High-affinity System Research Articles

Transport and metabolism of L-glutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes.

The intracellular glutamate concentration of oxygenated, isolated adult rat heart cells incubated with 0.15 mM glutamate amounts to 2.89 +/- 0.6 mM. Under these conditions the velocity of glutamate transport was 24.3 +/- 1.6 pmol.min-1.mg protein-1 and occurs via a high-affinity carrier characterized by an apparent affinity (K(m)) value of 0.18 +/- 0.03 mM. At high glutamate concentrations ( > 1mM) this high-affinity transport system is superimposed by additional uptake processes of a low affinity but a high capacity for glutamate. The 1.6-fold increased uptake of glutamate observed during 30 min of anoxic incubation of cardiomyocytes does not prevent an intracellular decrease in this amino acid to a concentration of 0.49 mM. After 15 min reoxygenation of cardiomyocytes the intracellular glutamate content increases to the control values of oxygenated cells. Only 2.4% of the glutamate increase after reoxygenation is due to the transport o glutamate from the incubation medium. The competitive inhibitor of transaminases, aminooxyacetate, prevents both the observed intracellular decrease in glutamate during anoxia and the increase in intracellular glutamate after reoxygenation of cardiomyocytes. Half of the amino groups needed for the synthesis of glutamate originate from intracellular alanine, which increases during anoxia and is metabolized during reoxygenation of cardiomyocytes. The velocity of the glutamate uptake of cardiomyocytes incubated in a medium containing 10 mM L-glutamate amounted to 728 +/- 140 pmol.min-1.mg protein-1. During anoxic incubation of cardiomyocytes at this high extracellular glutamate concentration, the intracellular glutamate breakdown may be compensated by a simultaneous uptake of this amino acid via the transport processes characterized by a high capacity

Starvation and endotoxin act independently and synergistically to coordinate hepatic glutamine transport.

Because hepatic glutamine transport is markedly enhanced during critical illness, we tested the hypothesis that nutrient starvation and endotoxemia act coordinately to augment transport activity. Fed or starved (48 hours) rats received Escherichia coli endotoxin (LPS, 10 mg/kg of body weight, intraperitoneally) or saline before hepatocyte isolation for measurement of glutamine transport. Hepatocytes were isolated from fed or fasted rats 4 hours after LPS treatment. [3H]glutamine uptake was measured and normalized to cellular protein. Data (mean +/- standard deviation, three separate determinations) were analyzed by Student's t test and analysis of variance. Starvation induced a 1.6-fold increase in glutamine transport, while LPS treatment of fed rats increased transport activity 2.6-fold. Treatment of fasted animals with LPS induced a sixfold increase in glutamine transport. Kinetically, this effect in endotoxemic starved rats was mediated by both an increase in System N Vmax and the induction of a high affinity System A amino acid carrier which transports glutamine. Starvation and endotoxemia regulate hepatocyte glutamine transport independently and synergistically. This hepatic response provides glutamine and other amino acids to support key metabolic pathways in the liver during critical illness.

Component characteristics of the vectorial transport system for taurine in isolated bovine retinal pigment epithelium.

1. A wide range of substrate concentrations (5-1600 microM) were used to screen for the presence of systems capable of transporting taurine into isolated and free-floating samples of bovine retinal pigment epithelium (RPE). Both high and low affinity systems displaying Michaelis-Menten saturation kinetics were identified. The high affinity system was characterized by a K(m) of 23 microM and a V(max) of 86.7 pmol (5 min)(-1) (4 mm disc of tissue sample)(-1). Similarly, the low affinity system was characterized by a K(m) of 507 microM and a V(max) of 344 pmol (5 min)(-1)(4 mm disc)(-1). 2. Ussing-type incubation chambers and double-label radiotracer techniques were used to assess the presence of specific taurine carriers on apical and basolateral surfaces of the RPE. High affinity carriers were shown to be present on both surfaces and the kinetic constants (K(m) and V(max)) for apical and basolateral systems were determined as 23.2 microM and 34.8 pmol (5 min)(-1) (4 mm disc)(-1) and 29 microM and 54.7 pmol (5 min)(-1)(4 mm disc)(-1), respectively. Both these high affinity systems were sodium dependent with a Hill coefficient of about 2.0 indicating that two sodium ions are required for the translocation of one molecule of taurine. The low affinity system was unevenly distributed over the two surfaces of the RPE, basolateral capacities being roughly twofold higher. The basolateral system was totally insensitive to sodium whereas the apical one with 50% sodium sensitivity suggested the presence of low affinity carrier heterogeneity. 3. A temperature-dependent mechanism for the release of pre-loaded taurine from bovine RPF was also demonstrated. 4. The effect of [K+]o trans-RPF gradients on the vectorial transport of taurine across the isolated preparation was also investigated. The results demonstrated that the direction and magnitude of taurine transport could be controlled by physiological variations in the extracellular concentration of potassium. 5. The determined kinetic parameters of the carriers were used to construct a working model of the vectorial pathway for the translocation of taurine across bovine RPE. This estimated the level of free intracellular taurine to be in the micromolar range. However, direct measurements of total RPE taurine by high performance liquid chromatography showed levels of 19.5 +/- 3.6 mM indicating that the major taurine pool in bovine RPE exists in the bound form. The model also showed that the magnitude and direction of net transport was a function of the transepithelial taurine gradient.

