Concentrations Of L-lysine Research Articles

Growth Inhibition of Microcystis Cyanobacteria by L-Lysine and Disappearance of Natural Microcystis Blooms with Spraying

Low concentrations of L-lysine and L-lysine-containing-peptides strongly inhibited the growth of axenic strains of 4 species belonging to the genus Microcystis (cyanobacteria). Inhibitory activities decreased in the order of L-lysine>tri-L-lysine>di-L-lysine>α-poly-L-lysine>L-lysyl-L-histidine>L-lysyl-L-alanine. The addition of 50 μM of L-lysine to growing cells of axenic strains of M. novacekii TAC20-1 and M. viridis NIES102 resulted in a loss of buoyancy, a decrease in chlorophyll a content and consequent cell lysis. The cells took up most of the L-lysine added to the culture medium of M. viridis NIES-102 in one day. However, the amino acid was released back into the medium with cell lysis 3 days after the addition. Laboratory results were confirmed in experimental ponds outdoors by spraying L-lysine onto natural Microcystis blooms in the summer of 1999 and 2000. The spraying of 50 μM L-lysine caused Microcystis colonies to vanish from the surface water within two days; there was a dramatic change in the color and transparency of the pond surface water. After the immediate disappearance of Microcystis sp., both Euglena sp. and/or Phormidium tenue appeared, the latter becoming the dominant species in the phytoplankton community of the pond. In the experiment in summer 2000, the L-lysine provided to the pond was assayed. The level decreased gradually and L-lysine was not detected in the pond water after 6 days. The mechanisms of growth inhibition by L-lysine and a possible mitigation of Microcystis blooms by the amino acid are discussed.

Biosynthesis of l-lysine by Corynebacterium glutamicum grown on fish silage

The microbial utilization of fish silage as a fermentation substrate for the extracellular production of l-lysine by Corynebacterium glutamicum ATCC 21543 was studied in Erlenmeyer baffled flasks. When culture media were supplemented with fish silage at 40 g/l the concentrations of biomass and l-lysine attained 10 and 30 g/l, respectively after 72 h of fermentation. Kinetic fermentation data, the specific rate of l-lysine production and the product yield reached maximum values of 0.096 g/g h and 0.30 g/g, respectively, when yeast extract was replaced by fish silage in culture media. Statistical analysis showed that there was not a significant difference ( P>0.05) between yeast extract (20 g/l) and fish silage (40 g/l) kinetic fermentation data.

Amino acid transport system y+L of human erythrocytes: specificity and cation dependence of the translocation step.

The transport specificity of system y+L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of L-[14C]-lysine. Some neutral amino acids, L-lysine and L-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external L-leucine and L-lysine increased the rate by 5.3 +/- 0.63 and 6.2 +/- 0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (L-tryptophan, L-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (L-alanine, L-serine). Half-saturation constants (+/- SEM) calculated from this effect (L-lysine, 10.32 +/- 0.49 microM and L-leucine, 11.50 +/- 0.50 microM) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na+, Li+ or K+ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na+ or Li+ were replaced by K+. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.

Loss of phosphatidylserine synthesis results in aberrant solute sequestration and vacuolar morphology in Saccharomyces cerevisiae

Null cho1 mutants of Saccharomyces cerevisiae are incapable of phosphatidyl-serine synthesis. They were more susceptible than wild-type strains to 100 mM CaCl 2, 3 mM ZnCl 2 or 1 mM MnCl 2, but not to MgCl 2 nor KCl. They were also susceptible to high concentrations of basic amino acids, l-lysine and l-arginine, and to an l-lysine analog, S-2-aminoethyl- l-cysteine. Their vacuolar pools of amino acids, especially those of basic ones, were decreased. Pigmentation of chol ade2 double mutants was obscured and vacuoles of cho1 mutants were considerably fragmented. These indicate that phosphatidylserine plays vital roles in normal vacuolar function and morphogenesis.

