High Concentrations Of Zn2 Research Articles

The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn2+ binding to this domain

The voltage-gated proton channel Hv1 is strongly sensitive to Zn2+. The H+ conduction is decreased at a high concentration of Zn2+ and Hv1 channel closing is slowed by the internal application of Zn2+. Although the recent studies demonstrated that Zn2+ interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn2+ binds to His244 and His266 residues. The thermodynamics signature of Zn2+ binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn2+ to His244 (mutant H266A) and His266 (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation

Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn2+, required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn2+ in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn2+ activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn2+ to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn2+ also occurred in the absence of Ca2+ in the incubation medium, and required the tmAC, indicating that Zn2+ activates a GPCR. The AR stimulated by Zn2+ is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

Expression, purification, and characterization of NADP+-dependent malic enzyme from the oleaginous fungus Mortierella alpina.

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)+ to NAD(P)H. The NADP+-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP+ as the cofactor. The K m values for L-malate and NADP+ were 2.19±0.01 and 0.38±0.02 mM, respectively, while the V max values were 147±2 and 302±14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn2+, Mg2+, Co2+, Ni2+, and low concentrations of Zn2+ rather than Ca2+, Cu2+, or high concentrations of Zn2+. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K i values were 0.08 and 0.6 mM, respectively.

Inhibitional effects of metal Zn²⁺ on the reproduction of Aphis medicaginis and its predation by Harmonia axyridis.

BackgroundContamination, including metals, can disturb the reproductive processes of many organisms, including both prey and predatory insects. However, there is virtually no information on the effects of high level Zinc (Zn) pollution on aphids and ladybirds. The high concentrations of Zn2+ or Zn pollution inhibit reproduction in the phytophagous aphid, Aphis medicaginis, and the predatory ladybird Harmonia axyridis could provide important information.ResultsIt was observed in this study that Zn concentrations in Vicia faba (broad bean) seeds and seedlings in all Zn2+ treatments were significantly higher than that in the control group, and increased with increasing Zn2+ concentrations in the solution. The rate of reproduction in A. medicaginis declined significantly (p<0.05) over time in the five groups fed on broad bean seedlings treated with different concentrations of Zn2+ solution compared with the control group. These results showed that higher concentrations of Zn2+ significantly inhibited the reproductive capacity of A. medicaginis. We also cloned and identified a gene encoding vitellogenin (Vg) from A. medicaginis, which has an important role in vitellogenesis, and therefore, reproduction was affected by exposure to Zn2+. Expression of AmVg was reduced with increasing exposure to Zn2+ and also in the F1–F3 generations of aphids exposed to different Zn2+ concentrations. Predation by H. axyridis was also reduced in aphids exposed to high-levels of Zn2+. Similarly, ovipositioning by H. axyridis was also reduced.ConclusionsOur results suggest that Zn2+ can significantly affect the reproductive capacity of both A. medicaginis and its predator H. axyridis, the former through effects on the expression of AmVg and the latter through avoidance of aphids containing high levels of Zn2+.

Zinc induced folding is essential for TIM15 activity as an mtHsp70 chaperone

BackgroundTIM15/Zim17 in yeast and its mammalian ortholog Hep are Zn2+ finger (Cys4) proteins that assist mtHsp70 in protein import into the mitochondrial matrix. MethodsHere we characterized the Zn2+ induced TIM15 folding integrating biophysical and computational approaches. ResultsTIM15 folding occurs from an essentially unstructured conformation to a Zn2+-coordinated protein in a fast and markedly temperature-dependent process. Moreover, we demonstrate unambiguously that Zn2+ induced TIM15 folding is essential for its role as mtHsp70 chaperone since in the unstructured apo state TIM15 does not bind to mtHsp70 and is unable to prevent its aggregation. Molecular dynamics simulations help to understand the crucial role of Zn2+ in promoting a stable and functional 3D architecture in TIM15. It is shown that the metal ion, through its coordinating cysteine residues, can mediate relevant long-range effects with the interaction interface for mtHsp70 coupling thus folding and function. ConclusionsZn2+ induced TIM15 folding is essential for its function and likely occurs in mitochondrial matrix where high concentrations of Zn2+ were reported. General significanceThe combination of experimental and computational approaches presented here provide an integrated structural, kinetic and thermodynamic view of the folding of a mitochondrial zinc finger protein, which might be relevant to understand the organelle import of proteins sharing this fold.

