Amino Acids L-lysine Research Articles

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids l-lysine, l-arginine, l-histidine and l-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicles consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly- l-ornithine or l-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly- l-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than l-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5–6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine / phosphatidylethanolamine / phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca 2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly( l-histidine)- and poly( l-lysine)-mediated fusion.

L-lysine production at 50 degrees C by mutants of a newly isolated and characterized methylotrophic Bacillus sp

The amino acid L-lysine was produced from homoserine auxotrophic and S-(2-aminoethyl)-L-cysteine-resistant mutants of a newly isolated gram-positive methylotrophic bacterium, capable of growth on methanol at 60 degrees C. The temperature optimum for growth was between 50 and 53 degrees C. These aerobic, gram-positive, endospore-forming, rod-shaped bacteria required biotin and vitamin B12 for growth. Extracts of the bacteria grown on methanol lacked hydroxypyruvate reductase and contained hexulose 6-phosphate synthase activity. Therefore, these bacteria were considered to be type I methylotrophic bacteria of the genus Bacillus. Fed-batch fermentations resulted in cell densities of 50 g of cell dry weight per liter. Biomass yields on carbon, nitrogen, phosphate, and sulfate were determined. Generation of homoserine auxotrophic and amino acid analog-resistant mutants resulted in L-lysine concentrations of nearly 20 g/liter in fed-batch fermentations.

Photoexchange products of cytosine and 5-methylcytosine with Nα-acetyl- l-lysine and l-lysine

The photoinduced exchange reactions of cytosine ( Ia) and 5-methylcytosine ( IIa) with Nα-acetyl- l-lysine, a derivative of the common amino acid l-lysine, were studied. These reactions of Ia and IIa at pH 7.5 produce, respectively, 2- N-acetylamino-6-(1-cytosinyl)hexanoic acid ( Ib) and 2- N-acetylamino-6-(1-(5-methylcytosinyl))hexanoic acid ( IIb) as major final products. In addition, small amounts of the corresponding deamination products were formed in the 5-methylcytosine- Nα-acetyl- l-lysine and cytosine- Nα-acetyl- l-lysine systems, namely 2- N-acetylamino-6-(1-thyminyl)-hexanoic acid and 2- N-acetylamino-6-(1-uracilyl)hexanoic acid. The compounds Ib and IIb were deacetylated by acid hydrolysis to yield the corresponding lysine products: 2-amino-6-(1-cytosinyl)hexanoic acid ( Ic) and 2-amino-6-(1-(5-methylcytosinyl))hexanoic acid ( IIc). The compound Ic was identified as a product in the photoreaction of cytosine with l-lysine at near neutral pH, while IIc is found as a product in the corresponding reaction of 5-methylcytosine. The occurrence of the above photoexchange reactions at, pH values near those found in physiological systems could have biological implications; in particular, our observations suggest that cytosine and 5-methylcytosine residues, contained in DNA, might react with the ϵ-amino groups of lysine residues in proteins upon UV irradiation of nucleosomes and other DNA-protein complexes under physiological conditions.

In situ metabolism of 1,omega medium chain dicarboxylic acids in the liver of intact rats as detected by 13C and 1H NMR.

The hepatic metabolism of 1,omega-dodecanedioic acid, a physiologically relevant representative of the medium-chain dicarboxylic acid family, has been studied by a combination of in vivo and in vitro 13C and 1H NMR spectroscopic techniques. Rats in different nutritional or hormonal situations were infused with [1,12-13C2]- or [1,2,11,12-13C4]dodecanedioic acid, and the kinetics of 13C label appearance as well as the final relative concentrations of metabolic products were measured noninvasively in the liver of the intact rat by 13C NMR spectroscopy. Perchloric acid and chloroform/methanol extracts of liver biopsies obtained at the end of the infusion period were further analyzed by high resolution 13C NMR and one-dimensional and two-dimensional COSY and J-resolved 1H NMR. [1-13C]- and [1,2-13C2]adipic acids were the main end products of the in vivo metabolism of [1,12-13C2]- or [1,2,11,12-13C4]dodecanedioic acids, respectively, indicating that the beta-oxidation pathway of medium-chain dicarboxylic acids proceeds in situ monodirectionally. [1-13C]Adipic acid, the main product of peroxisomal beta-oxidation, could also be detected in situ. This finding, together with the in vivo and in vitro absence of signals characteristic of intramitochondrial oxidation of [1-13C]acetyl-coenzyme A, provide a strong evidence supporting a predominant contribution of the peroxisomal beta-oxidation system to the overall oxidation of these compounds in vivo. Homonuclear two-dimensional COSY 1H NMR spectra of acid extracts from rat liver provided a convenient method of analyzing the metabolic repercussions of dicarboxylic acid accumulation, revealing a decrease in the hepatic concentration of beta-hydroxybutyrate and an accumulation of adipic acid and the amino acid L-lysine.

