Accumulation Of Lysine Research Articles

Phenylglyoxal suppresses cationic lysine/K+ symport under alkaline conditions in brush border membrane vesicles from larval Manduca sexta midgut.

The arginine-specific reagent, phenylglyoxal, decreases the initial rate of lysine/K+ symport (cotransport) as well as maximum lysine accumulation at pH 9.2, by brush border membrane vesicles obtained from the larval midgut of the lepidopteran, Manduca sexta. The symport of a neutral amino acid, leucine, remained unaffected. Following exposure to phenylglyoxal, the apparent dissociation constant for lysine increased by a factor of 2.5 whereas the maximum uptake rate decreased by a factor of 0.4. More than one arginine residue appears to react with phenylglyoxal. Apparently phenylglyoxal reacts preferentially with arginine residues on a symporter that is specific for positively charged lysine. Phenylglyoxal shows promise as a specific covalent label for the identification of a cationic amino acid symporter.

Lysine synthesis and catabolism are coordinately regulated during tobacco seed development.

The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine-ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.

Bacillus thuringiensis subsp. Aizawai δ-endotoxin inhibits the k+/amino acid cotransporters of lepidopteran larval midgut

Abstract 1. The δ-endotoxin of Bacillus thuringiensis subsp. aizawai inhibits in a dose-dependent manner the K+-driven accumulation of histidine and lysine as well as their equilibrative uptake into brush border membrane vesicles from the midgut of Bombyx mori larvae. 2. The decrease of uptake in the absence of a K+-gradient is neither due to a leakage of the labelled substrate from the vesicles nor to a reduction of the osmotically active space available, as a result of a detergent-like effect of the toxin. 3. The toxin acts as a non competitive inhibitor of the K+/amino acid cotransporters of the larval midgut of Lepidoptera, with no preference for a specific transport system.

Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum

The Gram-positive Corynebacterium glutamicum has the potential to synthesize L-lysine via a split pathway, where amino-ketopimelate is converted to the ultimate lysine precursor diaminopimelate either by reactions involving succinylated intermediates, or by one single reaction catalysed by D-diaminopimelate dehydrogenase. To quantify the flux distribution via both pathways, 13C-enriched glucose was used and specific enrichments in lysine and in pyruvate-derived metabolites were determined by 13C- and 1H-NMR spectroscopy. Using a system of linear equations, the contribution of the D-diaminopimelate dehydrogenase pathway was determined to be about 30% for the total lysine synthesized. This was irrespective of whether lysine-accumulating mutants or the wild-type strain were analysed. However, when the distribution was determined at various cultivation times, the flux partitioning over the dehydrogenase pathway in a producing strain decreased from 72% at the beginning to 0% at the end of lysine accumulation. When ammonium sulphate was replaced by the organic nitrogen source glutamate, the ammonium-dependent D-diaminopimelate dehydrogenase pathway did not contribute to total lysine synthesis at all. Additional experiments with varying initial ammonium concentrations showed that in Corynebacterium glutamicum the flux distribution over the two pathways of lysine synthesis is governed by the ammonium availability. This is thus an example where an anabolic pathway is directly influenced by an extracellular medium component, probably via the kinetic characteristics of D-diaminopimelate dehydrogenase.

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the gypsy moth (Lymantria dispar).

Brush border membrane vesicles were prepared from both fresh and frozen midguts of Lymantria dispar larvae by Mg/EGTA precipitation and differential centrifugation. The vesicles were enriched 10 to 13-fold, relative to the homogenate, in aminopeptidase and gamma-glutamyltransferase activity. No significant difference was found in enzyme enrichment of vesicles prepared from fresh and frozen midguts. Inwardly directed potassium salt gradients resulted in transient accumulation of leucine and lysine but not glutamic acid by the vesicles.

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked. Study of the inhibition properties of dihydrodipicolinate synthase and aspartate kinase activities in RAEC-1 x RLT 70 confirmed the heterozygote state of both mutations, because only half of their lysine-sensitive activity could still be inhibited by this negative effector. Analysis of the free amino acid pool during the growth of the double mutant revealed a major free lysine overproduction reaching up to 50% of the total pool, whereas the other aspartate-derived amino acids remained equally or even less abundant than in the wild type. An abnormal phenotype was clearly associated with such high levels of lysine accumulation, which points out the possible role of this amino acid in the developmental features of the plant. Comparison of the amino acid content, free and total (free + protein-bound), between the wild type, the two mutants, and the double mutant obtained by crossing them brings new insights on the regulation of the aspartate pathway, and on its implications in relationship to plant nutritional value improvement.