Lipid bilayer – DNA interaction mediated by divalent metal cations: SANS and SAXD study

The structure of aggregates formed due to DNA interaction with dipalmitoylphosphatidylcholine (DPPC) in presence of Ca2+ and Zn2+ is examined using small-angle synchrotron X-ray diffraction (SAXD) and small-angle neutron scattering (SANS). SAXD detected two structures: LC - condensed lamellar phase and LX - lamellar phase with DNA strands intercalated between the adjacent lipid bilayers, without regular packing at low cation concentration (∼1 mM). The high concentration of Zn2+ induces a macroscopic phase separation in mixtures. The SANS curves of DPPC+ions2+ vesicles evaluated using the strip function model have shown different modes of cations binding to the DPPC bilayers.

Electrodeposition of ZnO layers for photovoltaic applications: controlling film thickness and orientation

A systematic study of the effect of the zinc oxide (ZnO) electrodeposition parameters (concentration, temperature, potential and pH) on film morphology, thickness, transparency, roughness and crystallographic orientation is presented with the view of producing optimized thin, planar, and continuous ZnO films for photovoltaic applications. Electrochemical measurements of the deposition charge as a function of time are used to understand the mechanism of nucleation and textured growth. Continuous thin films of crystalline ZnO are obtained at temperatures below 100 °C without the need for subsequent annealing. The formation of continuous films is favoured by high concentrations of Zn2+ precursor (> 100 mM), high temperature (> 70 °C) and low potentials (< −1.1 V/AgAgCl). A low bulk solution pH is shown to be a key factor in obtaining thin continuous films and the crystallographic orientation of these films can also be controlled by the deposition parameters. The importance of orientation and thickness control on device performance is shown by using the electrodeposited films as electron extracting interlayers in a model organic photovoltaic system.

Highly Selective Ratiometric Emission Color Change by Zinc‐Assisted Self‐Assembly Processes

Color sense: 2-(Anthracen-9-ylethynyl)-1-methylbenzimidazole (BzIm-An) shows a ratiometric color change in emission from blue to light yellow to green with increasing Zn2+ concentration. The dimeric 3:1 complex [{(BzIm-An)3Zn2+}2] formed at low Zn2+ concentration is converted into the 2:1 complex [(BzIm-An)2Zn2+] at high concentrations of Zn2+. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Solvent Extraction of Indium, Gallium, and Zinc Ions with Acidic Organophosphates having Bulky Alkyl Groups

The extraction equilibria of In3+, Ga3, and Zn2+ with bis(4‐ethylcyclohexyl)phosphoric acid (D4ECHPA), bis(4‐cyclohexylcyclohexyl)phosphoric acid (D4DCHPA), and bis(2‐ethylhexyl)phosphoric acid (D2EHPA) were investigated in acidic aqueous sulfate media. The order of extractability of metal ions is D4DCHPA > D2EHPA > D4ECHPA, which corresponds to the lipophilicity (log P) of the extractants. The separation factors, β(In/Ga) and β(Ga/Zn), of D4ECHPA and D4DCHPA are greater or comparable than that of D2EHPA, because of the steric hindrance of the bulky cyclohexyl groups. In3+ can be therefore separated from simulated liquor containing a high concentration of Zn2+ by D4DCHPA.

Assisted spray pyrolysis production and characterisation of ZnO nanoparticles with narrow size distribution

Nano-sized ZnO particles with a narrow size distribution and high crystallinity were prepared from aqueous solutions with high concentrations of Zn2+ containing salts and citric acid in a conventional spray pyrolysis setup. Structure, morphology and size of the produced material were compared to ZnO material produced by simple spray pyrolysis of zinc nitrates in the same experimental setup. Using transmission electron microscopy and electron tomography it has been shown that citric acid-assisted spray pyrolysed material is made up of micron sized secondary particles comprising a shell of lightly agglomerated, monocrystalline primary ZnO nanoparticles with sizes in the 20–30 nm range, separable by a simple ultrasonic treatment step.

