High-affinity System Research Articles

Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae.

The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated. Xylose was taken up by means of low- and high-affinity glucose transport systems. In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected. Glucose strongly inhibited the transport of xylose by both systems. Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose. A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca. 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture. No activity converting xylitol to xylulose or vice versa was found in crude extracts. Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy. Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist. However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-[1-13C]xylose was labelled only in the C-2 position. Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.

Divergent Regulation of Fuel Utilization in Human Fibroblasts by Epidermal Growth Factor

Epidermal growth factor (EGF) is known to enhance wound healing, partly by stimulating fibroblast proliferation. We studied the effects of EGF on the cell membrane transport of glutamine (GLN) and glucose, the two major fuels utilized by fibroblasts. Human foreskin fibroblasts were grown to confluence on culture wells in Dulbecco's MEM. EGF (10 ng/ml) or vehicle was added to the cells; substrate uptake was assayed at various timepoints. As previously shown, GLN uptake was predominantly Na +-dependent and occurred via two distinct carriers, the low-affinity System A and the high-affinity System ASC. Activity of the high affinity system was assayed with [ 3H]GLN while blocking the low-affinity system with excess α-methylaminoisobutyric acid (MeAIB), a selective System A substrate. [ 3H]-MeAIB uptake was used to assay activity of the low affinity carrier. EGF caused a time-dependent increase in GLN transport by fibroblasts. The activity of the low-affinity GLN carrier (System A) increased by 60% after 18 hr of exposure to EGF. The increase in the high-affinity agency revealed an early increase at 1-2 hr and a late increase at 18-24 hr. The enhanced transport activity was due to an increase in V max with no change in carrier affinity. In contrast, glucose uptake was Na +-independent and EGF caused a decrease in glucose uptake at early time points. EGF treatment results in divergent regulation of glutamine and glucose transport in human fibroblasts. This response may be designed to provide GLN for both energy production and nucleotide biosynthesis to support cell metabolism and proliferation.

Characterization of L-arginine transport by pulmonary artery endothelial cells

The transport of L-arginine by porcine pulmonary artery endothelial cells (PAECs) was characterized. Uptake of 50 microM L-arginine was time dependent and linear in the presence and absence of sodium, with approximately 70% of uptake occurring via a carrier-mediated Na(+)-independent process. Kinetic studies of saturable Na(+)-independent transport revealed two transport components: a high-affinity transporter [Michaelis constant (Km) = 304 +/- 23 microM, maximal transport velocity (Vmax) = 679 +/- 34 pmol.mg protein-1.30 s-1], and a low-affinity carrier (Km = 3.9 +/- 1.0 mM, Vmax = 2.8 +/- 0.7 nmol/mg protein-1.30 s-1). Saturable Na(+)-independent uptake of 50 microM L-arginine transport showed no significant variation in uptake between pH 6.0 and 8.0 and was blocked by the system y+ substrates L-arginine, L-homoarginine, L-lysine, and L-ornithine. Na(+)-dependent L-arginine transport occurred via a single high-affinity system (Km = 62 +/- 3 microM, Vmax = 211 +/- 24 pmol.mg protein-1.30 s-1) which was significantly inhibited by L-arginine, L-lysine, L-ornithine, L-leucine, L-alanine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. Na(+)-dependent arginine uptake was pH and hormone insensitive, and lithium did not substitute effectively for sodium. These data are consistent with mediation of high-affinity arginine transport by PAECs via Na(+)-independent system y+ and Na(+)-dependent system BO,+.

Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes.

The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T3) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na(+)-independent, and efficiently inhibited by T3 (half-inhibition at approximately 2 microM). Two Na(+)-independent L-like systems (L1 and L2), common to leucine and aromatic amino acids, were characterized kinetically. System L2 had a low affinity for leucine and tryptophan (Km = 0.3-0.9 mM). The high-affinity system L1 (Km approximately 10 microM for both amino acids) was competitively inhibited by T3 with a Ki of 2-3 microM (close to the T3 transport Km). Several T3 analogues inhibited system L1 and the T3 transport system similarly. Glutamine uptake and alpha-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T3 had little effect on the uptakes of glutamine and alpha -(methylamino)isobutyric acid. The results indicate that the T3 transport system and system L1 are related.

