Inhibitory Effect of Glycine on the Production of Amylase and Proteinase by Bacillus subtilis

Cytological effects of glycine on Bacillus subtilis var. amyloliquefaciens were compared between the cells of the glycine-sensitive parent and resistant mutant. Glycine induced disruption of the protoplasts which had been prepared by treating the glycine-sensitive cells with lysozyme. This effect of glycine was almost completely prevented by preincubating the protoplasts with spermine. The protoplasts prepared from the resistant cells were markedly stable in the presence of glycine. In this mutant, neither cell lysis nor cessation of the enzyme production by glycine occurred, contrary to the results obtained with the glycine-sensitive parent. Between both type of cells little difference could be observed in the metabolic activity for glycine, but free amino acid content was higher in the glycine-resistant cells than in the parent ones.     

The Molecular Biology of Euglena gracilis IX. Amino Acid Pool Composition

The amino acid composition of the acid soluble fraction of Euglena gracilis was determined from cells grown in 4 different culture media. Glutamic acid is the major free amino acid. Hydrolysis of this fraction increases the amount of free amino groups, the major amino acids found are then glutamic acid, aspartic acid, glycine and arginine. The pattern of amino acid distribution is similar in all 4 culture media. L-arginyl-L-glutamine was isolated and identified in extracts from all 4 culture conditions. It was shown to be a metabolic intermediate by radioactivity chase experiments.     

Production of extracellular free amino acids by cyanobacteriumAnabaena siamensis Antarikanonda

The cyanobacteriumAnabaena siamensis Antarikanonda, isolated from rice paddies of Bangkok, Thailand, liberates substantial quantities of free amino acids into the external medium irrespective of whether it is growing on N₂, NH₄ ⁺, NO₃ ^– or under nitrogen-starved conditions. Addition of such combined nitrogen causes changes in both intracellular and extracellular free amino acid pool patterns. No overall relationship exists between the amino acid efflux and the intracellular pools. The most abundant free amino acids found in the external media of N₂, NO₃ ^–, NH₄ ⁺-grown and N-starved cultures were phenylalanine, threonine, glutamate, and glycine, respectively. These investigations suggest that amino acid liberation by the cyanobacterium is a selective diffusional process that is sensitive to environmental changes.     

Uptake of amino acids by the aquatic resurrection plant Chamaegigas intrepidus and its implication for N nutrition

Chamaegigas intrepidus is a poikilohydric aquatic plant that lives in rock pools on granitic outcrops in Central Namibia. The pools are filled intermittently during the summer rains, and the plants may pass through up 20 rehydration/dehydration cycles during a single wet season. Rehydrated plants also have to cope with substantial diurnal fluctuations in the pH and extreme nutrient deficiency. Ammonium concentrations are normally around 30 μM. Additional nitrogen sources are amino acids. Total free amino acids are up to 15 μM with glycine and serine as the predominant amino acids. Experiments on uptake of radiolabelled amino acids into roots of C. intrepidus showed high␣affinity (K _M= 16 μM) and low-affinity (K _M= 159 μM) uptake systems. The K _M of the high-affinity system is well in accordance with the free amino acid concentration found in the water of the pools. We conclude that amino acids, predominantly glycine and serine, can be utilised by C. intrepidus in its natural habitat. Since glycine uptake showed a strong reduction at pH 10, nitrogen uptake from glycine or serine should occur mainly in the morning when the pH of the pool water is slightly acid. Further experiments with ¹⁵N-labelled ammonium in combination with non-labelled glycine demonstrated high ¹⁵N values in plant tissues. Under experimental conditions C. intrepidus preferred ammonium as a nitrogen source. The implication of amino acids for nitrogen nutrition of C. intrepidus may depend on the relation of inorganic and organic nitrogen available in the pool water and the preferential utilisation of one or the other nitrogen source may change during the day corresponding with pH changes in the water. Received: 28 January 1998 / Accepted: 24 July 1998     

A Sensitive and Simple Method for the Determination of Fatty Acid Hydroperoxides with Horseradish Peroxidase