Biogenetic implication of lupin alkaloid biosynthesis in bitter and sweet forms of Lupinus luteus and L. albus

The concentrations of lupin alkaloids, l-lysine and cadaverine were determined by a capillary GC-MS and an amino acid analyser in bitter and sweet plants of Lupinus luteus and L. albus. The contents of total crude alkaloids were ca four-fold higher in bitter plants than those in sweet plants; by contrast, the alkaloid patterns were similar in bitter and sweet plants. However, the cellular concentrations of l-lysine and cadaverine, a precursor amino acid and a decarboxylated intermediate for biosynthesis of the alkaloids, did not differ markedly between bitter and sweet plants. The enzymatic activities of acyltransferases for formation of (-)- p-coumaroyllupinine and (+)- 13α-tigloyloxylupanine were also the same in the cell-free extracts of bitter and sweet plants. These results suggest that the biosynthetic steps of ring closure forming initial cyclic alkaloids such as (-)-lupinine and (+)-lupanine from cadaverine are blocked in sweet plants, but the steps prior to cadaverine and the later steps for modification of the cyclized alkaloids are not altere

Increase in susceptibility of Pseudomonas aeruginosa to carbapenem antibiotics in low-amino-acid media

The in vitro susceptibility of Pseudomonas aeruginosa PAO1 to carbapenem antibiotics, such as CS-533, was influenced by various concentrations of basic amino acids, i.e., L-lysine, L-histidine, and L-arginine, in agar media. P. aeruginosa PAO1 showed higher susceptibility to carbapenems in minimal medium than it did in rich media such as Mueller-Hinton II agar. The susceptibility was decreased by the addition of a basic amino acid to the minimal medium, whereas it was influenced less by other amino acids. The susceptibility of PAO1 to cephalosporins, piperacillin, quinolones, and gentamicin was not influenced by the addition of a basic amino acid to the minimal medium. A significant change in susceptibility to carbapenems by the addition of a basic amino acid was not observed with D2 protein-deficient mutants of PAO1. Clinical isolates of P. aeruginosa also showed an increase in susceptibility in minimal medium. L-Lysine in minimal medium did not have any influence on the production of D2 protein, beta-lactamases, or penicillin-binding proteins of PAO1 or on the chemical degradation of CS-533. These results strongly indicate that the increase in susceptibility of P. aeruginosa to carbapenems relates to less competition with basic amino acids for permeation through the D2 protein channel of P. aeruginosa.

Tissue culture isolation of a second mutant locus for increased threonine accumulation in maize

Regenerable maize (Zea mays L.) tissue cultures were selected for ability to grow in the presence of inhibitory (1.0-1.5 mM) concentrations of L-lysine plus L-threonine. Testcross kernels from one regenerated plant (LT20) segregated for wild-type and high free threonine concentration in a 1∶1 ratio consistent with a single dominant gene for high free threonine. Free threonine concentrations (nmol/mg dry weight) increased an average of 29-fold in bulked F2 kernel samples from heterozygous mutant plants, and the total (free plus protein-bound) threonine concentration increased 68%. Increases in protein-bound methionine, lysine and glycine concentrations were also noted, suggesting a possible effect of the mutation on protein concentration and composition. Allelism tests with a previously selected mutant line, Ltr (*)19, showed that two unlinked, codominant genes conditioned the high free threonine phenotype. Based on a separate study of aspartate kinase feedback inhibition characteristics in the two mutant lines, we propose that the mutant alleles [gene and allele designations are according to guidelines for maize genetic nomenclature (Burnham et al. 1975)] be designated Ask-LT19 and Ask2-LT20 for the Ltr (*)19 and LT20 mutants, respectively.

Enzymatic determination of l-lysine by flow-injection techniques

An enzymatic assay that is highly selective for l-lysine, based on flow-injection techniques combined with spectrophotometric detection, is presented. l-Lysine-α-oxidase (E.C. 1.4.3.14) from Trichoderma viride and horseradish peroxidase were used in a coupled enzyme assay. Peroxide produced in the first reaction was converted by peroxidase with phenol and 4-aminoantipyrine to a quinoneimine dye detectable at 500 nm. An analytical enzyme reactor filled with coimmobilized enzymes was incorporated in the flow-injection system. The assay has a measuring frequency of 30 samples h −1 and a response time of less than 2 min. To adapt the assay to high concentrations of l-Lysine and to minimize interferences, the injected sample volume was reduced to 2 μ-l, resulting in a linearity range of 1–16 mM l-lysine with a sensitivity of 6–7 mV 1 mmol −1, a limit of detection (3σ) of 1 mM and a reproducibility of 0.5% (repetitive injection of a 10 mM l-lysine sample). The enzyme cartridge is stable for several months and thousands of measurements.

