External Glycine Research Articles

Potentiation of neuronal activity by tonic GluD1 current in brain slices

Ion channel function of native delta glutamate receptors (GluDR) is incompletely understood. Previously, we and others have shown that activation of Gαq protein‐coupled receptors (GqPCR) produces a slow inward current carried by GluD1R. GluD1R also carries a tonic cation current of unknown cause. Here, using voltage‐clamp electrophysiological recordings from adult mouse brain slices containing the dorsal raphe nucleus, we find no role of ongoing G‐protein‐coupled receptor activity in generating or sustaining tonic GluD1R currents. Neither augmentation nor disruption of G protein activity affects tonic GluD1R currents, suggesting that ongoing G‐protein‐coupled receptor activity does not give rise to tonic GluD1R currents. Further, the tonic GluD1R current is unaffected by the addition of external glycine or D‐serine, which influences GluD2R current at millimolar concentrations. Instead, GqPCR‐stimulated and tonic GluD1R currents are regulated by physiological levels of external calcium. In current‐clamp recordings, block of GluD1R channels hyperpolarizes the membrane by ~7 mV at subthreshold potentials, reducing excitability. Thus, GluD1R carries a G‐protein‐independent tonic current that contributes to subthreshold neuronal excitation in the dorsal raphe nucleus.

A novel effect of glycine on the growth and starch biosynthesis of storage root in sweetpotato (Ipomoea batatas Lam.)

Sweetpotato (Ipomoea batatas Lam.) plays an indispensable role in feed, starch-based industries and ethanol biofuel production. Few studies have investigated on how external amino acids affect the growth and production of sweetpotato. In the study, we evaluated morphological, physiological and molecular effects of external glycine (Gly) on the root growth and starch metabolism of sweetpotato, Xushu16. At morphological level, the Xushu16 with Gly stimuli had larger plant biomass than that under control condition. At physiological level, the photosynthesis strength of the Xushu16 with Gly treatments showed significant differences relative to those under control condition. The relative content of plant hormone and starch in storage roots was higher under Gly conditions than that under control condition. At molecular level, a total of 4836 differentially expression genes were identified in the storage roots with different Gly treatments by RNA-Seq. Among them, as many as 1830 genes were involved in carbohydrate metabolism, which held maximum proportion among all the DEGs. Further, a few genes involved in starch biosynthesis were proved to be Gly-induced significantly by RT-qPCR. All the results indicated extrinsic Gly promotes the growth of storage roots by strengthening photosynthesis and increasing plant hormone, and enhances starch biosynthesis of storage roots by accelerating carbohydrate metabolism and regulating the expression of starch-related genes.

Integrated molecular, physiological and in silico characterization of two Halomonas isolates from industrial brine.

Two haloalkaliphilic bacteria isolated from industrial brine solutions were characterized via molecular, physiological, and in silico metabolic pathway analyses. Genomes from the organisms, designated Halomonas BC1 and BC2, were sequenced; 16S ribosomal subunit-based phylogenetic analysis revealed a high level of similarity to each other and to Halomonas meridiana. Both strains were moderate halophiles with near optimal specific growth rates (≥60% μ max) observed over <0.1-5% (w/v) NaCl and pH ranging from 7.4 to 10.2. Isolate BC1 was further characterized by measuring uptake or synthesis of compatible solutes under different growth conditions; in complex medium, uptake and accumulation of external glycine betaine was observed while ectoine was synthesized de novo in salts medium. Transcriptome analysis of isolate BC1 grown on glucose or citrate medium measured differences in glycolysis- and gluconeogenesis-based metabolisms, respectively. The annotated BC1 genome was used to build an in silico, genome-scale stoichiometric metabolic model to study catabolic energy strategies and compatible solute synthesis under gradients of oxygen and nutrient availability. The theoretical analysis identified energy metabolism challenges associated with acclimation to high salinity and high pH. The study documents central metabolism data for the industrially and scientifically important haloalkaliphile genus Halomonas.

