate values for the minimum and the maximum of the three-rotameric states of chi1. In addition, the conformation angles chi2 and chi3 have been addressed with the improved data set. The results reported here could be of use in protein modeling.     

The influence of amino acids on the lobeline production of Lobelia inflata L. hairy root cultures

The herb Lobelia inflata L. (Lobeliaceae) containspiperidine alkaloids. The main alkaloid is the pharmacologically-activelobeline. We have studied the effects of alkaloid precursor amino acids (lysineand phenylalanine) on the growth and alkaloid production of hairy root culturesof Lobelia inflata L. The hairy root clone 8009/h7transformed with Agrobacterium rhizogenes strain R 1601wascultivated on B5 solid medium containing lysine (Lys) and phenylalanine (Phe)both in the presence and absence of the growth regulators IAA and kinetin. Onthe medium containing hormones growth was delayed until day 14. The applicationof growth regulators to the B5 media containing amino acids either singly or incombination increased the biomass in all cases. The maximum dry weight wasachieved in a medium containing Phe+Lys and growthregulators. The highest lobeline level (36 g/g) was detectedintissues cultivated on hormone-free medium containing Phe. In hormone-freemedia,lobeline production increased in the presence of either Phe or Lys comparedwiththe control, but the addition of both greatly decreased synthesis. In contrast,the addition of both amino acids to media supplemented with IAA and kinetinincreased lobeline production.     

Differentiation of the photosynthetic apparatus of Chloroflexus aurantiacus depending on growth with different amino acids

The phototrophic green bacterium Chloroflexus aurantiacus was grown anaerobically in batch culture with different amino acids at 56°C and constant illumination of 25 klx. The composition of the photosynthetic apparatus was measured by quantitation of the bacteriochlorophylls (Bchl) a and c (representing the membrane-bound and the chlorosomal moieties, respectively). Ser added at concentrations up to 15 mM stimulated protein formation and Bchl a and c syntheses. A comparable stimulation was found with Glu and Ala. Coproporphyrin accumulation approached saturation at 5 mM of Ala, Asp, Orn, and Ser, while with Glu and Arg saturating concentrations were above 5 mM. Protein and tetrapyrrole syntheses became saturated at 2.5 to 5 mM of Asp, Ile, and Val. However, with Arg and Orn Bchl c synthesis was stimulated up to 2.5 mM, growth and Bchl a synthesis up to 5 mM. At higher Arg or Orn concentrations these activities were inhibited. Coproporphyrin accumulation was highest with Arg or Orn, at concentrations which inhibited growth and Bchl formation. Stimulation of Bchl synthesis took place preferentially at the level of Bchl c, while Bchl a was more sensitive toward inhibition. In both cases however, the ratio of Bchl c to Bchl a increased with higher amino acid concentrations. Nevertheless, each amino acid induced a typical effect. To understand different effects exerted by different amino acids, chemostat cultures were grown limited by either Ser or Glu. With Ser, steady state protein levels and specific Bchl a contents decreased slightly when increasing the dilution rate (D). Concomitantly Bchl c and coproporphyrin levels as well as the ratio of Bchl a/Bchl c increased. With Glu as the limiting substrate, all of the above mentioned parameters decreased. Since all of the Ser was consumed and increasing amounts of Glu remained unutilized in the spent medium, it is concluded that differences in the formation of the three pyrrole derivatives tested are due to differences in the affinities of uptake systems for Ser and Glu.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

Liberation of amino acids by heterotrophic nitrogen fixing bacteria

Summary. Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.     

A rapid method for isolation of purified,physiologically active chloroplasts,used to study the intracellular distribution of amino acids in pea leaves

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of ¹⁴CO₂ fixation and CO₂-dependent O₂ evolution (over 100 mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and -(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, -aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.