The properties of the preferential uptake of l-leucine by isolated intestinal epithelial cells

1.

1. Epithelial cells isolated from rat intestine have shown the ability to preferentially take up 1 mM l-leucine as compared to the d-isomer. The uptake was found to be concentrative. l-Leucine uptake was inhibited by neutral l-amino acids and basic l-amino acids but was not inhibited by d-valine or d-isoleucine. Galactose and α-methyl-d-glucose were inhibitory; glucose was significantly less inhibitory; and fructose activated uptake. Inhibitors of energy metabolism, sulfhydryl inhibitors, ouabain, and procedures which damaged the morphology of the cell all decreased l-leucine uptake. l-Leucine uptake was decreased in the absence of either Na⁺, K⁺, Ca²⁺, or Mg²⁺ and exhibited a broad ph optimum between 4 and 8. d-Leucine uptake was a linear function of time during the first 5 min of incubation. The apparent K_m for l-leucine uptake was 3.2 mM, and l-valine was competitive inhibitor of l-leucine uptake. Inhibitors of protein biosynthesis did not reduce l-leucine uptake. The efflux of l-leucine from the cells was inhibited by the cold.     

Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status

The characteristics of valine uptake by isolated microcolonies of Galaxea fascicularis (Linnaeus 1758) were studied under various conditions including light, dark and feeding. The results demonstrated the presence of: (1) a linear component which might represent either a diffusional transport or a low-affinity carrier-mediated transport (apparent carrier affinity >250 mol·l^–1), and (2) a high-affinity active carrier-mediated transport (apparent carrier affinity about 5 mol·l^-1). The latter is mediated by two different systems: (i) a Na⁺-dependent carrier, stimulated by light and operative in both fed and unfed polyps, and (ii) a Na⁺-independent carrier, light insensitive and present only in unfed polyps. Competition experiments with other amino acids show that the Na⁺-dependent carrier is highly specific for neutral amino acids, as indicated by the high inhibition constants of basic and acidic amino acids. Our results suggest that the energy supplied by zooxanthellae photosynthates is necessary for the process of amino acid uptake, and that the Na⁺-dependent carrier responsible for valine uptake by G. fascicularis is similar to the B^0,+ system.Abbreviations AA amino acid(s) - AC/HC ratio autotrophic/heterotrophic carbon - ASW artificial sea water - DOM dissolved organic material - HPLC high performance liquid chromatography - K ₁ apparent inhibition constant - K _m apparent affinity of the carrier - SE standard error - V _max maximal rate of absorption     

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Salinity dependence,uptake kinetics,and specificity of amino-acid absorption across the body surface of the oligochaete annelidEnchytraeus albidus

Enchytraeus albidus is able to absorb dissolved¹⁴C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to¹⁴C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,¹⁴C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S).     

Fungicidal Activity of Oxadiazolyl Sulfides

Glycyl-l-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases, Bestatin competitively inhibited dipeptide hydrolase activities in isolated cells with a K_i value of 10^?8 m, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10^?4 m bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.     

Amino acid uptake in skeletal muscle of rats bearing the Yoshida AH-130 ascites hepatoma

Rats bearing the Yoshida AH-130 ascites hepatoma show decreased activity of neutral amino acid transport in skeletal muscle measuredin vivo as the tissue accumulation of the analogue -amino [1-¹⁴C]isobutyrate (AIB). The decreased accumulation of AIB observed is not merely a consequence of the hypoinsulinaemia present in these animals (as a result of tumour burden) sincein vitro experiments carried out using incubations of isolated soleus muscles also showed a decreased uptake of neutral amino acids. In these preparations the addition of insulin results in similar increases in uptake both in the pair-fed controls and the tumour-bearing animals, thus suggesting similar insulin sensitivities. The decrease in amino acid uptake in soleus muscle is associated with a decrease in the activity of system A, while systems L and ASC show no particular changes as a result of the tumour growth. The kinetic characterisation of system A in the Yoshida-bearing rats shows a decrease in V_max together with a decrease in K_M in relation with the pair-fed animals.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

