Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity,low affinity,and histidine-facilitated pathways

Zinc is both a vital nutrient and an important toxicant to aquatic biota. In order to understand the interplay between nutrition and toxicity, it will be important to determine the mechanisms and the factors that regulate zinc uptake. The mechanism of apical intestinal Zn(II) uptake in freshwater rainbow trout and its potential modification by the complexing amino acid histidine was investigated using brush-border membrane vesicles (BBMVs). Following characterisation of the BBMV preparation, zinc uptake in the absence of histidine was both time- and concentration-dependent and consisted of two components. A saturable phase of uptake was described by an affinity constant of 57+/-17 microM and a transport capacity of 1867+/-296 nmol mg membrane protein(-1) min(-1). At higher zinc levels (>500 microM) a linear, diffusive component of uptake was evident. Zinc transport was also temperature-dependent, with Q10 values suggesting zinc uptake was a carrier-mediated process. Zinc uptake by vesicles in the presence of histidine was correlated to a mono-histidine species (Zn(His)+) at all Zn(II) concentrations examined.     

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout (Oncorhynchus mykiss)

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant (K_m) for Ni transport within the specifically described vesicular media was calculated as 17.9 ± 1.9 μM, the maximal rate of transport (J_max) was calculated as 108.3 ± 3.7 nmol mg protein⁻¹ min⁻¹, and the slope of the linear diffusive component was calculated as 0.049 ± 0.005 nmol mg protein⁻¹ min⁻¹ per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time (T_1/2) of 125.2 ± 7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni-histidine complexation was rapid, with a T_1/2 of 12.2 s describing the Ni-histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

Zinc transport across an endothelium includes vesicular cotransport with albumin

Bovine pulmonary arterial endothelial cells (BPAEC) were grown on permeable polycarbonate membrane filters suspended between two compartments representing the blood vessel lumen and the interstitium. This in vitro model of an endothelium was subjected to a battery of tests to unravel the mechanisms of zinc transport from the blood into peripheral tissues. Transport of ⁶⁵Zn across BPAEC from media containing zinc concentrations up to 50 μmol/L exhibited both saturable and nonsaturable kinetics. V_max of the saturable component was 246 ± 43 pmol/(h × cm²) and K_m was 2.3 ± 1.3 μmol/L. Transport was pH and temperature sensitive and substantially influenced by albumin and histidine concentrations, but not influenced by analogous minerals or metabolic inhibitors. Inhibition of coated vesicle formation by depletion of intracellular potassium reduced ⁶⁵Zn transport. Albumin carrying a zinc ion crossed the endothelium more rapidly than zinc-free albumin. When evaluated together, this body of evidence supports the existence of two major pathways of zinc transport across the pulmonary endothelium, but neither involves entry into the endothelial cells. One pathway involves receptor-mediated cotransport with albumin by transcytotic vesicles. The other is nonsaturable and involves cotransport with albumin and low molecular weight ligands, principally histidine, through intercellular junctions and nonselective, bulk-fluid transcytosis. © 1996 Wiley-Liss, Inc.     

Equivalent uptake of organic and inorganic zinc by monkey kidney fibroblasts, human intestinal epithelial cells, or perfused mouse intestine

Zinc (Zn) is recognized as an essential nutrient, and is added as a supplement to animal and human diets. There are claims that zinc methionine (ZnMet) forms a stable complex that is preferentially transported into tissues, and this has contributed to uncertainty about conflicting reports on the bioavailability of various Zn compounds. This study evaluated the cellular and intestinal uptake of inorganic and organic forms of Zn. Steady-state uptake of⁶⁵Zn by human intestine epithelial cells, and monkey kidney fibroblasts was not significantly different with zinc chloride (ZnCl₂), ZnMet, or zinc propionate (ZnProp) (P > 0.05). Uptake of⁶⁵Zn from zinc chelated with EDTA was significantly lower (P < 0.01). In live mice,⁶⁵Zn uptake by perfused intestine and deposition in intestine and liver showed no significant difference between ZnCl₂ and ZnMet. Equimolar [⁶⁵Zn]methionine and zinc[³⁵S]methionine were prepared according to a patented method that yields “ complexed” Zn. Cellular uptake of the radiolabeled methionine was <0.1% of the radiolabeled Zn from these complexes, indicating separate uptake of the Zn and methionine. Gel filtration did not distinguish between⁶⁵Zn in ZnCl₂, ZnProp, or reagent ZnMet, though feed-grade ZnMet containing >10% protein did give a higher-mol-wt form of⁶⁵Zn. Results of this study show equivalent uptake of Zn from inorganic and organic compounds, and support recent feed trials on Zn bioavailability.     