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles.

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.

Is leucine an allosteric modulator of the lysine transporter in the intestinal basolateral membrane?

The transport of the dibasic amino acid L-lysine was investigated using basolateral membrane vesicles prepared from rat jejunal mucosal scrapings. The majority of the carrier-mediated transport was unaffected by the presence of sodium in the incubation medium, but voltage clamping of the vesicles did increase lysine uptake, indicating an associated movement of charge. Kinetic analysis of lysine influx and efflux showed the system to be symmetrical, but although the Vmax was comparable to other amino acid transport systems in this membrane, the dissociation constant for the overall reaction (KT) was an order of magnitude larger. This low affinity for lysine would explain the relatively slow rate of transport of this amino acid across the basolateral membrane. Competition experiments indicated that this system has a relatively narrow specificity carrying only lysine, arginine, ornithine, and histidine. In contrast the presence of L-leucine caused a marked stimulation of lysine efflux and influx across the vesicles. This effect was observed with leucine concentrations as low as 0.1 microM. It is concluded that although the lysine transport system in the basolateral membrane is slow in its basal state it can be rapidly turned on by the presence of L-leucine. The remarkably low concentrations required to do this suggest a possible allosteric interaction between the transporter and this neutral amino acid.

Effect of environmental factors on the limpid composition membrane structure and permeability ofMicrosporum gypseum

Supplementation with unsaturated fatty acids, substitution of glucose by glycerol as carbon source and lowered growth temperature (20°C) increased the total phospholipid content ofMicrosporum gypseum spheroplasts. Levels of sterols increased with glycerol substitution and decreased in other growth conditions. Substantial changes were seen in the ratios of unsaturated to saturated fatty acids and phosphatidylcholine to phosphatidylethanolafne under all the experimental conditions. Changed lipid composition resulted in altered uptake of amino acids (L-lysine, L-aspartic acid and L-glycine) and increased number of binding sites for a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.

Poly-lysines as modifiers of one- and two-chain tissue-type plasminogen activator activity.

Poly-L-lysine and certain mixed polymers of L-lysine and other amino acids modify the activity of one- and two-chain tissue-type plasminogen activator (t-PA) towards its substrates. In particular the rate of plasminogen activation in the presence of optimal poly-L-lysine concentrations, is increased by approximately 100-fold. In contrast, activity towards a small synthetic substrate is inhibited by 85%. These effects are observed with both the one- and two-chain forms of t-PA. The use of poly-L-lysines in a coupled assay system optimised for t-PA and plasmin activities allows the reproducible assay of t-PA at the 10(-12) to 10(-13) molar level.

Kinetics and Specificity of Amino Acid Uptake by the DuckweedSpirodela polyrhiza(L.) Schleiden