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Dihydrodipicolinate synthase, the first enzyme unique to lysine biosynthesis in higher plants, was purified about 5100-fold from suspension-cultured cells of wheat (Triticum aestivum var Chinese Spring). The synthase has an average molecular weight of 123,000 as determined by gel filtration and exhibited maximum activity at pH 8.0. The kinetics of the condensation reaction are compatible with a "Ping Pong" mechanism in which pyruvate reacts first with the enzyme to form a Schiff base. Pyruvate and l-aspartic-beta-semialdehyde (ASA) have respective K(m) values of 11.76 and 0.80 millimolar. Allosteric inhibition was observed with increasing concentrations of l-lysine and its structural analogs, including threo-4-hydroxy-l-lysine and S-(2-aminoethyl)-l-cysteine, with respective I(0.5) values of 51, 141, and 288 micromolar. These amino acids were competitive inhibitors with respect to ASA and noncompetitive inhibitors with respect to pyruvate. We propose that the binding site for lysine overlaps with the ASA binding site, possibly by an attachment of the common alanyl moiety. The wheat enzyme was inhibited by Zn(2+), Cd(2+), and Hg(2+) and also by sulfhydryl inhibitors, p-(hydroxymercuri)benzoic acid and p-chloromercuribenzenesulfonic acid.

Digestive end products mobilize secretory proteins from subcellular stores in the pancreas.

Two digestive end products, D-glucose and L-lysine, produced substantial concentration-dependent release of amylase and trypsinogen, respectively, from subcellular storage pools into a postmicrosomal supernatant fraction of rat pancreatic tissue homogenate. This process was selective in that D-glucose did not lead to trypsinogen release, while L-lysine did not effect amylase. An analogue of D-glucose, 2-deoxy-D-glucose, was much less potent than D-glucose on an equimolar basis. Half-maximal release for both end-product enzyme pairs occurred at concentrations within the range of normal plasma values for these end products in the rat. Although amylase release reached an apparent plateau when the concentration of glucose was increased beyond the maximally effective level, lysine concentrations higher than that maximally effective resulted in a fall in trypsinogen release that ultimately returned (at 3.0 mM L-lysine) to the level seen in its absence. When isolated zymogen granules were exposed to the same concentrations of D-glucose or L-lysine, a similar pattern of release was seen, indicating that the zymogen granules are a source of the enzymes released from the particulate phase of the homogenates. These findings can be explained most simply by the selective movement of digestive enzymes across zymogen granule membranes in response to the presence of appropriate end products. They are also consistent with the concept that digestive end products can act rapidly and directly on the pancreatic acinar cell to regulate the mixture of enzymes secreted in response to the specific hydrolytic needs of a meal.

Fluorometric and electrochemical methods for assay of biological and environmental substances

The isolation, purification, and analytical use of enzymes from microbial sources are described: L-lysine decarboxylase fromE. coli, L-methionine ammonia lyase fromPseudomonasovalis, nitrate reductase fromChlorella vulgaris, nitrite reductase fromAzotobacter chroococcum, sulfate reductase fromDesulvibrio desulficans, and creatininase fromPseudomonas. Both electrochemical, fluorometric, and spectrophotometric methods are proposed for the assay of enzyme activity, and specific, sensitive, fast, and inexpensive methods are described for the assay of 10-1-10-1 M concentrations of L-lysine, L-methionine, nitrate, nitrite, creatinine, and triglycerides.

Transport of basic amino acids and regulation of aspartate kinase activity in thialysine and L-canavanine resistant strains of Serratia marcescens.

Growth of Serratia marcescens was not inhibited by high concentrations of L-lysine and its structural analogues, L-cancavanine and S-(2-aminoethyl)-L-cysteine (thialysine). This insensitivity was not caused by deficient transport of basic amino acids, unlike in mutant strains of Escherichia coli having the same properties. The tested strains showed a lack of regulation at the aspartate kinase level toward L-lysine and thialysine. The data indicate great intraspecific variability for aspartate kinase regulation in S. marcescens.