Evidence for a zinc/proton antiporter in rat brain

The data presented in this paper are consistent with the existence of a plasma membrane zinc/proton antiport activity in rat brain. Experiments were performed using purified plasma membrane vesicles isolated from whole rat brain. Incubating vesicles in the presence of various concentrations of 65Zn2+ resulted in a rapid accumulation of 65Zn2+. Hill plot analysis demonstrated a lack of cooperativity in zinc activation of 65Zn2+ uptake. Zinc uptake was inhibited in the presence of 1 mM Ni2+, Cd2+, or CO2+. Calcium (1 mM) was less effective at inhibiting 65Zn2+ uptake and Mg2+ and Mn2+ had no effect. The initial rate of vesicular 65Zn2+ uptake was inhibited by increasing extravesicular H+ concentration. Vesicles preloaded with 65Zn2+ could be induced to release 65Zn2+ by increasing extravesicular H+ or addition of 1 mM nonradioactive Zn2+. Hill plot analysis showed a lack of cooperativity in H+ activation of 65Zn2+ release. Based on the Hill analyses, the stoichiometry of transport may include Zn2+/Zn2+ exchange and Zn2+/H+ antiport, the latter being potentially electrogenic. Zinc/proton antiport may be an important mode of zinc uptake into neurons and contribute to the reuptake of zinc to replenish presynaptic vesicle stores after stimulation.     

Possible involvement of plasma histidine in differential brain permeability to zinc and cadmium

Zinc gets into the brain parenchyma across the blood-brain and the blood-cerebrospinal fluid barriers, while cadmium hardly gets into the brain parenchyma. Because histidine may be involved in zinc transport across the brain barrier systems, the binding to histidine was compared between zinc and cadmium to understand the difference in brain permeability to both metals. Sephadex G-10 gel filtration indicated that ¹⁰⁹Cd, unlike ⁶⁵Zn, does not bind to histidine. When the plasma incubated with ⁶⁵Zn or ¹⁰⁹Cd was dialyzed in physiological saline containing histidine (0-10 mM), ⁶⁵Zn concentration in the dialysate was increased with the increase of the histidine concentration, suggesting the transfer of zinc from plasma proteins to histidine. The low affinity of zinc to plasma proteins may be important for brain permeability to this metal. On the other hand, ¹⁰⁹Cd was not detected in the dialysate in the presence of 0.1 mM histidine, which is equal to the concentration in the plasma, suggesting no transfer of cadmium from plasma proteins to histidine. These results suggest that the avid binding of cadmium to plasma proteins is related to brain impermeability to this metal.     

Chlorsulfuron reduces rates of zinc uptake by wheat seedlings from solution culture

Wheat plants differing in zinc efficiency (Excalibur; Zn-efficient, Gatcher and Durati; Zn-inefficient) were grown in HEDTA chelate-buffered nutrient solution in controlled conditions and supplied with 0 or 40 g chlorsulfuron L^-1 . Zinc uptake rates of 12-d-old plants were measured over 80 or 90 minutes using⁶⁵ Zn added to nutrient solutions. Increasing the zinc concentration of the solution increased the rate of zinc uptake, while the percentage of zinc transported to shoots was decreased. Addition of chlorsulfuron to uptake solutions for 90 minutes did not influence rate of zinc uptake or transport of zinc to shoots. Pretreating plants with chlorsulfuron for 5 days decreased zinc uptake rates, but transport to shoots was proportionally increased. Three-day pretreatment with chlorsulfuron was the minimum required for significant differences in uptake and transport of zinc to occur. Plants exposed to chlorsulfuron for 3 days required a further 5 days of growth in chlorsulfuron-free solutions before uptake rates recovered to control plant rates. It is concluded that chlorsulfuron deleteriously but reversibly affects uptake of zinc across the plasma membrane after prolonged exposure.     

Absorption of copper and copper–histidine complexes across the apical surface of freshwater rainbow trout intestine

Bioavailability is integral in mediating the delicate balance between nutritive and potentially toxic levels of copper in fish diets. Brush-border membrane vesicles isolated from freshwater rainbow trout intestine were used to characterise apical copper absorption, and to examine the influence of the amino acid histidine on this process. In the absence of histidine, a low affinity, high capacity copper uptake mechanism was described. However, when expressed as a function of ionic copper (Cu²⁺), absorption was linear, rather than saturable, suggesting that the saturable curve was an artifact of copper speciation. Conversely, in the presence of l-histidine (780 μM) saturable uptake was characterised. The uptake capacity discerned (J _max of 354 ± 81 nmol mg protein⁻¹ min⁻¹) in the presence of histidine indicated a significantly reduced capacity for copper transport than that in the absence of histidine. To determine if copper uptake was achievable through putative histidine uptake pathways, copper and histidine were incubated in the presence of tenfold greater concentrations of amino acids proposed to block histidine transporters. Accounting for changes in copper speciation, significant inhibition of uptake by glycine and lysine were noted at copper levels of 699 and 1,028 μM. These results suggest that copper–histidine complexes may be transportable via specific amino acid-transporters in the brush-border membrane.     