Glycine, glycylglycine, glycine methyl ester and glycine ethyl ester were found to be effective for the production and release of γ-galactosidase by Escherichia coli. Addition of an appropriate concentration of glycine and glycylglycine to the culture increased total enzyme production 6 to 7-fold and extracellular enzyme production over 240-fold at 24 hr cultivation. The enzyme synthesis was stimulated even at the exponential period of growth, and 93% of enzyme was found in the culture fluid at 24 hr cultivation on addition of 1.2% glycine. A large amount of protein was also accumulated in the culture fluid. A micrograph showed glycine gave swollen and irregular cells, indicating that the cell surface was altered. Various amino acid analogues and some antibiotics had a small or no effect on the production and release of the enzyme as compared with glycine. Polypeptone or brain heart infusion was needed as a nitrogen source for efficient production of the enzyme.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Immobilization and mineralization of dissolved free amino acids by stream-bed biofilms

1. Radiolabelled (¹⁴C) amino acids were used to investigate the influence of sediment size as well as dissolved free amino acid (DFAA) concentration and composition on immobilization and mineralization of DFAAs by biofilms from a first-order stream. 2. Over time (240 min), biofilms on stony substrata immobilized a DFAA mixture more effectively than those on sandy substrata, however proportional mineralization of immobilized DFAAs was higher for sandy substrata (36 v 20%). 3. Using stony substrata, the DFAA mixture was immobilized more rapidly than glycine alone at ‘near-natural’ amino acid concentrations (c. 37 μgl^?1), as well as enriched concentrations (1 and 100 mg 1-^?1). Instantaneous rates of glycine immobilization and mineralization were not saturated at glycine enrichments of up to 980 mgl^?1. 4. With both the amino acid mixture and glycine alone, proportional mineralization of the immobilized amino acids increased on enrichment to Img 1-^?1 (DFAA mixture: from 25 to 37%; glycine alone: from 50 to 54%), but then fell on further enrichment to 100mgl^?1 (DFAA mixture: 11%; glycine alone: 7%). 5. Results are discussed in terms of the potential trophic utility of immobilized DFAAs as well as the apparent roles of biotic and abiotic immobilization mechanisms. Immobilization and mineralization responses to variables investigated in this study give an insight into potential variability of carbon immobilization and retention in stream-bed sediments. This is fundamental to an understanding of how DOC may become available to higher trophic levels.     

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.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

TRANSPORT OF TYROSINE, PHENYLALANINE, TRYPTOPHAN AND GLYCINE IN NEUROBLASTOMA CLONES

Amino acid transport was studied in three neuroblastoma clones, N-TD6, which synthesizes norepinephrine, N-T16, which synthesizes small amounts of serotonin, and N-S20Y, which synthesizes acetylcholine. All three clones exhibited high-affinity saturable transport systems for tyrosine, phenylalanine, tryptophan and glycine as well as systems unsaturated at amino acid concentrations of 1 mM in the external medium. Tyrosine, phenylalanine and tryptophan enter all three clones by rapidly exchanging transport systems which appear to be relatively insensitive to lowered external [Na⁺] or to the presence of 2,4-dinitrophenol (DNP). Glycine uptake was slower and was much more sensitive to lowered external [Na⁺] and to the presence of DNP in the medium. Glycine transport in N-T16 cells was decreased more markedly at low temperature than was transport of the three aromatic amino acids. K_m and V_max values found for saturable transport of tyrosine, phenylalanine and tryptophan were sufficiently low to suggest that, if similar amino acid transport systems exist in neuronal membranes, and if amino acid levels in brain extracellular fluid are similar to levels in plasma, such systems may serve, in conjunction with transport systems in cerebral capillaries, to limit the entry of amino acids into brain cells when blood amino levels are near the normal physiological range.     

Glucuronyl glycine,a novel N-terminus in a glycoprotein

Treatment of cutinase, an extracellular glycoprotein produced by Fusarium solani f. pisi, with NaB³H₄ at pH 7.0 generated labeled enzyme. Acid hydrolysis showed that all of the label was in an acidic carbohydrate which was identified as gulonic acid. The N-terminal amino group of the enzyme is blocked; the precursor of gulonic acid has a free reducing group and it is attached via a linkage resistant to β-elimination. Furthermore, pronase digestion of NaB³H₄-treated cutinase gave rise to a ninhydrin negative compound which contained the bulk of the ³H and this compound was identified as N-gulonyl glycine. These results strongly suggest that the amino group of glycine, the N-terminal amino acid of this enzyme, is in amide linkage with glucuronic acid.     