Glycine accumulation is toxic for the cyanobacteriumSynechocystissp. strain PCC 6803, but can be compensated by supplementation with magnesium ions

The observation that accumulation of the amino acid glycine is associated with strong growth inhibition or even death in cyanobacteria, plants and humans led to the hypothesis that glycine might act toxically if a certain threshold is exceeded. In this report, it is shown that Synechocystis PCC 6803 wild-type cells could sustain higher glycine addition than mutants impaired in enzymes using glycine such as the T-protein of the glycine decarboxylase complex or PurT involved in purine biosynthesis. A mutant defective in the glycine uptake system was barely influenced by external glycine. This shows that the intracellular level of accumulated glycine is critical. The toxic effect could be alleviated by addition of MgCl(2), suggesting that glycine might be toxic by reducing intracellular Mg(2+) ions, which are essential for many vital processes.

Regulation of compatible solute accumulation in Salmonella typhimurium: evidence for a glycine betaine efflux system

The regulation of glycine betaine accumulation has been investigated in Salmonella typhimurium. The size of the glycine betaine pool in the cells is determined by the external osmotic pressure and is largely independent of the external glycine betaine concentration. Analysis of the activity of the ProP and ProU transport systems suggests that other systems must be active in the regulation of the glycine betaine pool. Addition of p-chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulphonate (PCMBS) to cells that have accumulated glycine betaine provokes rapid loss of glycine betaine. The route of glycine betaine efflux under the influence of PCMB is independent of either the ProP or ProU transport systems. Rapid loss of the accumulated pool of glycine betaine in the presence of PCMB is specific to glycine betaine and proline; accumulated pools of serine and lysine are not significantly affected by the -SH reagent. A specific glycine betaine/proline efflux system is postulated on the basis of these data and its role in the regulation of glycine betaine and proline accumulation is discussed.

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108–116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na +/Cl −/glycine cotransport system is discussed.

Efflux and exchange of glycine by synaptic plasma membrane vesicles derived from rat brain

The influx and exchange of glycine were studied in synaptic plasma membrane vesicles isolated from rat brain. The vesicles were loaded with [U- 14C]glycine by active transport driven by an Na + as well as a Cl - gradient ( out > in ). Dilution-induced efflux requires the simultaneous presence of internal Na + and Cl -. As the efflux of glycine has been demonstrated to be strictly dependent on the presence of both ions (Mayor, F., Jr., Marvizón, J.G., Aragón, M.C., Giménez, C. and Valdivieso, F. (1981) Biochem. J. 198, 535–541), it can be concluded that the efflux of glycine is, in many aspects, symmetrical with its influx. Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being partially dependent on external sodium and not on external chloride. The parallelism observed in influx and efflux processes shows that the release of glycine (by efflux and homoexchange) occurs via the carrier system. These results suggest that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. Both sodium and chloride have to be present on the same side as glycine.

UPTAKE, RELEASE AND HOMO‐ AND HETERO‐EXCHANGE DIFFUSIONS OF INHIBITORY AMINO ACIDS IN GUINEA‐PIG CEREBELLAR SLICES

Abstract— GABA, taurine and β‐alanine are taken up by guinea‐pig cerebellar slices by both the high‐and low‐affinity uptake processes, whereas glycine is taken up only by the low‐affinity process. A considerable amount of labelled GABA loaded in the slice is released by unlabelled external GABA and a minute amount is released by external β‐alanine, glycine and taurine. External glycine and β‐alanine releases labelled glycine loaded in the slice. Labelled taurine loaded is effectively released by external taurine and β‐alanine, while labelled β‐alanine loaded is released only by external β‐alanine.It is suggested that hetero‐exchanges which are one‐directional in some cases also take place between the amino acids in addition to homo‐exchanges. Therefore, high‐affinity uptake processes observed with GABA and taurine could be the result of the homo‐exchange diffusions, while that of β‐alanine could be due to either the homo‐exchange or the hetero‐exchange diffusions or both.K+′‐evoked releases of GABA and to a lesser extent, taurine are partially dependent upon the presence of Ca+ in the superfusion media, whereas that of glycine and probably that of β‐alanine, are not, K+ ‐evoked releases of labelled GABA and taurine are larger when loaded by their high‐affinity uptake systems than by their low‐affinity uptake processes. The reverse is the case with labelled glycine and β‐alanine. These results do not rule out the possibility that taurine might act as a neurotransmitter in the cerebellum.

Salinity dependence, uptake kinetics, and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved14C-labeled neutral amino acids (glycine, L-alanine, L-valine,α-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloadedα-aminoisobutyric acid against external glycine orα-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non-α-amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to14C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,14C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 ‰ S, uptake increases markedly reaching maximum values at 30 ‰ S; these remain almost constant at 40 ‰ S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 ‰ S).