Kinetics of 2-Oxoglutarate Uptake by Synaptosomes from Bovine and Rat Retina and Cerebral Cortex and Regulation by Glutamate and Glutamine

2-Oxoglutarate (2-OG) is a metabolic precursor of glutamate and may be utilized to replenish the neurotransmitter pool. 2-OG is rapidly transported into neurons by a high-affinity carrier that is particularly prevalent in glutamatergic terminals. Here we report the kinetics of [U-14C]2-OG uptake by crude synaptosomal preparations from bovine and rat retina and brain, and the modulatory effects of glutamate and glutamine. In all four tissues, 2-OG uptake was mediated by a high-affinity system (Kt approximately 1 microM) that was subject to negative feedback control by glutamate and biphasic modulation by glutamine (another precursor of neurotransmitter glutamate).

Glycine betaine transport by Staphylococcus aureus: evidence for two transport systems and for their possible roles in osmoregulation

The transport of glycine betaine by Staphylococcus aureus was investigated. Two transport systems were found that could be differentiated on the basis of their affinity for glycine betaine and their activation by osmotic pressure. The high-affinity system was relatively independent of osmotic pressure and exhibited a Km of approximately 3 microM. This system was not inhibited by proline, for which a separate high-affinity transport system has been recently discovered. The low-affinity system was activated approximately 35-fold by an increase in osmotic pressure and exhibited a Km of approximately 130 microM for glycine betaine. This system is partially inhibited by excess proline and may be identical to the low-affinity system recently described for proline. Both glycine betaine transport systems are Na(+)-dependent.

Phosphate transport by capillaries of the blood-brain barrier.

Capillaries were isolated from bovine brain cortex and used for phosphate transport studies. The influx of phosphate through capillary membranes was studied by incubation with [32Pi]phosphate followed by a rapid filtration technique. Phosphate uptake by brain capillaries was mediated by a saturable high-affinity system which is independent of the sodium concentration in the incubation medium. The apparent half-saturation constant (Km) and maximal influx (Vmax) were estimated to 160 microM and 0.37 nmol/mg protein/30 s. Transport was inhibited by the phosphate analogues arsenate and phosphonoformic acid with apparent inhibition constants of 5 and 11 mM, respectively. The metabolic inhibitors cyanide and ouabain had no effect on the transport activity. Competition experiments showed that phosphate uptake was inhibited up to 41% by various anions (pyruvate, acetate, citrate, glutamate, and sulfate). In addition, phosphate uptake was significantly decreased by two selective inhibitors of anionic exchangers, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Chloride was not a substrate of the phosphate carrier as the replacement of external chloride, by nitrate, thiocyanate, or gluconate, did not increase phosphate transport. Aminohippuric acid and N'-methylnicotinamide, two specific substrates of anionic and cationic drug exchangers, did not compete with the phosphate carrier of cerebral capillaries. However, trans-stimulation with bicarbonate increased phosphate transport by 28%, and this stimulation was inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that the carrier of the cerebral capillaries could exchange phosphate with bicarbonate.

Identification of a new transport system (y+L) in human erythrocytes that recognizes lysine and leucine with high affinity.

1. The effect of neutral amino acids on the transport of L-lysine across the human erythrocyte membrane was studied. 2. All neutral amino acids tested (range 0.3-5 mM) inhibit the influx of L-[14C]lysine (1 microM). The inhibition pattern is biphasic, and tends to reach a maximum at approximately 50% of the original flux. The concentrations that give 25% inhibition are (mM): L-cysteine (2.7), L-alanine (1.3), L-serine (0.9), L-isoleucine (0.6), L-phenylalanine (0.35), L-methionine (< 0.3), L-leucine (< 0.3). L-lysine and L-arginine completely inhibit the rate at the highest concentration. 3. These results can be explained by assuming that L-lysine transport occurs through two independent transporters that differ in their affinity for neutral amino acids. A detailed kinetic analysis of the effect of L-leucine on L-lysine entry is consistent with this hypothesis. 4. Using a new experimental strategy, the substrate and inhibitor transport parameters for the two systems were determined. The half-saturation constants for lysine (+/- S.E.M.) are found to be: KmA, 0.014 +/- 0.002 mM and KmB, 0.112 +/- 0.017 mM. The maximum rates differ by a factor of 8.2 (VmaxB/VmaxA). The leucine inhibition constants are: KiA, 0.022 +/- 0.003 mM and KiB, 30.36 +/- 7.9 mM. If the sodium in the incubation medium is replaced by potassium, the apparent affinity for leucine (1/KiA) is reduced approximately 30-fold. 5. The maximum inhibition caused by leucine decreases as the lysine concentration is raised, showing that leucine acts upon the higher affinity system. 6. When added to the trans side, L-leucine, L-phenylalanine and L-isoleucine do not cause inhibition, but stimulate the flux by approximately 30%. This indicates that these analogues are also transported. 7. In conclusion, in the concentration range 1-100 microM, lysine crosses the red cell membrane through two distinct transport systems, one of which recognizes both neutral and cationic amino acids with high affinity.