Borstlap, A. C., Meenks, J. L. D., van Eck, W. F. and Bicker, J. T. E. 1986. Kinetics and specificity of amino acid uptake by the duckweed Spirodela polyrhiza (L.) Schleiden.—J. exp. Bot. 37: 1020-1035. Uptake of 14C-labelled amino acids by intact, axenically grown plants of Spirodela polyrhiza (L.) Schleiden was investigated. Experiments in which uptake was measured from the decrease in the amino acid concentration in the medium, indicated that saturable uptake conforms to the sum of two Michaelis-Menten terms, possibly corresponding with a high-affinity and a low-affinity system. Further experiments with L-leucine, L-glutamic acid, and L-lysine, in which uptake was measured by assaying the amount of 14C in the plants, showed the presence of a non-saturable component in addition to the dual saturable uptake. Uptake of L-glutamic acid precipitously declined between pH 4 0 and 6 0 and that of L-leucine between pH 4 0 and 8 0, whereas L-lysine uptake was optimal at pH 6 0. No evidence was found that the apparent high-affinity and low-affinity systems responded differently to changes in external pH or to the addition of CCCP. The non-saturable uptake component was not affected by changes in external pH or by adding CCCP, and might have been due to free space uptake. Mutual inhibition of uptake was found between acidic and neutral amino acids (L-leucine, L-methionine, L-glutamic acid) and between basic amino acids (L-lysine, L-ornithine). The basic amino acids had no effect on the uptake of L-leucine, L-methionine and L-glutamic acid, although the uptake of basic amino acids was inhibited by glutaminc acid and several neutral amino acids. It is suggested that the duckweed has a high-affinity transport system for neutral and acidic amino acids, and a distinct high-affinity system for basic amino acids. It is argued that the first system transports zwitterionic amino acids (z-system), and that the second system transports cationic amino

Taste responses to amino acids in the southern leopard frog, Rana sphenocephala

1. 1. Integrated taste recorclings of the glossopharyngeal (IX) nerve innervating the tongue of the southern leopard frog were studied in response to various amino acids and quinine hydrochloride. 2. 2. Amino acids and quinine hydrochloride elicited primarily phasic taste responses. 3. 3. Acidic ( l-aspartic and l-glutamic) and basic ( l-lysine and l-arginine) amino acids, adjusted to pH8, were effective taste stimuli. 4. 4. All glossopharyngeal nerve twigs that responded to amino acid stimuli also responded to quinine; however, not all quinine-sensitive IX nerve bundles were responsive to amino acids. 5. 5. Electrophysiological thresholds for amino acids were estimated to be 2.5–10 mM, whereas threshold for quinine hydrochloride averaged approximately 10 μM.

ChemInform Abstract: SYNTHESIS AND ANTITUMOR ACTIVITY OF L‐LYSINE NITROSOCARBAMATES

AbstractAus L‐Lysin (I) werden über dessen Kupferkomplex und weiter die Carbamate (III) die Nitrosocarbamate (IV) hergestellt, deren Hydrolysebeständigkeit, Toxizität und Antitumor‐Wirkung geprüft werden.

Synthesis and antitumorigenic activity of L-lysine nitrosocarbamates

After the high antitumorigenic activity of nitrosourea derivatives has been established on many experimental tumors, research on their structural analogs developed. Nitroso derivatives of alkylbiurets, alkylureas, carboxamides, alkylnitroguanidines, and alkylthioureas were obtained [I, 2]. In continuation of these studies, we synthesized nitrosocarbamates (Ia, b) in which the residue of the natural amino acid L-lysine (II) is the carrier of the cytotoxic nitrosocarbamate grouping.

Discrimination of parallel neutral amino acid transport systems in the basolateral membrane of cat salivary epithelium.

Transport of short-chain and long-chain neutral amino acids across the basolateral membrane of the epithelium in the perfused cat salivary gland has been studied using a rapid (less than 30 s) single circulation paired-tracer dilution technique. Amino acid uptake was measured by comparing the venous dilution profiles for a tritiated amino acid and D-[14C]mannitol (extracellular reference) following a bolus intra-arterial injection of a mixture containing both molecules. Unidirectional influx (v) was estimated from the maximal tracer uptake (Umax), the perfusate flow (F) and the perfusate amino acid concentration (Ca): v = [-F . ln (1-Umax)] . Ca. L-alanine influx was saturable and apparently mediated by a single entry system (Km = 0.83 +/- 0.11 mM and Vmax = 655 +/- 32 nmol/min . g). These kinetic constants were considerably lower than our previously reported values for L-phenylalanine: Km = 6.4 mM and Vmax = 1719 nmol/min . g. In cross-inhibition experiments performed over a wide range of concentrations (0.05-24 mM), influx of L-alanine and L-phenylalanine could be further discriminated, since both L-phenylalanine (Ki = 22 mM) and L-alanine (Ki = 19 mM) behaved as poor competitors. Removal of Na+ from the perfusate resulted in a selective inhibition of L-alanine and L-serine influx, whereas influx of the long-chain neutral amino acids L-leucine, L-phenylalanine and L-tryptophan remained unaffected. Although prolonged perfusion of glands with dinitrophenol (0.8 mM for 20-30 min) caused a variable but net inhibition of unidirectional uptake, it markedly enhanced the tracer efflux of L-leucine, L-phenylalanine, L-tyrosine and the basic amino acid L-lysine. It appears that at least two separate neutral amino acid transport systems are operative at the blood-tissue interface of the salivary epithelium: (i) a Na+-dependent alanine-serine-cysteine preferring type of carrier exhibiting a high affinity for amino acids with short, polar or linear side chains and (ii) a Na+-independent leucine preferring type of carrier selective for large neutral amino acids.

