Differential reactivity of individual zinc ions in clusters from bacterial metallothioneins

The bacterial metallothionein SmtA binds four zinc ions with high affinity and specificity in a Zn₄S₉N₂ cluster. We have explored the reactivity of these zinc ions towards the metal-chelator EDTA. Under pseudo-first-order conditions, initial break-down of zinc-thiolate bonds is rapid, followed by several slower phases. The reaction with stoichiometric amounts of EDTA is relatively slow and has been followed by ¹H NMR and mass spectrometry. Both methods reveal that partially metallated intermediates occur during the reaction. Three- and two-metal species are observed in only minor amounts, whereas the Zn₁ species is dominant during the mid stages of the reaction, before complete metal depletion occurs. These results suggest that the zinc finger site in SmtA is not only inert towards metal exchange, but also more resilient towards chelating agents. The greater inertness of this site may help to maintain the protein fold during metal depletion, and allow subsequent facile metal uptake. Conversely, it is likely that the protein fold is the major contributor to the observed persistence of Zn₁SmtA in this reaction. Mass spectrometric studies with His-to-Cys mutants of SmtA reveal that the primary site for EDTA attack is the His49-containing zinc site C, and that His40 has a major influence on the reactivity of three sites.     

Novel zinc finger nuclease created by combining the Cys2His2- and His4-type zinc finger domains

To improve the DNA hydrolytic activity of the zinc finger nuclease, we have created a new artificial zinc finger nuclease (ZWH4) by connecting two distinct zinc finger domains possessing different types of Zn(II) binding sites (Cys₂His₂- and His₄-types). The overall fold of ZWH4 is similar to that of the wild-type Sp1 zinc finger (Sp1(zf123)) as revealed by circular dichroism spectroscopy. The gel mobility shift assay demonstrated that ZWH4 binds to the GC box DNA, although the DNA-binding affinity is lower than that of Sp1(zf123). Evidently, ZWH4 hydrolyzes the covalently closed circular plasmid DNA (form I) containing the GC box (pBSGC) to the linear duplex DNA (form III) in the presence of a higher concentration (50 times) of the protein than DNA for a 24-h reaction. Of special interest is the fact that the novel mixed zinc finger protein containing the Cys₂His₂- and His₄-type domains was first created. The present results provide the useful information for the redesign strategy of an artificial nuclease based on the zinc finger motif.     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Interaction of arsenite with a zinc finger CCHC peptide: evidence for formation of an As-Zn-peptide mixed complex

The interaction of arsenite with a Cys₃His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of Zn^II was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K = 2083 M^− 1 and 442 M^− 1 at 25 °C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate.     

Nucleotide pyrophosphatase from yeast. The presence of bound zinc.

Nucleotide pyrophosphatase from yeast was inhibited by thiols, o-phenanthroline, 8-hydroxyquinoline, EDTA, and 8-hydroxyquinoline-5-sulfonic acid. The inhibition by chelating agents was time and concentration dependent. Inhibition by EDTA was decreased by complexing the EDTA with metal ions before addition to the enzyme. The effectiveness of the metal ions in preventing inhibition by EDTA paralleled the stability constants of the EDTA-metal complexes. Partial recovery of EDTA-inhibited enzyme activity was achieved with Zn²⁺, Co²⁺, Fe²⁺, and Mn²⁺. Analyses for zinc in the purified enzyme by atomic absorption spectroscopy and by titration with 8-hydroxyquinoline-5-sulfonic acid revealed the presence of approximately 1 g atom/mol of enzyme (M_r 65,000). The data indicate that yeast nucleotide pyrophosphatase is a metalloenzyme in which the zinc plays some role in activity.     

Characterization of the Antiviral Activity for Influenza Viruses M1 Zinc Finger Peptides

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn²⁺. Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID₅₀ of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.     