Characterization of the nitrate transporter in root plasma membranes of Cucumis sativus L

Nitrate uptake in right-side out plasma membrane vesicles isolated from cucumber roots was characterized. Nitrate uptake into vesicles was driven by an artificially imposed pH gradient. The uptake was strongly inhibited by phenylglyoxal, an arginyl residue modificator. Only a slight repression of NO ₃ ⁻ transport in vesicles was observed in the presence of NEM, a thiol group reagent. pCMBS, an other thiol reagent and DEPC, an effector of histidine residue, had no effect on the nitrate transport in plasma membranes. ATP-driven proton transport in vesicles was not significantly affected in the presence of both, phenylglyoxal and DEPC, whereas pCMBS and NEM abolished it almost completely. The possible role of the particular amino acids residues in the active nitrate transport is discussed. NO ₃ ⁻ uptake into vesicles isolated from both, nitrate-induced and nitrate-depleted plant material was higher than that observed in the vesicles obtained from uninduced plants. Thus, isolated vesicles reflect the well-known in vivo response of intact plants on the exogenous nitrogen regime.     

131I Induced Hematological Alterations in Rat Blood: Protection by Zinc

The present study was planned to determine the potential of zinc in attenuating the toxicity induced by ¹³¹I in rat blood. Female wistar rats were segregated into four main groups. Animals in Group I served as normal controls; Group II animals were administered a dose of 3.7 Mbq of ¹³¹I (carrier free) intraperitoneally, Group III was supplemented with Zinc in the form of ZnSo₄.7H₂O (227 mg/l drinking water), and Group IV was given a combined treatment of Zinc as well as ¹³¹I, in a similar way as was given to Groups IV and II animals, respectively. The effects of different treatments were studied on various parameters in rat blood including hemoglobin (Hb) levels, % hematocrit, zinc protoporphyrins (ZPP), activities of enzymes which included aminolevulinic acid dehydratase (δ-ALAD) and Na⁺ K⁺ ATPase and uptake of ⁶⁵Zn in blood. The study revealed an increase in the levels of hemoglobin, % hematocrit, activities of δ-ALAD, Na⁺ K⁺ ATPase and uptake of ⁶⁵Zn, 7 days after the ¹³¹I treatment. On the contrary, the levels of ZPP were found to be significantly decreased after ¹³¹I treatment. However, zinc treatment to ¹³¹I-treated animals significantly attenuated the various biochemical and hematological indices. Moreover, zinc treatment to the ¹³¹I-treated animals could significantly decrease the uptake of ⁶⁵Zn, which was increased after ¹³¹I treatment. Based upon these data, the present study suggests that zinc has the potential to attenuate ¹³¹I induced toxicity by restoring the altered hematological indices and biochemical changes.     

Effects of histidine on tissue zinc distribution in rats

Histidine has been reported to affect body zinc status by increasing urinary zinc excretion. The effects of experimental histidinemia on distribution of⁶⁵Zn in anesthetized rats were studied. Infusion ofl-histidine at a rate sufficient to raise plasma concentrations to approximately 2mm for 6h starting 48 h after a single intraperitoneal⁶⁵Zn injection did not alter⁶⁵Zn activities in a variety of tissues when compared with anesthetized uninfused animals. However, plasma⁶⁵Zn and erythrocyte⁶⁵Zn were decreased, and liver⁶⁵Zn was increased. If⁶⁵Zn was injected intravenously during histidine infusion, net accumulation of zinc by some tissues was increased, but uptake by others was reduced relative to uninfused animals. In all cases, however, uptake expressed relative to plasma⁶⁵Zn levels was increased when allowance was made for the more rapid fall in plasma⁶⁵Zn during histidine infusion. Similar infusions ofd-histidine produced quantitatively similar effects. Since enzymatic mechanisms and amino acid carriers would be expected to show stereoselectivity, such processes are unlikely to be involved in the zinc distribution changes described. The possibility of zinc transport by a hitherto unidentified carrier is discussed. These experiments confirm that histidinemia can affect zinc status, but any associated changes in urinary zinc excretion do not seem adequate to account for the tissue changes found.     