Acetate uptake by the unicellular cyanobacteria Synechococcus and Aphanocapsa

Acetate uptake by strains of Synechococcus and Aphanocapsa in short experiments required light, and was strongly inhibited by m-dichlorocarbonyl cyanide phenylhydrazone and dichlorophenyl dimethyl urea. Acetate carbon was distributed in amino acids and in the acyl portion of lipids in the same way as during growth experiments when CO₂ was available, but the reduced incorporation in the absence of CO₂ was primarily into the lipid fraction. An apparent K _m for uptake by Synechococcus and for Aphanocapsa 6308 of 20 and 180 M at pH 7.4 was obtained; corresponding V _max values were 6 and 11 nmol x min^-1 x mg protein^-1. Uptake with Synechococcus was affected by pH, with affinity decreased and maximal rate increase with rising pH. Acetate uptake was not affected by propionate or butyrate when both were added at the same time, but a light and concentration dependent inhibition developed if suspensions were preincubated with propionate. Acetate carbon moved rapidly into acid insoluble material, but after 10–15 s 75% or more of the recovered intracellular counts were in acetyl CoA. Counts in this compound were reduced by preincubation with propionate.Kinetic measurements of acetyl CoA synthetase in fractionated cell extracts gave values for K _m of about 50 M for acetate, 5 mM for propionate, 100 M for CoA and 0.38 mM for ATP. The internal pool of free CoA was measured to be about 20 M, and was reduced by preincubation with propionate. This suggests that the activity of CoA-mediated reactions may be regulated by the availability of this cofactor.Abbreviations Used CCCP m-Dichlorocarbonyl cyanide phenyl hydrazone - DCMU dichlorophenyl dimethyl urea - TCA trichloroacetic acid - Tris trishydroxymethyl amino methane - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid     

Amino-acid transport into cultured tobacco cells

Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca²⁺ for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not the result of exchange diffusion. Arginine uptake over a concentration range of 2.5 M to 1 mM was characterized by simple Michaelis-Menten kinetics with a K_m of 0.1 mM and a V_max of 9,000 nmol g^-1 fresh weight h^-1. Transport was inhibited by several compounds including carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, N,N-dicyclohexylcarbodiimide, and N-ethylmaleimide. Inhibition by these compounds was not the result of increased efflux resulting from membrane damage. A variety of amino acids and analogs, with the exception of D-arginine, inhibited transport, indicating that arginine transport was mediated by a general L-aminoacid permease. Competition experiments indicated that arginine and lysine exhibited cross-competition for transport, with K_i values similar to respective K_m values. Arginine transport and low-affinity lysine transport are probably mediated by the same system in these cells.Abbreviations BTP Bis Tris Propane - CCCP Carbonylcyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - DTT Dithiothreitol - NEM N-ethylmaleimide - MES 2(N-morpholino)ethanesulfonic acid - TCA trichloroacetic acid This paper is the third in a series on amino-acid transport into cultured tobacco cells. For parts I and II, see Harrington and Henke (1981) and Harrington et al. (1981)     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

AMINO ACID TRANSPORT IN NITZSCHIA OVALIS ARNOTT1,2

The marine diatom Nitzschia ovalis possesses at, least 3 amino acid uptake systems, specific for transport of acidic, polybasic, and, neutral amino acids. Maximum uptake velocity (V_max) for each, site is inversely related to the nitrogen content of the cell, and to the nitrogen available in the culture medium. Transport, of polybasic amino acids occurs throughout the course of growth in batch, culture, but the V_max increases dramatically as the culture ages and nitrogen/cell reaches a low value. K_s does not, change significantly. Acidic and neutral amino acids are taken up only by cells harvested from nitrogen-poor culture. It appears that amino acid transport is repressed by high concentrations of nitrogen in the medium. Under natural conditions, where nitrogen concentrations are low, the contribution of amino acid uptake to the nitrogen economy of Nitzschia populations may be significant.     

HIGH AFFINITY AMINO ACID TRANSPORT BY FROG SPINAL CORD SLICES

The characteristics of amino acid uptake by frog spinal cord slices was studied by in vitro incubations in appropriate media. The uptake mechanisms exhibited saturation; kinetic analysis demonstrated 2 distinct systems for the influx of the possible neurotransmitters: GABA, glycine, L-glutamic acid and L-aspartic acid. One system showed a comparatively high substrate affinity (K_m values, 10-26 μM) while the other system had a lower affinity (K_m, 0.4-1.6 mM).-Leucine, an amino acid presumably not a transmitter, was accumulated only by a low affinity mechanism (K_m 1.6 mM). The process responsible for high affinity uptake had many of the properties of an active transport mechanism. These included temperature sensitivity, energy dependence, requirement for Na⁺ ions and inhibition by ouabain. GABA and glycine uptake was inhibited only by closely related amino acids or structural analogues. The influx of L-glutamic acid was competitively inhibited by the presence of L-aspartic acid in the medium; the converse was also demonstrated. Thus, the high affinity uptake system for possible transmitter amino acids in the frog spinal cord closely resembles that described for mammalian CNS tissue. These results are compatible with the assumption that GABA, glycine, L-glutamic acid and L-aspartic acid are neurotransmitters in the amphibian spinal cord.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.