Soil isotopically exchangeable zinc: A comparison between E and L values

The objectives of the present paper were: (i) to determine isotopically exchangeable zinc using two isotopic exchange methods (E and L values) in a series of polluted and non-polluted Swiss agricultural soils, and (ii) to evaluate the ability of chemical extraction methods to estimate plant-available soil Zn using isotopic techniques. The surface horizon (0–20 cm) of seven polluted and non-polluted soils representing a wide range in physico-chemical properties and Zn contents were sampled. An isotopic exchange kinetics (IEK) approach was used to assess, in a batch experiment, the isotopically exchangeable Zn content (E value). In order to determine the L values, a pot experiment was carried out with Lolium multiflorum (cv. Axis) in a growth chamber using a ⁶⁵Zn-isotope dilution technique. Total Zn uptake and the isotopic composition (⁶⁵Zn/^stableZn) were determined in Lolium multiflorum for five successive cuts. The amounts of zinc extracted by different chemicals were compared with L values and regression parameters were estimated. The isotopic composition in soil extracted by DTPA and EDTAAc at the end of the pot experiment was also determined. Results showed that the equation describing the increase of isotopically exchangeable Zn with time could be extrapolated to three months for polluted and non-polluted neutral and acidic soils, and that the results were not different from the amount of isotopically exchangeable Zn experimentally determined with Lolium multiflorum (L value). In alkaline soils however, results suggest that either ⁶⁵Zn sorption occurred in the batch experiment or that the concentration of Zn in the soil solution had been overestimated, leading to an overestimation of the E value compared to the L values. Furthermore, the specific activities measured in DTPA and EDTA extractions at the end of the pot experiment were significantly different compared to the specific activity of the plant, showing that both these chelating agents extract neither all the available soil Zn nor only the available soil Zn for plants. Abbreviations: C _Zn– concentration of Zn in a soil water extract (mg Zn L^?1); C _Zn?Plant– concentration of Zn in plant shoots (mg Zn kg^?1 DM); DTPA – diethylene triamine pentaacetic acid; E _1\min– amount of Zn isotopically exchangeable within one min (mg Zn kg^?1 soil); E _(t)\exp– amount of Zn isotopically exchangeable after t min derived from experimental results (mg Zn kg^?1 soil); E _(t)pred– amount of Zn isotopically exchangeable after t min predicted using kinetic parameters derived from a 100 min long isotope exchange kinetic experiment together with C _Zn, and Zn_HNO3 (mg Zn kg^?1 soil); EDTA – ethylene diamine tetraacetic acid; IC_P– isotopic composition of Zn in plant shoots; IC_DTPA– isotopic composition of Zn in the soil DTPA extract; IC_EDTA– isotopic composition of Zn in the soil EDTA extract; IC_SE– isotopic composition of Zn in the soil extracts; IEK – isotope exchange kinetics; L value – amount of plant available Zn (mg Zn kg^?1 soil); Lolium multiflorum; TEA – Triethanolamine; Zn_DTPA– Zn extractable by 0.005 M DTPA + 0.01 M CaCl2 + 0.1 M TEA; Zn_EDTA?NH4Ac– Zn extractable by 0.5 M NH₄Ac, 0.02 M EDTA; Zn_{EDTA?Ca(NO3)2}– Zn extractable by 0.005 M EDTA, 0.01 M Ca(NO₃)₂; Zn_KCl– Zn extractable by 1 M KCl; Zn_CaCl2– Zn extractable by 0.01 M CaCl₂; Zn_NaNO3– Zn extractable by 0.1 M NaNO₃; Zn_HNO3– Zn extractable by 2 M HNO₃.     

Reactivity of metal chelates of sulphur containing ligands towards Lewis bases. Part III. Reaction of bis(1,2-dimethyl-and 1-phenyl-1,2-ethanediylidene)bis(S-methylhydrazinecarbodithioate)NN′SS′(−2)dizinc(II) with pyridines

The reaction of the dimeric zinc(II) chelates of the type I (R₁ = R₂ = CH₃, R₁ = H, R₂ = Ph) with pyridine, 2-methylpyridine, 3-methylpyridine and 4-methylpyridine afforded the monomeric monobase adducts. The isolated adducts were characterized by their electronic and ¹H NMR spectra, and a five coordinate square pyramidal structure was tentatively assigned for these adducts.The adduct formation reaction was followed spectrophotometrically and the reaction kinetics were studied using a stopped flow technique. From the available kinetic data, as well as the measured activated parameters (ΔH^#, ΔS^#), a mechanism for the adduct formation reaction is proposed.     