Substituted azomethine-zinc complexes: Thermal stability, photophysical, electrochemical and electron transport properties

A series of zinc complexes with salicylidene-aniline and its derivatives as ligands have been designed and synthesized for electron transport in organic light-emitting diodes (OLEDs). A systematic study on their thermal, photophysical, electrochemical and electron transport properties has been carried out and demonstrated that the substitution of −CH₃, −OCH₃, −CN and −N(CH₃)₂ on aniline ring of ligands can finely tune the properties of the corresponding zinc complexes. The density functional theory calculations of location and distribution of the frontier molecular orbital states unveiled the relationships between the substituents and the photophysical and electrochemical properties of these complexes. OLEDs with bis(salicylidene-p-methylaniline)zinc(II) (Zn(sama)₂) as the electron transport layer exhibited high current efficiency, indicating its great potential as a useful electron transport material for OLEDs.     

Kinetic identification of a mitochondrial zinc uptake transport process in prostate cells

Prostate cells accumulate high cellular and mitochondrial concentrations of zinc, generally 3-10-fold higher than other mammalian cells. However, the mechanism of mitochondrial import and accumulation of zinc from cytosolic sources of zinc has not been established for these cells or for any mammalian cells. Since the cytosolic concentration of free Zn(2+) ions is negligible (estimates vary from 10(-9) to 10(-15) M), we postulated that loosely bound zinc-ligand complexes (Zn-Ligands) serve as zinc donor sources for mitochondrial import. Zinc chelated with citrate (Zn-Cit) is a major form of zinc in prostate and represents an important potential cytosolic source of transportable zinc into mitochondria. The mitochondrial uptake transport of zinc was studied with isolated mitochondrial preparations obtained from rat ventral prostate. The uptake rates of zinc from Zn-Ligands (citrate, aspartate, histidine, cysteine) and from ZnCl(2) (free Zn(2+)) were essentially the same. No zinc uptake occurred from either Zn-EDTA, or Zn-EGTA. Zinc uptake exhibited Michaelis-Menten kinetics and characteristics of a functional energy-independent facilitative transporter associated with the mitochondrial inner membrane. The uptake and accumulation of zinc from various Zn-Ligand preparations with logK(f) (formation constant) values less than 11 was the same as for ZnCl(2;) and was dependent upon the total zinc concentration independent of the free Zn(2+) ion concentration. Zn-Ligands with logK(f) values greater than 11 were not zinc donors. Therefore the putative zinc transporter exhibits an effective logK(f) of approximately 11 and involves a direct exchange of zinc from Zn-Ligand to transporter. The uptake of zinc by liver mitochondria exhibited transport kinetics similar to prostate mitochondria. The results demonstrate the existence of a mitochondrial zinc uptake transporter that exists for the import of zinc from cytosolic Zn-Ligands. This provides the mechanism for mitochondrial zinc accumulation from the cytosol which contains a negligible concentration of free Zn(2+). The uniquely high accumulation of mitochondrial zinc in prostate cells appears to be due to their high cytosolic level of zinc-transportable ligands, particularly Zn-Cit.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 μg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ±13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 μg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 μg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10⁶ cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 μg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10⁶ cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 μg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 μg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 μg/mL Zn relative to 0 Zn (IVM alone) and 0.7 μg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.     

Utility of solid-phase spectrophotometry to determine trace amounts of zinc in environmental and biological samples

A solid-phase spectrophotometric analysis has been proposed for preconcentration and determination of Zn(II) in real samples. The procedure is based on sorption of zinc(II) as 5-(2-benzothiazolylazo)-8-hydroxyquinoline (BTAHQ) complex on dextran-type anion-exchange gel (Sephadex DEAE A-25). The influences of the analytical parameters, including pH of the aqueous solution, amounts of BTAHQ, and sample volume, were investigated. The absorbance of the gel at 675 and 750 nm, packed in a 1.0-mm cell, was measured directly. The molar absorptivities were found to be 2.50 × 10⁷ and 9.55 × 10⁷ L mol⁻¹ cm⁻¹ for 500 and 1000 ml, respectively. Calibration was linear over the range of 0.05–1.10 μg L⁻¹ with a relative standard deviation of less than 1.60% (n = 10). The detection and quantification limits of the 500-ml sample method were 12 and 40 ng L⁻¹ on using 50 mg. For the 1000-ml sample, the detection and quantification limits were 7.5 and 25 ng L⁻¹ using a 50-mg exchanger. Increasing the sample volume can enhance sensitivity. No considerable interferences were observed from other investigated anions and cations on the Zn(II) determination. The proposed method was applied to determine zinc in environmental samples, including natural water, food, certified reference materials, meat, and biological samples, comparing the results simultaneously with those obtained using a flame atomic absorption spectrophotometer, whereby the validity of the method was tested.     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

The Salmonella enterica ZinT structure,zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Background

In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT–ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim.

Methods

ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy).

Results

The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K_D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K_D < 20 nM) and one low affinity Zn(II) binding site (K_D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT–ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other.

Conclusions

ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA.

General significance

Knowledge of the ZinT–ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.