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn2+, CO2, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn2+ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn2+ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO2 concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO2 concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO2. The activity of extracellular CA at a CO2 concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO2 concentration was at 0.35 mL/L CO2. Light intensity from 4.0 mW/cm2 to 5.6 mW/cm2 was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn2+, CO2, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.

Imaging synaptic zinc: promises and perils

It is well established that some excitatory nerve terminals have high concentrations of Zn(2+) in their synaptic vesicles. For some time, it has been believed that synaptic Zn(2+) is released during neurotransmission and acts as a neuromodulator. Fluorescent Zn(2+) indicators that do not penetrate membranes offer the prospect of rendering the release of Zn(2+) visible. Here, I take a critical look at fluorimetric imaging experiments devised to determine whether Zn(2+) is released and show that they are particularly susceptible to artifacts. Moreover, I will argue that recent experiments suggest that, rather than being released, Zn(2+) is presented to the extracellular space firmly coordinated to presynaptic macromolecules.

Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)(-1) and with an average dissociation constant of approx. 5 microM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.

Growth and photosynthesis limitation of marine red tide alga Skeletonema costatum by low concentrations of Zn2+.

The specific growth rate, cell final yields and extracellar carbonic anhydrase activity of the red tide alga Skeletonema costatum increased with increasing concentrations of Zn2+ from 0 to 12 pM, but decreased when Zn2+ was over 24 pM. However, cells grown under high concentrations of Zn2+ had higher activities of intracellular carbonic anhydrase than those grown under low concentrations of Zn2+. Chlorophyll a-specific light-saturated photosynthetic rate (P(Chla)), dark respiration rate (R(chla)) and apparent photosynthetic efficiency (alpha(chla)) significantly increased with increasing concentrations of Zn2+ from 0 to 3 pM, but decreased when increasing concentrations of Zn2+ from 3 to 66 pM. Photorespiration is the lowest when cells cultured in 3 pM Zn2+. The results suggest physiological activity of Skeletonema costatum is very sensitive to the prevailing concentration of Zn2+.

The Treponema pallidum tro Operon Encodes a Multiple Metal Transporter, a Zinc-dependent Transcriptional Repressor, and a Semi-autonomously Expressed Phosphoglycerate Mutase

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E. coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm. The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo. Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T. pallidum encounters high Zn2+ levels.

Potentiation of inhibitory glycinergic neurotransmission by Zn2+: a synergistic interplay between presynaptic P2X2 and postsynaptic glycine receptors.

The divalent cation zinc is known to modulate chloride currents carried by native and recombinant mammalian glycine receptors (GlyRs). To unravel the effect of Zn2+ on glycinergic neurotransmission, inhibitory postsynaptic currents (IPSC) of rat spinal neurons grown in culture were analysed in the absence and presence of Zn2+. Low concentrations of Zn2+ (0.5 and 5 micro m) augmented the mean amplitude of miniature IPSCs by approximately 40% over values obtained in the absence of zinc, whereas higher concentrations of Zn2+ (50 micro m) significantly decreased mean amplitude values. Remarkably, low concentrations of Zn2+ also significantly increased the mean frequency of miniature IPSCs. This effect was blocked by the P2X receptor antagonists PPADS and suramin, indicating the presence of Zn2+-sensitive presynaptic P2X receptors on glycinergic terminals. Immunostaining with antibodies against different P2X receptor subtypes revealed that P2X2 receptors partially colocalize with the GlyR. Potentiating concentrations of Zn2+ also affected the kinetics of miniature and evoked IPSCs by significantly prolonging their decay time constants. Electrophysiological analysis of heterologously expressed glycine transporters (GlyT) revealed for GlyT2 zero, and for GlyT1 a modest (< 20%), reduction of glycine uptake in the presence of 5 micro m Zn2+, indicating that prolongation of glycinergic IPSCs by Zn2+ is not due to inhibition of glycine removal from the synaptic cleft. Together, these results suggest that Zn2+ is a potent modulator of glycinergic synaptic transmission which increases in a synergistic manner the agonist affinity of both presynaptic P2X2 receptors and postsynaptic GlyRs.

