L-leucine Transport Research Articles

Exchange diffusion of D-leucine in mouse Ehrlich ascites tumor cells.

The present investigation was undertaken to examine an evidence for the exchange diffusion of D-leucine as well as L-leucine and further to study the effect of temperature reduction on the exchange diffusion, in Ehrlich ascites tumor cells in vitro. The homoexchange diffusion for D-leucine was observed at 37 degrees C, the extent being nearly equal to that for L-leucine. The heteroexchange diffusion between D- and L-leucines also occurred at 37 degrees C. These findings were compatible with an assumption that there is a common pathway for D- and L-leucine transport in the tumor cells. It was demonstrated that the exchangeability of D-leucine was depressed remarkably at 0 degree C, while L-leucine still had a considerable exchangeability. This difference in exchangeability of D- and L-leucines in temperature reduction seems to reflect that the bulky hydrocarbon side chains of both the isomers are bound to the different site of carrier as described in the preceding paper.

Protoporphyrin-induced photodynamic effects on transport processes across the membrane of human erythrocytes

Previous studies have shown that illumination of erythrocytes with visible light in the presence of protoporphyrin results in cross-linking of membrane proteins and deterioration of several membrane functions, e.g. active transport of K + and Na +. In the present study it is shown that carrier-mediated transport of glucose, l-leucine, sulphate and glycerol is also inhibited by the photodynamic process, whereas non-specific permeability of glycerol and thiourea is increased. It is shown that these effects are not caused by lipid peroxidation, but by photooxidation of membrane proteins. The inhibition of carrier-mediated transport is caused either by photodynamic oxidation of susceptible essential amino acid residues of the carrier molecules, or by an aspecific perturbation of the membrane structure, leading to inhibition of carrier functions.

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K i of 0.085 mM, a value one-seventh that of melphalan ( K i, 0.635 mM).

Effect of chelating agents and phosphoramidon on the L-leucine transport system in microvillar vesicles from pig kidney

Effect of chelating agents and phosphoramidon on the L-leucine transport system in microvillar vesicles from pig kidney

Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.

L-Leucine-induced secretion of glucagon and insulin, and the "off-response" to L-leucine in vitro. I. Characterization of the dynamics of secretion.

The effects of L-leucine, D-leucine, and L-isoleucine upon the secretion of glucagon and insulin were investigated using the isolated, perfused rat pancreas. All experiments were conducted in the presence of 5.6 mM D-glucose. Ten-minute perfusions of 2, 5, and 10 mM L-leucine induced the release of glucagon and insulin in a dose-related manner. The removal of L-leucine was followed by renewed release of insulin ("off-response") but not of glucagon. The magnitude of the off-response was greater when L-leucine was perfused over longer periods. L-Isoleucine evoked the release of both glucagon and insulin. When L-leucine was administered during perfusion of L-isoleucine, L-leucine-induced release of glucagon was inhibited, that of insulin was augmented, and the insulin off-response prevailed. When the perfusion of L-leucine immediately preceded that of L-isoleucine, L-isoleucine-induced release of glucagon was abolished and that of insulin was augmented. D-Leucine evoked the release of glucagon but not of insulin, and no off-response occurred. When the perfusion of D-leucine followed that of L-leucine, D-leucine-induced glucagon release was inhibited; the insulin off-response to L-leucine was not altered. We reached the following conclusions. 1) Glucagon release induced by L-leucine, D-leucine, or L-isoleucine is likely to be related to the occupancy by these analogous amino acids of transport and/or receptor sites which they share. 2) The insulin off response to L-leucine seems to be evoked by events which take place during the period of administration of L-leucine; these events are not likely to be the release of insulin that occurs during perfusion of L-leucine or the transport of L-leucine into or out of the beta cell. 3) Structurally or chemically similar compounds which are secretagogues both for glucagon and insulin affect the release of these hormones in different ways; these differences are likely to be due to dissimilar mechanisms governing the secretion of the two hormones.

Digestive-absorptive function of the intestinal brush border in uremia

Amino acid absorption was studied in chronic uremic rats. Intestinal transport of l-leucine appears to be inhibited with mild uremic intoxication, whereas severe uremia enhances absorption. Brush border activity of intestinal maltase and disaccharidases is higher in rats with chronic renal insufficiency. The same holds for γ-glutamyl-transpeptidase activity.

Amino acid transport in suspension cultured plant cells: I. Methods and kinetics of L-leucine uptake

A method for studying amino acid transport into suspension-cultured Nicotiana tabacum. cv. Wisc. 38 cells has been developed. Results from experimental analysis of this method demonstrate that the method yields precise measurements of initial uptake rates. This method was employed to measure L-leucine transport and two systems have been identified. At the lower substrate levels investigated (0.08 mM to 20.0 mM) transport is via an energy dependent, saturable system while at the higher substrate levels (20.0 mM to 150.0 mM) uptake increases result from an energy independent, nonsaturable system. The saturable system is stereospecific for L-leucine and an Eadie-Hofstee plot gives a K m of 1.91 mM and a V max of 40.9 nM/ml HV/min.