Uptake of l-[35S]Cystine by Isolated Rat Brain Capillaries

Adult rat brain capillaries were isolated by a simplified procedure and showed an enrichment of the marker enzyme, γ-glutamyltranspeptidase. The uptake of [³⁵S]cystine at 37°C by this preparation can be divided into two components, a sodium- and energy-dependent transport process for the free amino acid pool, with an apparent K_m of 36 μm , and a binding process, with an apparent K_m of 1.13 mm . Chemical analysis of the amino acid pool indicates that cystine is the major form of intracapillary ³⁵S. Cystine transport was not inhibited by lysine, but glycine, α-methylaminoisobutyric acid and β-2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid were inhibitory to a small extent.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Overproduction of amino acids and the activity of methanol dehydrogenase of protaminobacter thiaminophagus mutants

The activity of methanol dehydrogenase of Protaminobacter thiaminophagus ATCC 21371 and its 10 mutants which where able to overproduce amino acids from methanol was studied. It was found that the activity of methanol dehydrogenase depended on the used strain varied from 56.1 to 100.6 mU/mg d. w. of cells. Specific production of amino acids was between 4.3 × 10^?7 and 13.0 × 10^?7 μg/CFU. Statistical analysis confirmed expected high positive correlation (r = 0.93) between activity of methanol dehydrogenase and specific production of amino acids. Based on the measurement of methanol dehydrogenase activity a rapid method of estimation of amino acid production ability of Protaminobacter thiaminophagus mutants was developed.     

THE FREE AMINO ACIDS OF HUMAN SPINAL FLUID DETERMINED BY ION EXCHANGE CHROMATOGRAPHY

(1) The free amino acids in human CSF from eighteen subjects have been determined. The analyses were performed on 0-75 ml of CSF by an ion exchange chromatographic method which is capable of detection to the 10^?10 mole level. (2) The amino acids always found in readily detectable amounts were: taurine, threonine, serine, glutamine, glutamic acid, citrulline, glycine, alanine, α-NH₂-n- butyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ethanolamine, ornithine, lysine, histidine and arginine. Urea was present. Aspartic acid and cystine, though always present, occurred in small or trace amounts. Proline was found in four cases and tryptophan in thirteen cases. In addition, twelve unknown peaks were nearly always evident in every chromatogram. (3) Filtrates 10 times more concentrated than those used regularly were prepared from pooled CSF and analysed. These analyses clearly confirmed the presence of those amino acids which were normally in very low concentration and they also served to distinguish the twelve unknown compounds from confusion with baseline artifacts. (4) The distribution of free amino acids in CSF was different from their distribution in blood plasma. (5) Despite a variety of neurological conditions and a wide age span few marked deviations were found in any of the amino acid concentrations.     

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     

Amino acid specificity of the Na/alanine cotransporter in pancreatic acinar cells

The method of tight-seal whole0cell recording was used to study the amino-acid specificity of the Na⁺/alanine cotransporter in pancreatic acinar cells. Single cells or small clusters of electrically coupled cells were obtained by enzymatic dissociation of mouse pancreas. Inward currents were measured under ‘zero-trans’ conditions, i.e., at finite concentrations of Na⁺ and amino acid at the extracellular side and vanishing concentrations at the cytoplasmic side. The cotransporter, which corresponds to ‘system A’, as previously defined in the literature, was found to exhibit a wide tolerance to neutral amino acids (l-cysteine, l-serine, l-alanine, glycine, l-phenylalanine). Competition experiments with 2-methylaminoisobutyric acid (MeAIB) indicate that for glycine a second electrogenic transport system exists in pancreatic acinar cells.     

Untersuchunge über Stoffwechselbeziehungen zwischen Parasit und Wirt am Beispiel von Puccinia graminis var. tritici auf Weizen

Summary Rust infected wheat plants were incubated with different ^14C-labelled amino acids. Uredospores that were formed during the incubation contained ¹⁴C-activity. By analysis of these spores it was investigated whether the parasitic mycelium of Puccinia graminis takes amino acids from the host. It could be demonstrated that the applicated amino acids were taken up directly from the wheat leaf. The carbon sceletons of applicated lysine and arginine showed only little randomization of ¹⁴C-activity. Glutamic acid, alanine and glycine isolated from uredospore protein showed a very strong alteration of the original label. The pools of free amino acids in the host and the parasite are in isotope equilibrium. This demonstrates, that synthesis of amino acids in the mycelium is quantitatively not important. By following the kinetics of incorporation of an amino acid it could be demonstrated that the amino acids enter the parasite as free amino acids and not in the form of proteins.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.