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

SummaryA major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein‐bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.

Isolation and properties of threonine-producing mutants with both dihydrodipicolinate synthase defect and feedback-resistant homoserine dehydrogenase from Brevibacterium flavum.

Threonine-producing mutants of Brevibacterium flavum with HDR or DPS defect were selected by resistance to less than 8g/l of AHV, a threonine analogue, in our previous studies. Growth of a mutant with HDR, BK 29, was still inhibited by 12 g/1 of AHV. Eighteen AHV-resistant mutants derived from strain BK29 under these conditions produced more than 12 g/1 of threonine. All the best five strains tested showed much lower levels of or no DPS activity in addition to HDR. The best threonine producer, strain DB185, produced 16.5 g/1 of l-threonine, about twice that by the parent, but the accumulation of lysine decreased to 3.8 from 10 g/1 as the HC1 salt by the parent. The threonine production and growth were stimulated by DAP. On the other hand, growth of a mutant with DPS defect, DK330, was not inhibited by 12 g/1 of AHV. However, addition of lysine enhanced the growth inhibition by AHV to be almost as complete as that of strain BK29. The best strain BD122 among mutants resistant to AHV plus lysine derived from...

Amino acid transport by membrane vesicles of an obligate anaerobic bacterium, Clostridium acetobutylicum.

Membrane vesicles were isolated from the obligate anaerobic bacterium Clostridium acetobutylicum. Beef heart mitochondrial cytochrome c oxidase was inserted in these membrane vesicles by membrane fusion by using the freeze-thaw sonication technique (A. J. M. Driessen, W. de Vrij, and W. N. Konings, Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985) to accommodate them with a functional proton motive force-generating system. With ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine-cytochrome c as the electron donor, a proton motive force (delta p) of -80 to -120 mV was generated in these fused membranes. This delta p drove the accumulation of leucine and lysine up to 40- and 100-fold, respectively. High transport activities were observed in fused membranes containing Escherichia coli lipids, whereas the transport activities in fused membranes containing mainly soybean lipids or phosphatidylcholine were low. It is suggested that branched-chain amino acids and lysine were taken up by separate systems. The effects of the ionophores nigericin and valinomycin indicated that lysine and leucine were translocated in symport with a proton.

Prostacyclin-inhibition of lysine accumulation by the rat left ventricle

1. 1. Left ventricular slices of male Sprague-Dawley rats were incubated with a fixed concentration of 0.5 μCi/ml 3H-lysine and several concentrations of unlabelled lysine ranging from 0.2 to 5.0 mM in control and prostacylin-treated experiments. The time of incubation ranged from 0.5 to 90 min. 2. 2. Left ventricular slices were cut to have an optimal thickness of 0.47 ± 0.09 mm. 3. 3. Lysine was taken up against a concentration gradient. Saturation was reached at 0.5 mM and steady state accumulation of lysine was attained within 60 min. 4. 4. Prostacyclin in concentrations ranging from 1.2 × 10 −8 to 4.8 × 10 −8 M inhibited lysine transport in left ventricular slices significantly ( P < 0.01).

Lysine uptake by rat renal brush-border membrane vesicles.

Lysine uptake by isolated rat renal brush-border membrane vesicles occurs via a single saturable system plus a significant diffusion component with indication of a single energy of activation on an Arrhenius plot. Initial uptake is not sodium dependent, and intravesicular accumulation of lysine at longer time points is greatest in the absence of sodium. Accumulation levels differ in the presence or absence of NaCl or choline chloride and are specific for the cation used. Lysine uptake is membrane potential sensitive and inhibitable by cystine, dibasic amino acids, and cycloleucine. Heteroexchange diffusion of lysine with cystine and lysine with arginine occurs, but no heteroexchange occurs with cycloleucine, indicating that lysine shares a transport system with cystine and dibasic amino acids but not with cycloleucine.