Low Micromolar Zinc Accelerates the Fibrillization of Human Tau via Bridging of Cys-291 and Cys-322

A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau_244–372 and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn²⁺ dramatically accelerate fibril formation of wild-type Tau_244–372 under reducing conditions, compared with no Zn²⁺. Higher concentrations of Zn²⁺, however, induce wild-type Tau_244–372 to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn²⁺ has been chelated by EDTA. Unlike wild-type Tau_244–372, low micromolar concentrations of Zn²⁺ have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn²⁺ binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.     

Macrocyclic dinuclear Zn(II) complexes: synthesis, structure and hydrolysis of tris(p-nitrophenyl) phosphate

A series of mono- and dinuclear zinc complexes of 3,6,9,17,20,23-hexaaza-29,30-dihydroxy-13,27-dimethyl-tricyclo[23,3,1^11,15]triaconta-1(28),11,13,15(30),25,26-hexaene (H₂L or BDBPH) have been defined in solution by potentiometry. The crystal structure of [Zn₂C₂₆H₄₀N₆O₂(CH₃OH)₂]·Br₂ has been determined by X-ray. Each zinc ion is coordinated to three nitrogen atoms, a bridged-phenolic oxygen atom, and a methanolic oxygen atom, which define a six-coordinated octahedron. Bond lengths of ZnN are in the range of 2.104(3)-2.120(3) Å and distances between Zn and O (bridged-phenolic oxygen) are 2.052(2), 2.062(2) Å, respectively. The dinuclear complexes: [Zn₂L]²⁺ and [Zn₂L(OH)]⁺ play crucial roles in hydrolytic reaction of tris(4-nitrophenyl)phosphate. A possible mechanism showed that [Zn₂L(OH)]⁺ acts as a nucleophile and [Zn₂L]²⁺ stabilizes the formation of the intermediate: [Zn₂L-BNP].     

Zinc and cadmium in 5-aminolevulinic acid dehydratase. Equilibrium,kinetic, and 113Cd-nmr-studies

5-Aminolevulinic acid dehydratase (ALAD) from bovine liver contains zinc that is partially lost during the isolation of the enzyme. ALAD has its maximal activity at 10^?5 M ZnCl₂. It binds 7.4 Zn per octameric protein with an association constant of 5.3 × 10⁶ M^?1. ALAD is inactivated by 1,10-phenanthroline or ethylenediaminetetraacetic acid (EDTA) but not by monodentate anions like cyanide or sulfide. After removal of zinc by chelating agents, the enzyme activity may be restored by Zn²⁺ or Cd²⁺. Removal or zinc by EDTA increases K_M 60-fold and decreases V_max to about $\text{1}\text{2}$ of its original value. The ¹¹³Cd nuclear magnetic resonance spectrum of the enzyme reconstituted with ¹¹³Cd-acetate exhibits a single sharp resonance signal at 79 ppm. It does not change by the addition of substrate but disappears when the inhibitor lead acetate is added. Therefore, an immediate interaction between the metal ion of the enzyme and the substrate is excluded, whereas lead changes the environment of cadmium and probably of zinc too.     

Isolation of GIF from porcine brain and studies of its zinc transfer kinetics with apo-carbonic anhydrase

Neuronal growth inhibitory factor (GIF) of porcine brain, was isolated and purified by a similar procedure which was used on the isolation of human and bovine GIF. The native porcine protein with stoichiometry of 4Cu⁺, 3Zn²⁺ was obtained for the first time. The kinetics of zinc transfer from Cu₄Zn₃MT-3 to apo-carbonic anhydrase were studied, and zinc transfer rate constants and thermodynamic parameters were obtained. It is found that like other MTs, porcine Cu₄Zn₃MT-3 can also transfer its zinc atom to apoCA, even much easier than other MTs. A possible association mechanism has been proposed, the formation of Cu₄Zn₃MT3-apoCA complex may be the rate-determining step. The obtained data indicate besides its neuronal growth inhibitory function, GIF might play a role in cellular Zn homeostasis in brain.     