Fine structure mapping and complementation studies of the metD methionine transport system in Salmonella typhimurium.

A fine structure deletion map of the metD region of the chromosome of Salmonella typhimurium responsible for a high-affinity methionine transport system has been constructed. Complementation tests involving the introduction of metD+DNA contained in a pUC8 vector into metD strains indicated the presence of four complementation groups in the metD region. This suggested that the methionine system belongs to the osmotic shock-sensitive class of transport system, and therefore should possess a periplasmic methionine-binding protein and several membrane proteins. But a deletion mutation covering all known metD point mutations did not affect the level of a methionine binding activity in osmotic shock fluids, suggesting either that the deletion did not extend into the gene encoding the binding protein, or that the binding activity is not associated with the metD system. Possible reasons for the failure to isolate mutations in the gene for the binding protein are discussed.

Determinants of substrate affinity for the oligopeptide/H+ symporter in the renal brush border membrane.

We and others have shown previously the existence of high and low affinity systems for oligopeptide transport in kidney brush border membrane vesicles (BBMV). In the present study we investigated the relationship between the structure of substrates and their affinity for interaction with the high-affinity oligopeptide/H+ transporter in kidney BBMV. Based on competition experiments using [3H]Gly-Gln as a probe we determined the Ki values for more than 60 selected peptides. For a high-affinity interaction with the carrier site the following structural features of substrates are required: (a) both a free amino and carboxyl terminus; (b) the amino group and peptide bond nitrogen located in the alpha-position; (c) a trans peptide bond rather than the cis configuration; (d) L-alpha-amino acid isomers in both COOH and NH2 termini, although D-isomers of hydrophobic amino acids are acceptable in the NH2 terminus; and (e) a backbone of less than 3 amino acid residues. A striking finding of the present study is that, for peptides satisfying these minimal structural requirements, the primary determinant of affinity is hydrophobicity. The fact that there is a highly significant (p less than 0.001) correlation between Ki and hydrophobicity allows the prediction of the affinity for any di- or tripeptide composed of alpha-amino acids in the L-form.

Ubiquitin immunoreactivity of multiple polypeptides in rat brain synaptic membranes.

The low molecular weight polypeptide ubiquitin is commonly found conjugated to other protein species. The ubiquitin moiety has a well characterised role in targetting many of these proteins for rapid degradation [reviewed in I]. Some ubiquitin conjugates however, such as the ubiquitinated histones H2A and H2B, appear to be long-lived and formed reversibly [2]. There is limited evidence for the ubiquitination of cell-surface proteins, for instance, the lymphocyte homing receptor LECAM-1 [reviewed in 31. Moreover, anti-ubiquitin antibodies inhibit several high-affinity neurotransmitter uptake systems when incubated with intact rat synaptosomes [4]. We have therefore investigated the possible ubiquitination of plasma membrane proteins in the mammalian central nervous system. Subcellular fractions from adult rat brain were prepared by a modification of the procedure of Cotman and Taylor [5] as previously described [6]. All operations were carried out in the presence of the following cocktail of protease inhibitors; 0.2mM phenylmethylsulphonyl fluoride (PMSF), and lpglml each of leupeptin, pepstatin, chymostatin and antipain. Affinity-purified rabbit anti-ubiquitin antisera have been prepared by a modification of the method of Haas and Bright [7]. Western blotting of brain fractions was carried out by standard protocols [8] using a wet blotting system.

Choline transport inFusarium graminearumA 3 5

Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM).