Renal tubular transport of delta-aminolevulinic acid in rat.

delta-Aminolevulinic acid (ALA) interferes with cell membrane and metabolic functions in a variety of tissues. To determine if ALA interacts with renal tubular transport functions, we examined concentrative transport of this heme precursor in rat kidneys. ALA was accumulated against a concentration gradient in rat renal cortical slices. Section freeze-dry autoradiography demonstrated selective accumulation in cells of proximal tubules. Concentrative uptake of ALA was inhibited by KCN, probenecid and p-aminohippurate (PAH). ALA inhibited slice uptake of PAH but failed to block slice accumulation of galactose, cycloleucine, lysine, glycine, proline, or alpha-aminoisobutyric acid and did not alter O2 utilization. Massive intraperitoneal injection of ALA did not increase 24 hr fractional excretion of amino acids in vivo. Concentrative transport of ALA in proximal tubules does not lead to generalized renal tubular transport defects but ALA appears to share the organic acid secretory system in rat kidney.

Effect of dietary and extracellular potassium on lysine metabolism in the rat

Previous studies have shown that potassium (K) depletion leads to the accumulation of lysine and other cationic amino acids in tissues. The metabolic nature of this interaction is unknown but is presumably related in part to a unique aspect of these amino acids, possibly their charge. These studies were completed to evaluate the nature of the effect of K on lysine metabolism in the rat. In the first study, 42 rats were maintained on K-deficient or K-adequate diets for 10 days, after which blood and tissue samples were taken from 12 rats for electrolyte, amino acid and blood gas analyses. Tissue slices of liver, kidney and segments of diaphragm were prepared from the remaining 30 rats (15 per group) and incubated in medium containing either 3 or 6 mM K, and effects of dietary and medium K concentrations on lysine catabolism were determined. In the second study, the effects of 0, 3, 6 or 12 mM K on 14C-lysine uptake, catabolism and incorporation into protein were evaluated. Dietary K deficiency did not influence blood pH, bicarbonate concentration or base excess of blood; however, it decreased the concentration of plasma K and caused accumulation of lysine and arginine in muscle and lysine in liver. K deficiency resulted in a 36% reduction in the rate of lysine catabolism in kidney. Much of the dietary K effect may be related to diminished plasma K concentration, as low K concentration in vitro caused similar, although less dramatic, decreases in lysine oxidation, diminished incorporation of lysine into protein in kidney and increased lysine uptake in both liver and kidney. K deficiency appears to conserve lysine in the free form in the intracellular pool by enhancing cellular uptake and by decreasing the degradation of lysine and the utilization of lysine in protein synthesis.

Ornithine accumulation and metabolism in rat lens

The non-protein amino acid ornithine is readily accumulated into rat lenses cultured in TC-199 bicarbonate medium. This accumulation, measured by the lens water/lens medium ratios of radiolabeled ornithine, appears to be the result of an apparent energy-dependent basic amino acid transport system. Moreover, competition studies suggest that increased levels of ornithine can depress the concomitant lenticular accumulation of arginine and lysine. Examination of the trichloroacetic acid protein precipitates of these cultured lenses indicates that the radiolabel from ornithine can also be incorporated into lens proteins as proline. This results from the conversion by ornithine aminotransferase (EC 2. 6. 1. 13) of radiolabeled ornithine to delta 1-pyrroline-5-carboxylic acid (P5C), which is subsequently converted to proline by P5C reductase (EC 1. 5. 1. 2). This incorporation of radiolabel into cultured lens proteins is reduced upon addition of either 1 m m proline or P5C to the culture medium or inhibited by the addition of the protein synthesis inhibitor, puromycin.

Characterization of Cultured Tobacco Cell Lines Resistant to Ethionine, a Methionine Analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110x), threonine (18x), and lysine (5x). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K(m) values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.

Microbial Production of Lysine and Threonine from Whey Permeate

Extracellular accumulation of lysine and threonine was investigated in modified whey permeate by using Brevibacterium lactofermentum ATCC 21086 and Escherichia coli ATCC 21151. Whey permeate was prepared from whey by membrane ultrafiltration, and lactose was hydrolyzed by treating permeate with HCl or beta-galactosidase. The highest amount of lysine (3.3 g/liter) was produced from a mixture of acid-hydrolyzed whey permeate and yeast extract (0.2%). The highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH(4))(2)SO(4) (1.4%), yeast extract (0.1%), and Na(2)CO(3) (0.3%).