Membrane transport by guinea pig peritoneal exudate leukocytes: effect of phagocytosis on hexose and amino acid transport.

Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-D-glucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre-phagocytic value of 20 nmoles/mg protein/90 seconds to a post-phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.

The transport of L-leucine into the brain of the rat in vivo: saturable and non-saturable components of influx.

The influx of L-leucine into the brain was measured under normal conditions, and also when its concentration in the circulation was progressively increased. The transport system carrying L-leucine into the brain could be saturated, but the results only conformed to Michaelis kinetics if a nonsaturable component was first subtracted from the observed influx data. The proportion of the total influx due to this non-saturable component was small, but it increased as the concentration of leucine in the blood was raised above the normal level. The saturable component of the influx of leucine could be inhibited by raising the concentration of L-valine in the blood, but the influx of leucine into the brain could not be reduced to zero. The residual influx of leucine in the presence of the inhibitor was in part, due to leucine competing with the inhibitor, valine, for the shared carrier, but it was also in part due to the non-saturable component of leucine influx, which is unaffected by the inhibitor. Thus there are two components in the influx of leucine into the brain: a major component, which is a carrier-mediated transport process, and a minor component which is non-saturable. The possibility of a nonsaturable component of the transport of amino acids into the brain has not previously been considered. The non-saturable component may be due to diffusion through the cerebral capillaries in small regions of the brain where the endothelium is permeable; osmotic effects may also contribute. The findings may be of importance in the treatment of aminoacidaemias.

Effect of semistarvation on kinetic parameters for the in vitro accumulation and transport of L-leucine in rat small intestine.

The effects of semistarvation on the kinetics of the tissue accumulation and the transmural transport of l-leucine were assessed in vitro in the rat jejunum. The Kt calculated for the tissue accumulation was slightly reduced, but the value for transmural transport differed insignificantly after semistarvation. The Vmax’s were unanimously enhanced in semistarvation for both the accumulation and the transport, regardless of the basis of expression. Semistarvation caused a profound increase in the diffusive component of the transport when assessed using carbonyl cyanide m-chlorophenylhydrazone. Energy-dependent, active component of the transport was however even significantly greater in semistarved intestine than in fed control. These results are compatible with the notion that semistarvation improves intestinal transport capacity through increase in the Vmax, which probably correlates with ‘carrier’ population, and concomitantly in the diffusive permeability across brush border and/or intestinal wall.

Evidence for carrier-mediated transport of L-leucine into isolated pea (Pisum sativum L.) chloroplasts

The uptake of leucine into isolated, intact, pea chloroplasts was investigated using the silicone oil centrifugation technique. The internal: external ratio of leucine exceeded unity at low external leucine concentrations. Uptake of leucine at different external concentrations showed passive diffusion and carrier-mediated transport components. Competition for uptake was shown between leucine and isoleucine but not between leucine and glycine. Rates of diffusion of leucine were found to be low compared with glycine, however, fast carrier-mediated transport of leucine assumed more importance at physiological concentrations.

Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12.

Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction. Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map. The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems. Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels. The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis

l-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na + dependent and presumably coupled to Na + gradient. Our results indicate that progesterone (10 −6 M) blocks abruptly, around the germinal vesicle breakdown, the saturable transport of l-leucine. p- Chloromercuribenzoate (10 −4 M) induces maturation and after a short lag of time strongly inhibits l-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.

Leucine transport in Escherichia coli. The resolution of multiple transport systems and their coupling to metabolic energy.

The multiple active transport systems mediating L-leucine accumulation in Escherichia coli strain 7 (K12) and ML 308-225 have been examined. In addition to the previously characterized osmotic shock-sensitive LS (L-leucine-specific) and LIV-I (L-leucine; L-isoleucine-, and L-valine-specific) activities, a third system (designated LIV-II) has been detected, confirming a report by Rahmanian et al. (RAHMANIAN, M., CLAUS, D.R., and OXENDER, D. L. (1973) J. Bacteriol. 116, 1258-1266). This third system transports L-leucine, L-isoleucine, and L-valine with a relatively low affinity (apparent transport Km equals 4 muM for L-leucine) and it is resistant to repression by cell growth on L-leucine. Exploitation of these properties and of the differential sensitivity of the three transport activities to inhibition by L-leucine analogues permits estimation of the contribution by each system to the total transport activity under varying conditions. Such experiments show that, unlike systems LS and LIV-I, system LIV-II is resistant to osmotic shock. The L-leucine, L-iosleucine, and L-valine transport activity in membrane vesicles from strain ML 308-225 has the properties of system LIV-II. Although the L-leucine transport activities in strains 7 and ML 308-225 are in all other respects similar, membrane vesicles from strain 7 do not transport L-leucine, L-isoleucine, or L-valine. L-leucine transport under various conditions of energy supply has been measured in strain ML 308-225 and the corresponding Mg-2+-ATP-ASE-DEFICIENT STRAIN, DL-54. These measurements support the view that the osmotic shock-sensitive LS and LIV-I activities depend on the synthesis of ATP, while the osmotic shock-resistant LIV-II activity depends on the energized membrane state generated by electron flow but not on ATP synthesis, per se. This conclusion is not supported by the inhibitory effects of the energy poisons arsenate and 2,4-dinitrophenol, but these compounds may have secondary chemical effects on the transport systems.