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.

The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.

Phenobarbital transiently stimulates uptake of 2-aminoisobutyric acid in hepatocytes.

Phenobarbital (PB) is a classical inducer of drug metabolizing enzymes and known to stimulate liver growth transiently in rodents. Previous studies have shown that regenerative liver growth after a partial hepatectomy is accompanied by the induction of the amino acid transport system A. In the present study we investigated whether amino acid transport is also increased by treatment of rats with PB. Na(+) -dependent hepatic uptake of the non-metabolizable amino acid 2-aminoisobutyric acid (AIB), which proceeds largely via transport system A, was studied in isolated hepatocytes from PB treated and untreated rats. Uptake of AIB (100 microM) was maximally induced (2.5-fold) 8 h after the beginning of PB treatment. Within 4 days, transport rates decreased to values similar to those determined in hepatocytes from untreated animals, despite the continuation of PB treatment. In contrast, induction of the PB-inducible cytochromes P450 2B1/2 was markedly increased during the entire experiment, as determined with the isoenzyme-selective substrate pentoxyresorufin. Kinetic analysis of AIB uptake revealed a "high" and a "low" affinity transport system. It is most likely that the high affinity system represents amino acid transport system A. Treatment with PB increased the V(max) value but did not affect the apparent Km value of the high affinity system. The present data suggest that the hepatic mitogen PB transiently induces amino acid transport system A.

Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.

Vitamin D target systems in the brain of the green lizard Anolis carolinensis.

Autoradiographic mapping criteria were employed to identify and localize specific high affinity binding sites (receptors) for the steroid hormone 1,25-dihydroxyvitamin D3 (1,25-D3) in the brain of Anolis carolinensis. In female and male lizards binding of tritiated 1,25-D3 occurred in identical regions of the fore-, mid-, and hindbrain, similar to findings in other species. There was a band of intensely labeled neurons forming a continuum from the n. accumbens, n. striae terminalis, the striatum, and extending into the amygdala. Target areas with high to intermediate labeling intensities were present in many other regions of the brain and single, small target cells were found throughout the organ. Some cells in the pituitary and pineal were labeled and also cells associated with the meninges, choroid plexuses and ependyma. The differential labeling suggests the existence of different 1,25-D3-responsive systems. One of the conspicuous "high capacity-high affinity systems" is found in the n. accumbens-n. striae terminalis and the amygdala. Most of the cerebral target regions for vitamin D correspond to those known for gonadal steroids, and the seasonal steroid 1,25-D3 may therefore act in conjunction with gonadal steroids in this seasonally breeding reptile.

Selection for mutants with low nitrate uptake ability in rice (Oryza sativa)

Screening for mutants deficient in the high affinity system of nitrate uptake was performed using mutagenized M2 population of rice (Oryza sativa, cv. Nipponbare or Kinmaze). For selecting mutants, M2 seedlings were transferred individually to 10 ml solution containing 250 μM potassium nitrate and 500 μM calcium sulphate at 20 or 28°C. After 6 or 24 h, nitrate concentration of the solution was determined with a nitrate selective electrode and the seedlings showing impaired nitrate uptake were selected as nitrate uptake deficient variants. Of 74 variants, three were confirmed to be mutants with low nitrate uptake ability in the M3 generation. Potassium uptake ability also decreased in the mutants. Three mutants were divided into two groups based on the analysis of nitrate reductase (NR, EC 1.6.6.1) activity and chlorate resistance. Two, NUE13 and NUE36, had a lower level of NR activity than the original cultivar and were not resistant to chlorate, while the seedlings of NUE50 had the same level of NR activity as the original cultivar and were more resistant to chlorate than the original cultivar. All mutants were resistant to cesium, a toxic ion analogue for potassium, suggesting that the decreased levels of both nitrate and potassium uptake were coupled to the change of plasma membrane H+‐ATPase activity.