Characteristics of lysine transport across the serosal pole of the anuran small intestine.

The transport of the dibasic amino acid L-lysine across the serosal pole of the intestinal epithelium has been studied using the vascularly perfused anuran small intestine. The exit of pre-loaded lysine into the vascular bed was inhibited by L-ornithine (2 mM) and L-arginine (10 mM) when pulsed through the lumen during the wash-out, while 2-aminoisobutyric acid (AIB), L-histidine, L-citrulline and L-cystine had no effect. Luminal L-leucine and L-alanine at a concentration of 10 mM markedly stimulated the unloading of lysine into the vascular bed and sarcosine, L-proline and beta-alanine also did so to a lesser extent. The instantaneous rate constant for lysine exit into the vascular bed was increased by the presence of L-arginine, L-ornithine, L-citrulline, L-histidine, AIB, L-leucine and L-alanine at a concentration of 10 mM in the vascular bed. L-proline had no effect. The simultaneously measured efflux of lysine into the lumen was unaffected by the presence of the other amino acids in the vascular bed. The uptake of lysine into the epithelium from the vascular bed was accelerated by L-ornithine and slightly by L-arginine when they were present in the lumen, while L-leucine, L-alanine, beta-alanine, L-proline, L-citrulline, sarcosine, L-histidine and AIB had no effect. The instantaneous rate constant for lysine wash-out into the vascular bed was transiently increased by the presence of L-leucine in the vascular bed at concentrations of 10, 0.10 and 0.01 mM. The steady-state transfer of lysine from the lumen to the vascular bed was stimulated in a biphasic manner by 5 mM-leucine in the lumen and by 0.5 mM-leucine in the vascular bed. The mechanisms for these interactions between lysine transport across the basolateral membrane of the enterocyte and other amino acids are discussed and a possible role for neutral amino acid stimulation of lysine exit is proposed.

Vasopressin release in response to intracerebroventricular L-alanine and L-arginine, and its dependence upon CSF NaCl concentration.

Influences on renal water, electrolyte, and arginine vasopressin (AVP) excretions of 1 h infusions (20 microliters/min) of a neutral (L-alanine) and two basic (L-lysine and L-arginine) amino acids into the lateral cerebral ventricle were studied in hydrated goats, and were compared to effects of control infusions of hypertonic (0.25 M) NaCl. L-alanine (0.11 M) dissolved in hypotonic NaCl caused more pronounced inhibition of the water diuresis and greater increase in AVP excretion than did the control infusions, but, in comparison to the latter, the responses developed very slowly. The effects were further delayed and were much attenuated when L-alanine was administered in isotonic glucose, but became considerably accentuated when isotonic NaCl was used as the solvent. L-lysine (0.09 M) in hypotonic NaCl did not inhibit the water diuresis or cause any apparent AVP release, whereas the corresponding L-arginine infusions caused inhibition of the water diuresis and increase in AVP excretion of approximately the same magnitudes and time courses as the control infusions. Like for L-alanine, these effects became accentuated when L-arginine was dissolved in isotonic NaCl, and became delayed and much attenuated when isotonic glucose was used as the solvent. L-arginine induced a more pronounced increase in renal Na excretion than did L-alanine and 0.25 M NaCl. Since transport together with Na (increasing the Na influx) generally is much more important for cellular uptake of neutral than of basic amino acids, the possibility is discussed that L-alanine here might have caused AVP release by increasing transmembrane Na transport of juxtacerebroventricular Na sensors regulating the AVP secretion--a suggestion supported by the lack of response to the basic L-lysine. The antidiuretic effect of the other basic amino acid, L-arginine, can not be explained along this line. However, with regard to the characteristic differences observed between the responses to L-alanine and L-arginine, the possibility is discussed that the latter might not have acted at a sensory level, but on the final neuronal link in the release of neurohypophyseal hormones, the hypothalamic neurosecretory cells. In contrast to L-alanine and L-arginine, L-lysine appeared to stimulate the appetite of the goats.