Dipeptide transport in isolated intestinal brush border membrane

Transport of glycyl- l-leucine into isolated brush border membrane vesicles was studied. On the basis of the following observations it was postulated that glycyl- l-leucine was transported intact by a specific dipeptide mechanism. 1. (1) The differing time course and Na + stimulation of glycine, l-leucine and glycyl- l-leucine. 2. (2) The failure of glycine and l-leucine to inhibit glycyl- l-leucine transport. 3. (3) Initial presence of dipeptide within the vesicle. 4. (4) Inhibition of glycyl- l-leucine uptake by other dipeptides. 5. (5) The occurrence of accelerated amino acid uptake in the presence of the dipeptide.

A kinetic analysis of the changes in membrane permeability induced by fertilization in the egg of the sea urchin Sphaerechinus granularis

Fertilization-induced modifications in egg membrane permeability to labeled adenine and l-leucine substrates have been studied by the initial rate-pulse technique, using the sea urchin Sphaerechinus granularis. Kinetic parameters have been determined for unfertilized eggs as well as for fertilized eggs at various developmental stages. Samples were usually processed according to a centrifuge-wash technique, since Millipore filtration was found to be inadequate in the case of leucine. Several arguments are presented which support the view that the uptake of adenine occurs through a process of simple physical diffusion across the cell membrane. It has been demonstrated, on the other hand, that the transport of l-leucine involves a carrier-mediated exchange-diffusion process. Kinetic data point to the existence of major fluctuations in general egg membrane permeability which appear to be largely independent of the mitotic cycle and of the mechanisms involved in the transport of the substrates studied. A unitary hypothesis is presented to account for these fertilization-induced changes in membrane permeability and which does not involve the synthesis or recruitment of previously inactive carriers.

Effect of Previous Water Stress on Ion Uptake and Transport in Barley Seedlings

Measurements of ion transport through roots and ion uptake into roots were made following a brief period of water stress induced by wilting in air. The basic processes of photosynthesis, respiration and protein sinthesis did not appear to be affected by this treatment, and uptake of 86Rb and L-leucine to the roots was not reduced. However, transport of 86Rb and L-leucine from root to shoot wasinhibited. Thus the response of the plants to water stress resembles the response to inhibitors of protein synthesis and to abscisic acid, which has been found to be produced in plants subject to water stress.

Interaction between hexoses and amino acids for transport in rat small intestine

1. 1. A time course study of l-leucine transport in rat jejunum was undertaken. 2. 2. d-Galactose failed to inhibit leucine transfer until after 2 min incubation but the degree of inhibition of galactose on leucine transport increased as the incubation time was extended. 3. 3. l-Methionine produced a marked reduction of leucine transfer and this effect was independent of the length of incubation. 4. 4. These findings suggest that the inhibitory effect of galactose on leucine transport is not due to an interaction at the carrier level.

Effect of weak acids on amino acid transport by Penicillium chrysogenum: evidence for a proton or charge gradient as the driving force.

A variety of weak acids at and below their pK(a) are potent inhibitors of transport in Penicillium chrysogenum. The effective compounds include sorbate, benzoate, and propionate (common antifungal agents), indoleacetate (a plant hormone), acetylsalicylate (aspirin), hexachlorophene, and a yellow pigment produced by the mycelia under nutrient-deficient conditions, as well as the classical uncouplers 2,4-dinitrophenol, p-nitrophenol, and azide. The results suggest that a proton gradient or charge gradient is involved in energizing membrane transport in P. chrysogenum. The unionized form of the weak acids could discharge the gradient by diffusing through the membrane and ionizing when they reach an interior compartment of higher pH. Experiments with 2,4-dinitrophenol and p-nitrophenol established that the ionized species are not absorbed by the mycelium to any great extent. The transport inhibitors also caused a decrease in cellular adenosine 5'-triphosphate (ATP) levels, but there was no constant correlation between inhibition of transport and suppression of cellular ATP. A decrease in aeration of the mycelial suspension had the same effect on transport and ATP levels as the addition of a weak organic acid. The effects on transport rates and ATP levels were reversible. The instantaneous inhibition of [(14)C]l-leucine transport by NH(4) (-) (and vice-versa) in nitrogen-starved mycelia at pH values of 7 or below can be explained by competition for a common energy-coupling system. The inhibition is not observed in carbon-starved mycelia in which the NH(4) (+) transport system is absent or inactive (but the general amino acid transport is fully active), or in iodoacetate-treated mycelia in which the NH(4) (+) transport system has been differentially inactivated. At pH values greater than 7.0, NH(3) and HPO(4) (2-) inhibit transport, presumably by discharging the membrane proton or charge gradient. Aniline counteracts the inhibitory effect of NH(3) and HPO(4) (2-) possibly by acting as a proton reservoir or buffer within the membrane.