Transport interaction of sugars and amino acids in mammalian kidney

Mutual inhibition of transport between the non-metabolized amino acid, α-aminoisobutyric acid, as well as other amino acids, and the non-metabolized hexose, α-methyl- d-glucoside, was observed in rat kidney cortex slices. The inhibition occurs at the influx stage of the transport process, since the effect could be demonstrated after short incubation periods and was not accompanied by accelerated efflux. Analysis of the interaction showed a non-competitive inhibitory pattern that did not appear completely linear in respect to inhibitor concentration. Accelerated efflux of the sugar could not be induced by external glycine at a concentration (20 mM) which markedly diminishes the steady-state distribution ratio. In respect to mechanism, these findings can be interpreted as competition for a common energy supply or as allosteric inhibition by related, yet separate “carrier” systems.

L'utilisation de la glycine pour la synthèse protéique dans la glande séricigène de Bombyx mori au cours du cinquième stade larvaire

The uptake of 3H glycine by the posterior silk gland of the silkworm, Bombyx mori, incubated in vitro, has been studied in various conditions. The incorporation with age into proteins varies with the medium composition. The amino acid concentration and that of glycine are two important factors. The exogenous glycine remaining in the amino acid soluble pool depends only upon the external glycine concentration.

Transport of Glycine by Hemolyzed and Restored Pigeon Red Blood Cells: Symmetry properties, trans effects of sodium ion and glycine, and their description by a single rate equation

Abstract The aim of the present work was to devise a reasonably detailed transport model and derive an equation from it which would describe the effects on unidirectional glycine transport of the substrates, Na+ and glycine, simultaneously present on both sides of the membrane. The model is a transition model rather than a static site model, and is passive in that energy for glycine-active transport comes solely from the Na+ gradient. Glycine entry and exit kinetics, including Na+ dependence, were compared; unidirectional glycine influx kinetics under steady state and equilibrium conditions were compared; inhibition of glycine exit by various external Na+ concentrations in the absence of external glycine was determined; the relief of this inhibition by external glycine was measured. With most of the measurements, six of seven indispensable parameters of the equation were evaluated as functions of the seventh. Expected values for the three remaining measurements could be calculated from the equation with the six evaluated parameters since kinetic behavior is insensitive to the seventh parameter, although individual rate and equilibrium constants strongly depend on it. Agreement between predicted and measured values was fair to good. In addition to the general fit of experiments to the model, several special properties were found. The system is asymmetrical in that glycine exit and entry kinetics are not mirror images. The asymmetry does not arise from the active character of glycine transport. Formation of a complex containing more than one Na+ must occur. Membrane-crossing rate constants for empty, Na+-loaded, and Na+ plus glycine-loaded mechanism are different. That for the fully Na+-loaded form (ENa2) is very much smaller than the other two, if not zero. Transport kinetics for hemolyzed and restored cells are very similar to those for intact cells.

Glycine Uptake in Escherichia coli: I. GLYCINE UPTAKE BY WHOLE CELLS OF ESCHERICHIA COLI W+ AND A D-SERINE-RESISTANT MUTANT

Abstract These experiments were designed to investigate the defect in amino acid uptake in a d-serine-resistant mutant (WS) of Escherichia coli W that is deficient in the uptake of glycine. Under conditions in which there is insignificant metabolism of glycine (i.e. 0° under nitrogen), the wild type took up significant amounts of glycine, whereas the WS mutant did not equilibrate with the external glycine pool. The initial rate of glycine uptake by the wild type as a function of the external glycine concentration was nonlinear, increasing rapidly from 1.3 x 10-5 m to 2.6 x 10-4 m and decreasing at concentrations above 2.6 x 10-4 m. The WS mutant took up glycine at an initial rate which was 3- to 4-fold less than that of the wild type. The defect could not be attributed to an increased exit process in the WS mutant. Although the WS mutant was able to take up glycine from low external concentrations to the same extent as the wild type, it did so at a much slower rate. Membrane preparations from the wild type cells were found to take up glycine, and membrane preparations from the WS mutant were found to exhibit the defect in glycine uptake. It is concluded that the defect manifested by this d-serine-resistant mutant of E. coli is in the passage of glycine through the cell membrane.

Glycine Uptake in Escherichia coli: II. GLYCINE UPTAKE, EXCHANGE, AND METABOLISM BY AN ISOLATED MEMBRANE PREPARATION

Abstract In this paper, glycine uptake by isolated membrane preparations of Escherichia coli W (W+) and the d-serine-resistant mutant (WS) is examined in detail. Glycine uptake by W+ membrane preparations is dependent on pH and on the presence of Mg++ and an energy source. In W+ membranes, glycine is first taken up into an exchangeable pool, from which it is subsequently removed as the incubation proceeds. The exchangeable pool is built up within 30 sec and remains constant with time, although glycine uptake by the membranes continues. Glycine uptake into the exchangeable pool of W+ membranes is independent of temperature. Uptake and exchange in W+ membranes are specific for glycine, dl-alanine, dl-serine, and dl-threonine. Membrane preparations from the WS mutant are unable to catalyze either the uptake of glycine or the exchange of glycine between the incubation medium and the pool. Glycine uptake by W+ membranes is inhibited 50 to 70% by anaerobiosis, incubation at 0°, or incubation in the presence of 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, NaN3, KAsO4, KCN, iodoacetamide, and p-chloromercuriphenylsulfonate. No inhibition is oberved with NaF, sodium Amytal, hydroxyquinoline N-oxide, antimycin A, Chloromycetin, or ouabain. Although glycine uptake at 30 min is inhibited by the conditions listed above, there is no diminution of glycine uptake when measured at 30 sec. Under none of the conditions studied is the uptake (at either 30 sec or 30 min) decreased to the values found with WS membranes at 37° or 0°. W+ membranes incubated at 37° convert about 94% of the glycine carbon taken up into compounds that have the properties of phosphatidylethanolamine and phosphatidylserine. Under all of the conditions studied, the concentration of glycine in the intramembranal pool approximates that of the incubation medium. Incubation at 0° or in the presence of dinitrophenol or iodoacetamide inhibits the conversion of glycine to phospholipids, and the amount of glycine in the intramembranal pool is increased above that found for uninhibited (control) preparations. WS membranes convert significant amounts of glycine to phospholipids, but the concentration of glycine in the intramembranal pool is only 10 to 20% of that in the external pool. When the external glycine concentration is raised so that the rate of glycine uptake by WS membranes is comparable to the rate exhibited by W+ membranes, the WS membranes convert as much glycine to phospholipids as do W+ membranes, but the concentration of glycine in the intramembranal pool remains only 10 to 20% of that in the external pool. Hydroxylamine almost quantitatively inhibits the conversion of glycine to phospholipids, and in the presence of this inhibitor the WS membranes manifest the same defect.

Glycylglycine uptake in streptococci and a possible role of peptides in amino acid transport

Group D streptococci possess an amino acid transport system specific for glycine, alanine, and serine which functions as an exchange process in which an amino acid in the pool leaves the cell in counterflow to the entering amino acid. In the present work uptake of labeled glycylglycine was studied, and it was shown that this peptide enters the cell by a pathway different from that of the free amino acid. A peptidase splits the peptide into free glycine, and this latter compound accumulates in the pool. This intracellular glycine derived from glycylglycine readily exchanges with external glycine, serine, or alanine. In addition, external glycylglycine stimulates the uptake of glycine. It is thus proposed that the peptide plays a role in amino acid transport by entering the cell, becoming hydrolyzed, and then exchanging for free amino acid outside the cell.

An amino acid transport system in Streptococcus faecium

Streptococcus faecium possesses an amino acid transport system which acts on glycine and l-alanine. It also probably functions with l-serine, and to a lesser extent with threonine, l-valine, and l-cysteine. d-Amino acids, peptides, esters, and amides are not competitors for glycine uptake. The system is temperature dependent but shows little glucose requirement and is not inhibited strongly by various metabolic inhibitors. Using uptake at 1 min. incubation as a measure of the initial rate, the system can be shown to obey Michaelis-Menten kinetics. Glycine and l-alanine show identical K m values, and as competitive inhibitors of each other show the same K I values. The uptake process is an exchange reaction, and the entry and exit reactions are probably coupled. Cells can synthesize alanine and retain it within their free amino acid pool. Alanine in the pool apparently exchanges with external glycine, serine, or threonine needed for growth. In this way the organism is able to obtain from its environment amino acids it requires in exchange for one it can make. Inhibitory inter actions of these amino acids may also involve reaction with the transport system.