Jejunal water and electrolyte secretion induced by L-arginine in man.

In this study a perfusion technique has been used to investigate jejunal secretion in response to the dibasic amino acid L-arginine. L-arginine at 5, 15, and 40 mmol/l in isotonic saline solutions induced net intestinal secretion of water and Na+. The structurally similar dibasic amino acid L-lysine caused net absorption at 5 and 15 mmol/l, and only modest net secretion of water and Na+ at 40 mmol/l, although absorption rates of the two amino acids were similar. D-arginine (15 mmol/l) was without effect on net water and Na+ absorption. L-arginine 15 mmol/l inhibited glucose-stimulated water and Na+ absorption when perfused in the same intestinal segment, but was without effect when perfused in separate jejunal or ileal segments. Parenteral chlorpromazine inhibited L-arginine induced jejunal water and Na+ secretion. Jejunal secretion induced by L-arginine thus appears not to be due to passive osmotic water flow, nor to release of circulating secretagogues. Stimulation of a mucosal secretory process is most likely to be the mechanism.

Kinetics of neutral amino-acid transport by isolated gill tissue of the bivalve Mya arenaria (L.)

An in vitro technique was used to examine the absorption by the gill of Mya arenaria (L.) of six neutral l-amino acids chosen for differences in their side chains, viz., short chain — glycine and alanine, long chain — leucine, sulphur containing — methionine, aromatic — phenylalanine, hydroxylic — serine. The uptake of all these substrates was active and carrier-mediated, and was analysed by Michaelis-Menten kinetics. Values of K t , the transport constant, decreased with increasing length of the side chain for glycine, l-serine, l-alanine, l-methionine, l-phenylalanine, and l-leucine, while values for V max , the maximum velocity of uptake, decreased as chain length increased, except in the case of l-serine. Inhibition experiments suggested that at least one transport locus was common to all the neutral amino acids examined, but homogeneity of transport was only demonstrated in the case of methionine and leucine. The transport of the basic amino acid l-lysine overlapped with several of the-neutral amino acids. These results emphasize the need to consider the mutual inhibitory effects between amino acids absorbed from sea water, when calculations are made of the value of this source of nutrition to marine invertebrates.

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18 hr show high nuclease activity, low rates of incorporation of amino acids and nucleosides into macromolecules, and high rates of spontaneous lysis. Addition to the leaf flotation medium of the senescence retardants cycloheximide or kinetin, of the dibasic amino acids L-lysine or L-arginine, or of the diamines putrescine or cadaverine reduces the rise in nuclease activity and spontaneous lysis of protoplasts, and increases the rate or extent of presumptive protein and nucleic acid synthesis. The diamines, which also retard chlorophyll degradation in the excised leaves, appear to act both on the membrane and on systems controlling macromolecular synthesis and breakdown. By contrast, the senescence promoter L-serine hastens chlorophyll degradation from excised leaves and does not improve protoplasts derived from those leaves.

INHIBITION OF INTESTINAL UPTAKE OF AMINO ACIDS BY UNCONJUGATED BILE SALT

The unconjugated bile salt, sodium deoxycholate, at a concentration of 0.5 mM was shown to inhibit the intestinal uptake of the amino acids L-glycine, L-leucine, L-proline, L-lysine and L-tyrosine in rats in vitro. This effect was acutely reversible except for the basis amino acid L-lysine and is therefore not simply due to tissue damage. These results, and the recent finding that sodium deoxycholate inhibits intestinal absorption of amino acids in vivo, suggest that impaired intestinal amino acid transport may contribute to hypoproteinaemia in patients with bacterial overgrowth in the upper small intestine in whom deoxycholate is present in the small intestinal lumen in excessive concentrations.