Intensification of the initial stage in amino acid metabolism in germinating cereal seeds with exogenous glutamine and proline

The initial stage of amino acid metabolism was intensified in germinating wheat seeds with exogenous glutamine and proline. Exogenous glutamine and proline accumulated over 2 h at 4°C in swelling seeds were spent at different rates over the following 2 h at 20°C, thus compensating for insufficiency of these amino acids during the initial stage of development. Creation of additional pools of glutamine and proline during the initial stage of amino acid metabolism had positive effects on the seed germination and vital activity of the plants.     

Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine

The amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5 degrees and 45.0 degrees C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5 degrees C and for 30 min at 45 degrees C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5 degrees C with a T1/2 = 15 min. At 45 degrees C, the decrease was 36%. Thermotolerance developed at 40.5 degrees C with a T1/2 = 30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4-10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.     

Changes in amino acid pools in the silkworm,Bombyx mori during embryonic life: Alanine accumulation and its conversion to proline during diapause

Alanine accumulated in silkworm eggs at the onset of diapause. When the eggs were kept at 4°C during diapause, this alanine was converted to glutamate, glutamine and especially proline. On resumption of development at 25°C after diapause, proline was used as an energy source for protein synthesis. In HCl-treated diapause eggs, which develop like non-diapause eggs, most amino acids showed similar developmental changes to those in eggs in resumption of embryogenesis after diapause. However, the proline level increased until the middle of embryonic development and then decreased. Continuous incubation of diapause eggs at 25°C after day 10 of oviposition caused a decrease in alanine with increases in glutamine and proline, while the levels of most other amino acids either decreased slightly or remained unchanged until day 80, when most eggs died. These results show that diapause eggs have a metabolic complex coupled with carbohydrate and amino acid metabolism inclusive of the 2-oxoglutarate-glutamate shuttle. Under conditions when embryogenesis proceeded, the level of phosphoethanolamine decreased rapidly.     

Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.     

Non-targeted metabolomic evaluations during seed germination and seedling growth in Salicornia brachiata (Roxb.) under saline conditions

Experimental studies were conducted for metabolomic profiling during seed germination and seedling development in Salicornia brachaita under saline conditions. The results revealed accumulation of sucrose, mannose, glycerol, methionine, tryptophan, glycerol, protocathechoic acid, and mannonic acid in germinating seeds. Abundance of rhamnose, glucose, glutamine, fructose, ornithine, quininic acid, proline and ketoglutaric acid were recorded during emergence of radical (EoR) and cotyledonary stage (CS) at 50% strength of seawater (SW) salinity. Higher levels of myo-inositol, ethanolamine, isoleucine and talose at 48 hours (hrs) of imbibition, EoR and CS stages; while glycine, tyrosine and turanose were so at CS stage only. Under 200 mM NaCl, richness of stearic acid, quercetin, leucine, erythritol and psicose were noted at 48 hrs of imbibition followed by EoR stage. Fructose, ornithine, mannitol, asparagine, mallic acid, glucose and citric acid were abundant at EoR whereas aminobutanoic acid, hexanedioic acid and tyramine were so at CS stage. Among detected metabolites maximum number of metabolites showed hits with amioacyl-tRNA biosynthesis pathway and the amino acid biosynthesis pathway had maximum impact during seedling development. Role of metabolic pathways (including amino acid metabolism) and differential expression of genes related to these pathways are suggested in meeting the energy needs for varied biological activities during seed germination and subsequent seedling development in S. brachiata.     