The mechanism of zinc uptake in plants

The mechanism of zinc influx was investigated using giant algal cells (Chara corallina Klein ex Will.esk. R.D. Wood), in which it was possible to discriminate clearly between tracer zinc bound in the cell wall and actual uptake into the cell. It was shown that despite lengthy desorption, retention of zinc in slowly exchanging zinc pools in the cell wall can invalidate tracer influx measurements. A comparative study of zinc desorption from isolated cell walls of wheat (Triticum aestivum L.) roots indicated exchange characteristics similar to that of Chara. Fractionation of Chara internodal cells taken directly from cultures showed that most of the cell-associated zinc was in the cell walls. The cytoplasmic and vacuolar zinc concentrations were 56 mmol·m−3 and 32 mmol·m−3, respectively, for cells grown in a zinc concentration of 0.1 mmol·m−3. Influx of 65Zn in Chara was linear over several hours, with rapid transfer to the vacuole, but only slow efflux. Influx occurred in a biphasic manner, which was tentatively attributed to the operation of two separate transport systems, a high-affinity system which is saturated at 0.1 mmol·m−3 and a low-affinity system which showed a linear dependence on concentration up to at least 50 mmol·m−3. Only the low-affinity system was examined in detail. Influx through this system showed a strong dependence on external pH with an optimum around 7 and was also stimulated by cytoplasmic acidification. Influx was sensitive to metabolic inhibition, but not to blockers of Ca2+ and K+ channels. Other characteristics included a slight sensitivity to Mn2+ and Fe2+ but little sensitivity to high concentrations of K+ or Na+. Influx was independent of membrane potential difference in cells voltage-clamped at − 65 to − 300 mV.

Uptake of inorganic carbon in the cyanobacterium Synechocystis PCC6803: physiological and genetic evidence for a high-affinity uptake system.

Synechocystis PCC6803 displays two inorganic carbon-uptake processes, a low-affinity one (apparent Km: 300-400 microM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60 +/- 12 microM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high-affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high-affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two-component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high-affinity transport process. It would belong to the subclass of these molecules lacking the DNA-binding domain.

Comparison of the transport characteristics of ceftibuten in rat renal and intestinal brush-border membranes

The transport characteristics of ceftibuten, a dianionic cephem antibiotic, in rat renal and intestinal brush-border membranes were compared. Ceftibuten transport was mediated by two transport systems in the renal brush-border membrane and by one transport system in the intestinal brush-border membrane. The apparent kinetic parameters for the uptake of ceftibuten by the renal brush-border membrane vesicles, respectively, were: K m 1 , K m 2 , values of 26 and 1946 μM and V max 1 , V max 2 values of 105 and 1400 pmol/mg protein per 30 s. The apparent kinetic parameters for the uptake by the intestinal brush-border membrane vesicles were: K m of 425 μM and V max of 1701 pmol/mg protein per 30 s. In the renal brush-border membrane, l-Ala- l-Pro was partially competitive and competitive inhibitor for the uptake by the high and low affinity systems, respectively. However, l-Ala- l-Pro was a non-competitive inhibitor for the uptake by the intestinal brush-border membrane vesicles. l-Carnosine was a specific and competitive inhibitor for the high affinity system in the renal brush-border membrane, while it had no effect on the low affinity system of the kidney or on the transport system of the intestine. It was concluded that the transport characteristics of ceftibuten in the renal and intestinal brush-border membranes are similar in some aspects but they are not identical.

Kinetics of NO3- Influx in Spruce.

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.

Identification of a high affinity taurine transporter which is not dependent on chloride.

Taurine transport by lactating gerbil mammary tissue has been examined. Taurine uptake is mediated by a high-affinity system which is specific for beta-amino acids. The uptake of taurine is Na(+)-dependent but appears not to be obligatory dependent upon Cl-. Thus, replacing Na+ with choline almost abolished taurine uptake. Substituting Cl- with NO3- had no effect whereas SCN- induced a small but significant increase in taurine influx. Taurine uptake was Na(+)-dependent under conditions where Cl- had been replaced with NO3-. However, it is apparent that the Na(+)-dependent taurine transport system requires the presence of a permeable anion because replacing Cl- with gluconate markedly reduced taurine uptake. Cell-swelling, induced by a hyposmotic challenge, increased the efflux of taurine from gerbil mammary tissue via a pathway sensitive to niflumic acid.

Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate

Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V max) are dependent on the dilution rate (D). At D=0.12 h−1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h−1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.