Effects of Exogenous Factors on Enzymes of Carbon Metabolism in Thermoacidophilic Bacteria of the GenusSulfobacillus

Aerobic thermoacidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans1269Tand Sulfobacillus thermosulfidooxidanssubsp. asporogenes41 were shown to be resistant to stress factors, including high concentrations of Zn2+(0.8 M) and supraoptimal concentrations of H+(pH 1.2). The growth and biomass gain rates decreased, but bacteria retained their functions. The activity of nearly all enzymes involved in carbon metabolism decreased. Glucose was primarily metabolized via the Entner–Doudoroff pathway. The activity of tricarboxylic acid cycle enzymes decreased compared to that in cells grown under normal conditions. After saturation of the growth medium with 5 vol % CO2, sulfobacteria utilized glucose by the Embden–Meyerhof and pentose phosphate pathways under mixotrophic conditions.

Zinc-induced changes in ionic currents of clonal rat pancreatic -cells: activation of ATP-sensitive K+ channels.

The effects of zinc (Zn2+) on excitability and ionic conductances were analysed on RINm5F insulinoma cells under whole-cell and outside-out patch-clamp recording conditions. We found that extracellular application of 10-20 microM Zn2+ induced a reversible abolition of Ca2+ action potential firing, which was accompanied by an hyperpolarisation of the resting membrane potential. Higher concentrations of Zn2+, in the tens to hundreds micromolar range, induced a reversible reduction of voltage-gated Ca2+ and, to a lesser extent, K+ currents. Low-voltage-activated Ca2+ currents were more sensitive to Zn2+ block than high voltage-activated Ca2+ currents. The Zn2+-induced hyperpolarisation arose from a dose-dependent increase in a voltage-independent K+ conductance that was pharmacologically identified as an ATP-sensitive K+ (KATP) conductance. The effect was rapid in onset, readily reversible, voltage independent, and related to intracellular ATP concentration. In the presence of 1 mM intracellular ATP, half-maximal activation of KATP channels was obtained with extracellular application of 1.7 microM Zn2+. Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open-state probability with no change in the single channel conductance. Our data support the hypothesis that Zn2+ binding to KATP protein subunits results in an activation of the channels, therefore regulating the resting membrane potential and decreasing the excitability of RINm5F cells. Taken together, our results suggest that Zn2+ can influence insulin secretion in pancreatic beta-cells through a negative feedback loop, involving both KATP and voltage-gated conductances.

Study on the separation of Co2+ from Zn2+, Cd2+ by anion-exchange chromatography

The separation of Co2+ from Zn2+, Cd2+ by anion-exchange chromatography was discussed. The chromatographic column containing anion resin 201 × 7 which was saturated with a solution of ammonium chloride. The effects of the eluant acidity and eluant composition on the separation were investigated. The results indicate that this anion-exchange chromatography is suitable to the separation of Co2+ from Zn2+, Cd2+, and the condition of separation is simple and convenient. When the column is saturated with NH4Cl solution (2.0 mol/L, pH=4.0), the separation can be completed effectively. Zn2+ and Cd2+ can also be separated when different eluants are used and the pure solution with high concentration of Zn2+, Cd2+ respectively can be obtained easily.

Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.

During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST). After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme. Mg2+ stabilized the oligomeric state but was not essential for octamer formation. Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity. EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+. At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme. Pb2+, Cd2+, and Hg2+ did not restore activity. A stimulatory effect of monovalent ions was observed. A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found. pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2). These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis. Some PBGS inhibitors were characterized. Finally, we succeeded in obtaining well-ordered crystals of P. aeruginosa PBGS complexed with the substrate analogue levulinic acid.