Aspartokinase isoenzymes of the fruiting myxobacterium Myxococcus xanthus

Two isoenzymes of aspartokinase can be found in extracts of the differentiating bacterium Myxococcus xanthus. Aspartokinase I is repressed by L-lysine and feedback is inhibited by meso-diaminopimelate and by low concentrations of l-lysine. However, the inhibition by l-lysine is no longer observed at high concentration of this amino acid. Aspartokinase II is repressed and feedback inhibited specifically by l-threonine. Both enzymes are stimulated significantly by L-methionine and l-isoleucine; the effect is greater with aspartokinase I. The role of these enzymes in relation to growth conditions of the organism is discussed and a correlation with life cycle activity is indicated.

Control of lysine biosynthesis in Bacillus subtilis: inhibition of diaminopimelate decarboxylase by lysine

Diaminopimelate decarboxylase has been characterized in extracts of Bacillus subtilis and resolved from aspartokinases I and II. Under certain conditions, the enzyme is specifically inhibited by physiological concentrations of L-lysine, but less specificity and altered kinetics of inhibition are observed if lower ionic strengths are employed in the assay procedure. Diaminopimelate decarboxylase can be desensitized to lysine inhibition by either lowering the pH or diluting the enzyme in Tris buffer in the absence of pyridoxal phosphate. Evidence is presented to incidate that, under proper conditions, lysine inhibition involves an interaction of the amino acid with the enzyme rather than competition for available pyridoxal phosphate in the assay. Lysine, by affecting the level of meso-diaminopimelate, may thus regulate its biosynthesis through sequential feedback inhibition. Analysis of the diaminopimelate decarboxylase of 15 revertants of mutants that had originally lacked diaminopimelate decarboxylase activity indicates that as little as 5% of the specific activity of enzyme observed in the wild-type strain is sufficient to permit normal growth rates. In the growing cell, diaminopimelate decarboxylase may therefore exist largely in an inhibited state.

Saturation of a shared mechanism which transports L-arginine and L-lysine into the brain of the living rat.

1. The rate of entry of L-arginine and L-lysine into the brain of the rat was measured in vivo by a direct method in which the amino acid concentration was at a constant level in the blood plasma over the period of the experiment.2. Both L-arginine and L-lysine enter the brain by a transport mechanism which can be saturated by a high concentration of the same amino acid in the bloodstream. The rate of entry can be explained by Michaelis-Menten kinetics.3. The entry into the brain of L-arginine can be inhibited by raised plasma concentrations of L-lysine or L-ornithine and the entry of L-lysine by raised concentrations of L-arginine.4. The inhibition of entry of an amino acid is most severe when its own concentration in the blood plasma is low and that of the inhibitor is high. The inhibition appears to be basically competitive in type, suggesting that common transport systems are shared by three dibasic amino acids.5. It is suggested that the raised levels of amino acids found in various disorders of amino acid metabolism are likely to reduce the rate of entry into the brain of other amino acids and a way is suggested in which the dietary treatment of hyperlysinaemia may be made more effective.

Effects of Exogenous Amino Acids on the Development of Marchantia polymorpha Gemmalings

The effects of various nitrogenous compounds in concentrations ranging from 10-4 to 10-3 m on the growth and morphology of gemmae of Marchantia polymorpha L. were investigated under axenic conditions. Only nine of the 24 compounds tested were effective in altering the normal developmental pattern of this plant. l-Isoleucine, l-leucine, l-methionine or l-threonine resulted in a disruption of the apical regions at concentrations of 10-3 m. The addition of lower concentrations of l-arginine, l-histidine, l-hydroxyproline, l-lysine or l-tryptophan to the nutrient medium resulted in morphological irregularities. These latter abnormal growth patterns were highly specific for the individual amino acids and ranged from lack of air chamber development to complete thallus disorganization. Both the formation and duration of abnormal developmental patterns were dependent upon the concentration of the amino acids employed. Although the experimental results are inconsistent with certain physiological mechanisms such as a general nitrogen toxicity, alternative possibilities are discussed.