Choline transport in Fusarium graminearum A [formula omitted

Fusarium graminearum A 3 5 possesses a high affinity system ( K m=32 ± 8 μM; mean ± SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine ( K i = 198 ± 29 μM), betaine aldehyde ( K i = 95 ± 14 μM) and chlorocholine ( K i = 352 ± 40 μM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor ( K IES = 1.9 ± 0.6 μM; K IE = 3.6 ± 1.9 μM).

Transport and metabolism of D-[3H]adenosine and L-[3H]adenosine in rat cerebral cortical synaptoneurosomes.

The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D-[3H]adenosine and L-[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D-[3H]adenosine, high- and low-affinity accumulation processes were present. For the high-affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D-[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 microM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an adenosine deaminase inhibitor, and 5'-iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D-[3H]adenosine to 6% with 5-s and 9% with 15-s incubations. This resulted in significantly higher KT values: 36 microM at 5 s and 44 microM at 15 s. At 10-min incubations in the presence of these inhibitors, metabolism of accumulated D-[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L-[3H]adenosine, apparent KT and Vmax values for 20-s incubations were 38.7 microM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L-[3H]adenosine was observed only at incubations of greater than 30 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.

D-glucose transport systems in rat jejunal brush border membrane: Influence of ageing

Jejunal brush border membranes were isolated from rats of different ages (very young, young, adult and old); the γ-GT specific activity and the vesicle volumes were unaffected by ageing, whilst protein content was significantly reduced in brush border from old rats. Vesicles were used to investigate the kinetics of Na-glucose cotransport under voltage-clamped and zero-trans conditions over a wide range of d-glucose concentrations (0.005–70 mM). Results provide evidence that in all the ages tested d-glucose can cross the brush border membrane both by a passive diffusional component and by two-Na-dependent saturable transport systems, namely one with high-affinity and low-capacity and the other with low-affinity and high-capacity. However in some old rats only one saturable and a very small passive component occur. The two Na-dependent transport systems were analyzed to define the stoichiometry of coupling between Na and glucose fluxes. In all the ages tested the Na:glucose ratio is higher in the high-affinity system than in the low-affinity one. Accordingly the effect of a superimposed membrane potential is more evident for the high-affinity transport mechanism. In conclusion, d-glucose transport systems seem to be unaffected by ageing from very young to adult rats; only in old animals age-related alterations can be observed.

The high affinity system of calcium (Ca 2+) transport by the luminal membrane of distal tubule (DT) is a Ca 2+ channel sensitive to parathyroid hormone and vitamin D

The high affinity system of calcium (Ca 2+) transport by the luminal membrane of distal tubule (DT) is a Ca 2+ channel sensitive to parathyroid hormone and vitamin D

Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe.

Mutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, KT, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher KT for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.

Neurochemical evidence for a neuronal GABAergic system in the rat sympathetic superior cervical ganglion.

Some characteristics of gamma aminobutyric acid (GABA) uptake and release in rat superior cervical ganglion (SCG) were investigated. Kinetic analysis of GABA uptake indicated the existence of both high affinity (Km = 18.6 microM) and low affinity (Km = 485 microM) uptake systems. 3H-GABA influx was decreased by inhibitors of glial (beta-alanine), neuronal (2,4-diaminobutyric acid, DABA), or glial and neuronal GABA uptake (nipecotic acid). 3H-GABA efflux was elicited by K+ depolarization in a dose-dependent manner, an effect unaltered by severing the preganglionic nerve fibers. Superfusion of SCG explants with DABA or beta-alanine resulted in increased 3H-GABA efflux from tissue, an effect amplified by the absence of calcium in the superfusion medium. 3H-GABA loading in the presence of DABA, but not in the presence of beta-alanine, resulted in abolition of K(+)-elicited 3H release. At 20 mM, but not at 50 mM K+, the release of 3H-GABA was inhibited by replacing Ca2+ by Mg2+ and by adding EGTA, or by incubating SCG in the presence of the Ca(2+)-channel blocker verapamil. Veratrine evoked GABA release in Ca(2+)-independent manner. None of several putative SCG autacoids or agonists (nicotine, muscarine, norepinephrine, dopamine, serotonin, baclofen, muscimol) significantly modified GABA release.

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R33 of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K T=0.22 mmol/L) and the glucose carrier (K T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H+ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H+ symporter.