Transcriptome atlas of glutamine family amino acid metabolism‐related genes in eight regenerating liver cell types

To explore glutamine family amino acid metabolism of eight liver cell types in rat liver regeneration, eight kinds of rat regenerating liver cells were isolated by using the combination of Percoll density gradient centrifugation and immunomagnetic bead methods, then Rat Genome 230 2.0 Array was used to detect the expression profiles of the genes associated with metabolism of glutamine family amino acid in rat liver regeneration and finally how these genes involved in activities of eight regenerating liver cell types were analysed by the methods of bioinformatics and systems biology. The results showed that in the priming stage of liver regeneration, hepatic stellate cells and sinusoidal endothelial cells transformed proline and glutamine into glutamate; hepatocytes, hepatic stellate cells, sinusoidal endothelial cells and dendritic cells catabolized glutamate to 2‐oxoglutarate or succinate; hepatic stellate cells and sinusoidal endothelial cells catalysed glutamate into glutamyl‐tRNA for protein synthesis; urea cycle, which degraded from arginine, was enhanced in biliary epithelia cells, sinusoidal endothelial cells and dendritic cells; synthesis of polyamines from arginine was enhanced in biliary epithelia cells, sinusoidal endothelial cells, Kupffer cells and dendritic cells; the content of NO was increased in sinusoidal endothelial cells and dendritic cells; degradation of proline was enhanced in hepatocytes and biliary epithelia cells. In the progress stage, biliary epithelia cells converted glutamine into GMP and glucosamine 6‐phosphate; oval cells converted glutamine into glucosamine 6‐phosphate; hepatic stellate cells converted glutamine into NAD; the content of NO, which degraded from arginine, was increased in biliary epithelia cells, oval cells, pit cells and dendritic cells. In the termination stage, oval cells converted proline into glutamate; glutamate degradation, which degraded from arginine, was enhanced in hepatocytes and dendritic cells; the content of NO was increased in oval cells, sinusoidal endothelial cells, pit cells and dendritic cells. The synthesis of creatine phosphate was enhanced in hepatocytes, biliary epithelia cells, pit cells and dendritic cells in both progress and termination stages. In summary, glutamine family amino acid metabolism has some differences in liver regeneration in different liver cells.     

Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte.     

Factors affecting carbohydrate and free amino acid content in overwintering larvae of Enosima leucotaeniella

Amounts of several metabolites were measured in overwintering larvae of Enosima leucotaeniella acclimated to temperatures between -5 and 15 degrees C for 30days. In the diapausing stage, cold hardiness, as shown by the survival rate, began rising below 15 degrees C. Glycogen content decreased as the temperature decreased from 10 to 0 degrees C. Trehalose content rose as the temperature decreased from 15 to 5 degrees C, but remained unchanged as the temperature decreased from 5 and 0 degrees C. Twenty-eight free amino acids were detected in the haemolymph; levels of proline, glutamine and glutamic acid increased at high temperatures, but alanine increased at low temperatures, especially as temperature decreased from 5 to 0 degrees C. Lipid content was unchanged by the different acclimation temperatures. The effects of temperature, diapause and aerobic conditions on the levels of carbohydrates and amino acids in overwintering larvae were analyzed. Alanine levels rose at low temperature only when the larvae were in the diapausing stage. The level of trehalose rose at low temperature in both the diapausing and post-diapausing stages, although it was higher at aerobic conditions in the post-diapausing stage. These results suggest that efficient trehalose synthesis occurs under the combination of low temperature and aerobic conditions of the post-diapausing stage, so that cold hardiness in overwintering E. leucotaeniella larvae may rise to a high level in winter.     

Amino Acid Pools in Herbaceous Plants at the Wintering Stage and at the Beginning of Growth

Free amino acids in 40 herbaceous perennial plants were analyzedunder natural conditions. From the major amino acid contentat the wintering stage, the pools were separated into the followingfive types: 1) a group which accumulated arginine (20 plantsout of 40); 2) a group which accumulated arginine and proline(9 plants); 3) a group which accumulated glutamate and glutamine(3 plants); 4) a group which accumulated asparagine (4 plants);and 5) a group which accumulated proline (4 plants). Changesin the amino acid pools in the plants occurred under snow duringwintering for about five months. Particularly, asparagine wasno longer the major amino acid in the group which had accumulatedit in fall. There was a tendency for the glutamine content toincrease, suggesting that NH₃ is utilized for the synthesisof the amide. Also, the relative concentrations of almost allthe free amino acids increased several-fold, which was indicativeof the occurrence of biosynthetic processes of general aminoacids during wintering. As the mobile fractions of stored nitrogen,the amino acids appeared to contribute to the initial stageof rapid growth in early spring. (Received August 4, 1986; Accepted November 17, 1986)     

Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

The role of proline in thidiazuron-induced somatic embryogenesis of peanut

Summary Peanut seeds germinated on media supplemented with thidiazuron [TDZ: N-phenyl-N′-(1,2,3-thiadiazol-yl)urea], formed somatic embryos at the hypocotyledonary notch region by Day 35 of the culture period. Supplementation of the culture media with proline, thioproline, or glutamine reduced the total number of embryos formed, but the resulting embryos were larger, greener and had a more synchronous development than the regenerants formed on media containing TDZ alone. Analysis of the endogenous amino acid content of the germinating seeds during the induction phase of somatic embryogenesis revealed accumulation of proline to 6% of the dry seed weight. Concurrent with the emergence of the radicle, the proline concentration remained significantly elevated throughout the expression phase of embryogenesis. Several other amino acids including alanine, aspartate, asparagine, glutamate, glutamine, γ-aminobutyrate (GABA), hydroxyproline, isoleucine, threonine and valine accumulated to peak values approximately 10-fold higher than those of the controls. These results indicate that proline plays a key role in directing the route of TDZ-induced somatic embryogenesis and that TDZ effectively stimulates a cascade of metabolic events resulting in the production of specific metabolites, including amino acids, required for the regenerative process.     

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.     

Changes in the Contents of Free and Protein Amino Acids in Tobacco Seeds and Placentae during Maturation

Changes in the levels of protein and free amino acids in theseeds and placentae of Nicotiana tabacum were studied duringseed development. Seed maturation was completed 24 days afteranthesis. During maturation, protein rapidly accumulated inthe seeds between the 6th and 18th day, along with an appreciablecompositional change in the protein amino acids as the proportionsof glutamic acid and arginine increased. The amount of freeamino acids in the seeds gradually decreased throughout maturation.The major free amino acid on the 6th day after anthesis wasglutamine, which then drastically decreased between the 6thand 12th day with increases of glutamic acid, proline, arginineand alanine. The latter amino acids decreased thereafter untilthe 24th day. On the other hand, the amount and composition of the proteinsin the placentae did not change significantly throughout seedmaturation. In the early stage of development, the major freeamino acids in the placentae were glutamine, asparagine andglutamic acid, while in the later stage asparagine was mostabundant. (Received March 12, 1982; Accepted August 16, 1982)     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Priming relieves dormancy in lettuce seeds independently of changes in osmotic constituents

The relationship was studied between germination and dormancy of lettuce seeds ( Lactuca sativa L. cv. Musette) and both soluble amino nitrogen metabolism and osmotic potential. Germination at 15°C in darkness coincided with a rise in the levels of free amino acids and total soluble amino nitrogen compounds and in the activity of glutamine synthetase (GS, EC nr. 6.3.1.2). In further experiments GS activity was used as indicator of soluble amino nitrogen metabolism. GS activity increased after the start of growth indicated by an increasing intolerance to desiccation. At 30°C seeds did not germinate, unless dormancy was broken beforehand during incubation at 2° or 15°C (priming). The alleviation of dormancy occurred much earlier than the rise in the activity of GS. Priming at 15°C in polyethylene glycol instead of water retarded the breaking of dormancy and at –1.28 MPa even stimulated the induction of secondary dormancy, but did not prevent a continued rise in the activity of GS. GS activity was also not reduced during induction of secondary dormancy by dehydration of primed seeds, which antagonized the beneficial effect of priming. Psychrometric measurements showed that osmotic potential (Ψ_π) of the seeds remained constant during prolonged priming in polyethylene glycol at 15°C. During incubation in water, Ψ_π increased both prior to and after the moment of germination to less negative values. It is concluded that changes in the level of dormancy in lettuce seeds occur independently of soluble amino nitrogen metabolism and of changes in Ψ_π.     