Escherichia coli periplasmic protein FepB binds ferrienterobactin

Most high-affinity systems for iron uptake in Gram-negative bacteria are thought to employ periplasmic-binding-protein-dependent transport. In Escherichia coli, FepB is a periplasmic protein required for uptake of iron complexed to its endogenously-synthesized siderophore enterobactin (Ent). Direct evidence that ferrienterobactin (FeEnt) binds to FepB is lacking because high background binding by FeEnt prevents use of the usual binding protein assays. Here the membrane localization vehicle LppOmpA [Francisco, J.A., Earhart, C.F. & Georgiou, G. (1992). Proc Natl Acad Sci USA 89, 2713-2717] was employed to place FepB in the E. coli outer membrane. Plasmid pTX700 was constructed and shown to encode, under lac operator control, the 'tribrid' protein LppOmpAFepB; the carboxy-terminal FepB portion lacks at most two amino acids of mature FepB. After short induction periods, most of the tribrid was in the outer membrane. A number of LppOmpAFepB species could be detected; some were degradation products and some may be related to the multiplicity of FepB forms previously observed in minicells and maxicells. Outer membrane harbouring the tribrid and lacking FepA, the normal outer membrane receptor for FeEnt, bound approximately four times more FeEnt than outer membrane from uninduced cells, from cells lacking pTX700 and from cells expressing only an LppOmpA 'dibrid'. Similarly, whole UT5600(fepA)/pTX700 cells induced for tribrid synthesis bound FeEnt and this binding was not affected by energy poisons. The results demonstrated that FepB can bind FeEnt, thereby definitely placing FeEnt transport in the periplasmic permease category of transport systems, and that the LppOmpA localization vehicle can be used with periplasmic binding proteins.

Effect of Iron Depletion on Protein Profiles of Bilophila wadsworthia

Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained <0.04 tM Fe+++. Iron was added to a concentration of 100 iM in preparation of the ironcontaining medium. The final pH was adjusted to 7.0-7.1. Four strains of B. wadsworthia originally isolated from appendicitis case specimens were studied, including the reference strain ATCC 49260. They were all incubated for 14 days in 200-mL flask cultures (in media with and without iron) at 37?C in an anaerobic atmosphere. Detection of siderophores in culture supernatants was attempted by means of the method described by Schwyn and Neilands [6]. Preparation of the outer membrane was accomplished according to the method described by Sprott et al. [7]. Cells harvested by centrifugation were disrupted ultrasonically. Unbroken cells were discarded by centrifugation. The supernatant was treated with 2% Triton

ATP-dependent Efflux of 2,4-Dinitrophenyl-S-glutathione: PROPERTIES OF TWO DISTINCT TRANSPORT SYSTEMS IN INSIDE-OUT VESICLES FROM L1210 CELLS AND A VARIANT SUBLINE WITH ALTERED EFFLUX OF METHOTREXATE AND CHOLATE

The transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) into inside-out vesicles from L1210 cells was employed to identify and characterize ATP-dependent efflux routes for DNP-SG. Measurements of ATP-dependent uptake at varying concentrations of [3H]DNP-SG revealed the presence of two distinct transport systems. Transport at low substrate concentrations occurred predominantly via a high affinity system (Km = 0.63 microM), whereas a low affinity system (Km = 450 microM) predominated at high concentrations of substrate. The high affinity system was characterized by a potent inhibition by the glutathione conjugates of bromosulfophthalein (Ki = 0.09 microM) and ethacrynic acid (Ki = 0.44 microM), leukotriene C4 (Ki = 0.20 microM), and the taurate diconjugate of bilirubin (Ki = 0.10 microM). The low affinity transport system for DNP-SG exhibited a high affinity for bilirubin ditaurate (Ki = 1.8 microM), indoprofen (Ki = 3.0 microM), and biphenylacetic acid (Ki = 5.9 microM). Different results were obtained with an L1210/C7 variant which has a defect in the efflux of methotrexate and cholate. Vesicles from the latter cells contain the same low affinity transport activity as parental cells, but the high affinity route is absent and has been replaced by a system with an intermediate affinity for DNP-SG (Km = 4.5 microM). These results indicate that L1210 cells contain two unidirectional efflux pumps for DNP-SG with substantial differences in inhibitor sensitivity. The high affinity system shows a binding preference for glutathione conjugates but can also accommodate large anionic conjugates, whereas the low affinity system has a binding preference for large organic anions. Results with the variant cells support the hypothesis that the high affinity transport system for DNP-SG also mediates the unidirectional efflux of methotrexate and cholate in intact L1210 cells.

Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli

The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins.

The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae

Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate. We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system. This requirement has enabled a characterization of the low affinity system. Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate. We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein. FET4's predicted amino acid sequence contains six potential transmembrane domains. Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity. In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity. Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity. These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast. Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways.

Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.

We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins.