Neutrophils as a source of branched-chain,aromatic and positively charged free amino acids

Neutrophils release branched-chain (valine, isoleucine, leucine), aromatic (tyrosine, phenylalanine) and positively charged free amino acids (arginine, ornithine, lysine, hydroxylysine, histidine) when adhere and spread onto fibronectin. In the presence of agents that impair cell spreading or adhesion (cytochalasin D, fMLP, nonadhesive substrate), neutrophils release the same amino acids, except for a sharp decrease in hydroxylysine and an increase in phenylalanine, indicating their special connection with cell adhesion. Plasma of patients with diabetes is characterized by an increased content of branched-chain and aromatic amino acids and a reduced ratio of arginine/ornithine compared to healthy human plasma. Our data showed that the secretion of neutrophils, regardless of their adhesion state, can contribute to this shift in the amino acid content.

Abbreviations: BCAAs: branched-chain amino acids; Е2: 17β-estradiol; LPS: lipopolysaccharide from Salmonella enterica serovar Typhimurium; fMLP: N-formylmethionyl-leucyl-phenylalanine.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Tyrosine availability prevents tyramine-induced tachyphylaxis in the isolated rat heart

Isolated rat hearts perfused with Chenoweth-Koelle solution exhibit tachyphylaxis to tyramine. This tachyphylaxis was prevented if tyrosine was included in the perfusion solution. Other amino acids, including glycine, serine, phenylalanine, valine, tryptophan, glutamate, aspartate, arginine, lysine and -tyrosine, failed to prevent the tyramine-induced tachyphylaxis. Hearts from reserpinized animals showed increased chronotropy after tyramine only when tyrosine was present in the medium. This response could be blocked by , m-hydroxybenzyl-hydrazine and d,l-propanolol, indicating that it was mediated by the synthesis and release of catecholamines. These experiments suggest that sympathetic nerves in the isolated rat heart exhibit a requirement for tyrosine when catecholamine release is enhanced.     

Trypanosoma gambiense: membrane transport of amino acids.

Trypanosoma gambiense absorbed ¹⁴C-labeled lysine, arginine, glutamate, phenylalanine, methionine, threonine, glycine, and alanine by mediated transport systems. The interactions of these compounds as inhibitors or stimulators formed complex patterns of uptake which suggested the presence of five binding and/or transport loci: Locus A bound glutamate, arginine, and lysine, and the binding of glutamate or arginine stimulated the transport of lysine. Locus B transported threonine, glycine, and alanine and appeared to be partially sensitive to ouabain and Na⁺. Locus C transported glutamate, locus D transported phenylalanine and methionine, and locus E transported lysine and arginine.     

Feeding and arginine deficient diets differentially alter free amino acid concentrations of hindlimb muscle in young rats

Summary The objective of these experiments was to examine short- and long-term (7 d) effects of arginine-deficient diets on free amino acid concentrations in hindlimb muscle of rats. In rats fed the control diet containing arginine (+Arg), muscle alanine and methionine concentrations were higher 1 and 2h after feeding compared to food-deprived rats, whereas branched-chain amino acids, arginine and asparagine concentrations were lower postprandially. In Experiment 1, rats were fed an arginine-deficient (–Arg) diet with glutamate (+Glu) substituted for arginine; alanine (+Ala), ornithine (+Orn) or citrulline (+Cit) were substituted for arginine in Experiment 2. In Experiment 1, arginine concentrations decreased in blood but not in muscle. This contrasts with rats fed –Arg/+Ala or –Arg/+Orn diets which had muscle arginine concentrations less than half the concentrations in controls or in rats fed the –Arg/+Cit diet. Muscle essential amino acids in Experiment 2 did not differ by diet, but muscle branched-chain amino acids were elevated relative to controls in the rats fed –Arg/+Ala or –Arg/+Orn diets; however, rats fed the –Arg/+Cit diet had levels similar to the controls. Also, muscle branched-chain amino acids were correlated with glutamine concentrations in both blood and muscle. The measurements in the post-meal period suggest that muscle amino acid concentrations may more closely reflect dietary amino acid patterns than do blood amino concentrations.Abbreviations BCAA branched-chain amino acids - BCKADH branched-chain ketoacid dehydrogenase - EAA essential amino acids - LNAA large neutral amino acids - NEAA nonessential amino acids - PDV portal-drained viscera - SELSM standard error of least squares means - SSA 5-sulfosalicylic acid - TAA total amino acids Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Effect of local x-ray irradiation of the abdominal area on the amino acid content of the blood plasma and their urinary excretion in dogs and rats

A study was made of the content of amino acids in blood plasma of dogs and rats and daily excretion thereof from rat body in urine after local X-irradiation of abdomen with a dose of 13.5 Gy. As the gastrointestinal syndrome developed the content and excretion of taurine increased, glucogenic amino acid content of plasma decreased, and the ratio of phenylalanine to tyrosine and branched-chain amino acids to aromatic amino acids changed. The ratio of arginine to ornithine and urea to ammonia sharply dropped in animals, which died later on, indicating that the urea-producing function of liver was impaired and hyperammonemia developed.     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

Interactions in the Metabolism of Glutamate and the Branched-Chain Amino Acids and Ketoacids in the CNS

Glutamatergic neurotransmission entails a tonic loss of glutamate from nerve endings into the synapse. Replacement of neuronal glutamate is essential in order to avoid depletion of the internal pool. In brain this occurs primarily via the glutamate-glutamine cycle, which invokes astrocytic synthesis of glutamine and hydrolysis of this amino acid via neuronal phosphate-dependent glutaminase. This cycle maintains constancy of internal pools, but it does not provide a mechanism for inevitable losses of glutamate N from brain. Import of glutamine or glutamate from blood does not occur to any appreciable extent. However, the branched-chain amino acids (BCAA) cross the blood–brain barrier swiftly. The brain possesses abundant branched-chain amino acid transaminase activity which replenishes brain glutamate and also generates branched-chain ketoacids. It seems probable that the branched-chain amino acids and ketoacids participate in a “glutamate-BCAA cycle” which involves shuttling of branched-chain amino acids and ketoacids between astrocytes and neurons. This mechanism not only supports the synthesis of glutamate, it also may constitute a mechanism by which high (and potentially toxic) concentrations of glutamate can be avoided by the re-amination of branched-chain ketoacids.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

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.     

Metabolism of 2-oxoacid analogues of leucine,valine and phenylalanine by heart muscle,brain and kidney of the rat

[1-¹⁴C]-Labelled 2-oxoacid analogues of leucine, valine and phenylalanine were used to study the metabolism of these 2-oxoacids in the brain, kidney and heart muscle of rats. By following the ¹⁴CO₂ release during 30–60 min of incubation at 37°C the decarboxylation rate was determined and measurement of the ¹⁴C-incorporation into the corresponding amino acid yielded the transamination rate. From these rates, decarboxylation/transamination ratios could be calculated which are indicative for the metabolic fate of the 2-oxoacid in the various organs. The results obtained show that all three tissues are capable of utilizing the 2-oxoacid analogues of leucine, valine and phenylalanine, however, to a different extent: kidney > heart muscle > brain. The decarboxylation/transamination ratios reveal that the branched-chain 2-oxoacids are predominantly decarboxylated in kidney and heart muscle while in brain they are mainly transaminated. The ratios calculated for phenylpyruvate in all tissues are within 0.19 and 0.36, indicating that this 2-oxoacid is preferentially transaminated. The results are discussed with respect to possible dietary alterations of enzymes involved in 2-oxoacid metabolism in order to improve transamination of these compounds.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

Growth and spore germination factors of various strains of Bacillus sphaericus

The work was aimed at studying the requirements of sixteen Bacillus sphaericus strains with a different larvicidal activity in amino acids and some other compounds necessary for their growth and spore germination. Most of the strains were found to require arginine, glutamate, methionine, threonine, serine, glycine, alanine and lysine, but they did not assimilate phenylalanine and proline. Arginine, methionine and glutamate were shown to be the most effective inductors of spore germination. Specific differences were detected in the requirements of virulent and avirulent strains. Glucose repressed both spore germination and spore formation.     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

Effect of the composition of branched chain amino acids, taurine, and tryptophan on the amino acid metabolism in experimental models of alcoholism

Ethanol withdrawal after forced alcoholization of rats according to Majchrowicz led to the development of amino acid imbalance in the pool of free amino acids in the liver (increasing levels of alanine, aspartate, glutamate, glutamine and histidine, decreasing levels of glycine, lysine, threonine and taurine) and blood plasma (increasing levels of tyrosine and alanine, decreasing levels of most glycogen aminoacids, branched-chain aminoacids and Lys). Less profound changes were observed after prolonged alcohol intoxication (decreasing levels of alanine, ornitine, citrulline and increasing level of Glu in liver, increasing levels of sulfur-containing compounds, Asp and Lys in blood plasma). Amino acid mixture which contained branched-chain amino acids, taurine and tryptophan administered intragastrically was found to correct levels of sulfur-containing amino acids, threonine, lysine and isoleucine after ethanol withdrawal and to eliminate disorders in urea cycle, exchange of threonine, glycine and phenylalanine after prolonged alcohol intoxication.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.