Enzymatic inhibition of lysine transport across rat and turtle small intestine

1. 1. Trypsin, Phospholipase- d and Neuraminidase at different concentrations inhibited significantly ( P < 0.001) lysine accumulation in rat intestinal cells, whereas Chymotrypsin did not show any significant effect. 2. 2. Trypsin, Chymotrypsin and Phospholipase- d did not show any significant change in lysine accumulation when incubated with mucosal strips isolated from turtle intestine; however, Neuraminidase caused a significant decrease in lysine uptake by these strips. 3. 3. Unidirectional lysine influx across the rat small intestine was significantly reduced ( P < 0.01) by the presence of Trypsin, Phospholipase- d and Neuraminidase in the preincubation solution, and its influx across the turtle small intestine was significantly inhibited ( P <0.01) by Neuraminidase only. 4. 4. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with any of the enzymes in both animals.

Effect of Plasma-membrane Phospholipid Unsaturation on Solute Transport into Saccharomyces cerevisiae NCYC 366

A comparison was made of kinetics of solute accumulation by Saccharomyces cerevisiae NCYC 366 grown anaerobically under conditions that lead to enrichment of the plasma membrane with ergosterol and either oleyl or linoleyl residues. Values for K T and V max were identical for accumulation of l-asparagine, l-glutamine, H2PO4 2-, Ca2+ and SO4 2-, while for accumulation of d-glucose, the value differed slightly but not significantly. Values for K T for accumulation of l-lysine, by both the low-and high-affinity systems, decreased when oleyl residues were replaced by linoleyl residues. Under these conditions, V max values for the high-affinity system decreased while that for the low-affinity system increased. An Arrhenius plot for accumulation of lysine by the high-affinity system revealed a discontinuity when membranes were enriched in linoleyl residues. However, no discontinuity was evident on plots of lysine accumulation when membranes were enriched in oleyl residues. Similar plots for accumulation of l-asparagine, which was used as a control, showed that substitution of linoleyl for oleyl residues significantly raised the transition temperature, but had little effect on the activation energy at temperatures below the discontinuity. When palmitoleyl residues were incorporated into the yeast plasma membrane, the K T value for l-lysine accumulation by the high-affinity system was hardly altered, although the V max value was lowered, as compared with organisms with membranes enriched in oleyl residues. Replacement of oleyl by palmitoleyl residues lowered both the K T and V max values for accumulation of l-asparagine. A modified statistical method is described for calculating confidence limits for transition points on Arrhenius plots.

Prostaglandin E1 inhibition of lysine transport across the rat, rabbit and turtle small intestine

1. 1. Prostaglandin E 1 in relatively large pharmacological doses caused a significant inhibition ( P < 0.01) of lysine accumulation in mucosal strips isolated from rat, rabbit and turtle intestines. 2. 2. Unidirectional lysine influx across the rabbit and turtle small intestines was significantly reduced ( P < 0.001) by the presence of PGE 1 in the preincubation solution. 3. 3. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with PGE 1 in the three animal species.

Activation of amino acid accumulation in delayed implantation mouse blastocysts

Delayed implantation blastocysts remained in a diapausing-like state when incubated in Na+ -depleted (55 mM) medium. Accumulation of radioactive amino acids remained low when embryos were in low Na+ medium, but increased in the manner characteristic of these embryos when placed in high (124 mM) Na+ medium. Differentiation of blastocysts was apparently arrested in medium containing only 55 mM Na+ since no progress toward formation of trophoblastic outgrowths was made in this medium even when it contained all other compounds required for this process. Low Na+ may prevent uptake of amino acids, such as lysine, essential to outgrowth formation. Accumulation of lysine was Na+ -dependent in blastocysts even though this amino acid is not transported via a Na+-symport system in most cell types. Glucose deprivation did not affect the increase in the capacity of delayed implantation blastocysts to take up amino acids when incubated in high Na+ medium, and glucose accumulation was not Na+-dependent. The capacity of delayed implantation blastocysts to take up amino acids was inversely related to the time before these embryos began forming outgrowths in vitro. The latter process occurred more rapidly if diapausing blastocysts had been stimulated in utero by injecting overiectomized mice with progesterone and estrogen. However, these hormones slowed outgrowth formation slightly when present in the culture medium in vitro.