Combination of BCAAs and glutamine enhances dermal collagen protein synthesis in protein-malnourished rats

Skin collagen decreases in protein-malnourished states. Amino acids regulate protein metabolism, glutamine stimulates collagen synthesis through the conversion process to proline and provides 75 % of the intracellular free proline in fibroblasts. However, the impact of these amino acids on collagen synthesis under malnutrition has not been examined. We investigated the effect of amino acids on dermal tropocollagen synthesis in protein-malnourished rats. The fractional synthesis rate (FSR, %/h) of dermal tropocollagen was evaluated by the incorporation of l-[ring-²H₅]-phenylalanine after 4 h infusion of each amino acid and the stable isotope. None of the infused 12 single amino acids (glutamine, proline, alanine, arginine, glutamate, glycine, aspartate, serine, histidine, lysine, phenylalanine and threonine) significantly increased the FSR (P = 0.343, one-way ANOVA). In contrast, amino acid mixtures of essential amino acids + glutamine + arginine (EAARQ) and branched-chain amino acids + glutamine (BCAAQ) significantly increased the FSR compared to saline, but the branched-chain amino acids (BCAAs) and amino acid mixture of collagen protein (AAC) did not alter the FSR (saline, 0.96 ± 0.24 %/h; EAARQ, 1.76 ± 0.89 %/h; BCAAQ 1.71 ± 0.36 %/h; BCAAs, 1.08 ± 0.20 %/h and AAC 1.39 ± 0.35 %/h, P < 0.05, Tukey’s test). Proline conversion from glutamine represented only 3.9 % of the free proline in skin, as evaluated by the primed-constant infusion of l-d7-proline and l-α-15N-glutamine in rats. These results suggested that the combination of BCAAQ is a key factor for the enhancement of skin collagen synthesis in protein-malnourished rats. The contribution of extracellular free glutamine on de novo proline synthesis and collagen synthesis is very low in vivo compared to the contribution in vitro.     

Involvement of ABA in induction of secondary dormancy in barley (Hordeum vulgare L.) seeds

At harvest, barley seeds are dormant because their germination is difficult above 20 degrees C. Incubation of primary dormant seeds at 30 degrees C, a temperature at which they do not germinate, results in a loss of their ability to germinate at 20 degrees C. This phenomenon which corresponds to an induction of a secondary dormancy is already observed after a pre-treatment at 30 degrees C as short as 4-6 h, and is optimal after 24-48 h. It is associated with maintenance of a high level of embryo ABA content during seed incubation at 30 degrees C, and after seed transfer at 20 degrees C, while ABA content decreases rapidly in embryos of primary dormant seeds placed directly at 20 degrees C. Induction of secondary dormancy also results in an increase in embryo responsiveness to ABA at 20 degrees C. Application of ABA during seed treatment at 30 degrees C has no significant additive effect on the further germination at 20 degrees C. In contrast, incubation of primary dormant seeds at 20 degrees C for 48 and 72 h in the presence of ABA inhibits further germination on water similarly to 24-48 h incubation at 30 degrees C. However fluridone, an inhibitor of ABA synthesis, applied during incubation of the grains at 30 degrees C has only a slight effect on ABA content and secondary dormancy. Expression of genes involved in ABA metabolism (HvABA8'OH-1, HvNCED1 and HvNCED2) was studied in relation to the expression of primary and secondary dormancies. The results presented suggest a specific role for HvNCED1 and HvNCED2 in regulation of ABA synthesis in secondary seed dormancy.