Zinc homeostatic proteins in the CNS are regulated by crosstalk between extracellular and intracellular zinc

Release of Zn²⁺ from presynaptic glutamatergic terminals has long been considered the principle challenge necessitating the existence of zinc homeostatic proteins (ZHP) in the mammalian nervous system. It is now known that neural cells also possess an intracellular zinc pool, termed here [Zn²⁺]_i, which functions in a cell signaling context. A major challenge is characterizing the interaction of these two populations of zinc ions. To assess the relationship of this Zn²⁺ pool to cellular ZHP production, we employed immunofluorescence and immunoblot analysis to compare the expression of ZHP's ZnT‐1 and MT‐I/II in olfactory bulb and hippocampus of wild‐type and ZnT‐3 KO mice, which lack synaptic Zn²⁺. In both areas, the respective distribution and concentration of ZnT‐1 and MT‐I/II were identical in ZnT‐3 KO and control animals. We subsequently examined ZHP content in ZnT‐3 KO and WT mice treated with a membrane‐permeable Zn²⁺ chelator. In both olfactory bulb and hippocampus of the KO mice, the ZHP content was significantly reduced 15 h after chelation of [Zn²⁺]_i compared to WT controls. Our findings support the conclusion that ZHP expression is regulated by crosstalk between synaptic and intracellular pools of Zn²⁺. J. Cell. Physiol. 224: 567–574, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence for presence of Zn-binding site in acetylcholinesterase

The purified electric eel acetylcholinesterase (AChE) was able to bind to the Zn⁺²-chelate-Sepharose affinity column only on treatment with EDTA. However goat brain and cobra venom AChE were binding to the column even without EDTA treatment. But all these enzymes were eluted from Zn⁺²-chelate-Sepharose affinity column by EDTA. Modification of histidine residues of AChEs by diethylpyrocarbonate resulted in abolition of its binding to Zn⁺²-chelate-Sepharose affinity column. Further the thermal stability of such EDTA-treated enzymes was decreased at 37 °C. Dot-blot studies involving ⁶⁵Zn⁺² further demonstrated the zinc binding property of AChE proteins. These results confirm the presence of Zn⁺²-binding site on AChE and further removal of metal from binding site with chelators resulted in loss of its catalytic function. Presence of metal-binding sequences HXXE…H in AChE, similar to many other metal containing enzymes supports our findings.     

Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis

Streptomyces naraensis was inoculated into 100 ml of culture broth, containing 50 µCi of ⁶⁵Zn, diluted with ZnCl₂ solution to make 10^-4 m Zn²⁺ ion, at 27°C for 5 days with shaking. ⁶⁵Zn-labeled neutral proteinase from Streptomyces naraensis was prepared by the method described previously. The preparation was homogeneous by disc electrophoresis and contained 1 g-atom of zinc per mole of enzyme in calculation by radioactivity.

It was suggested that the protein-bound zinc of neutral proteinase was not essential for enzymatic activity. Thus, this zinc was an essential component for the higher order structure of the protein, and the removal of zinc treated with EDTA* inactivated the enzyme. The enzymatic activity was maintained in the presence of calcium ion.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.     

Bio fertilizers and zinc effects on some physiological parameters of triticale under water-limitation condition

In order to study bio fertilizers and zinc effects on some physiological parameters of triticale under a water-limitation condition, a factorial experiment was conducted based on randomized complete block design with three replications in 2014 and 2015. Experimental factors consisted of three irrigation treatments [normal irrigation (W₀); moderate water limitation (W₁) and severe water limitation (W₂)]; four bio fertilizers’ levels [(no bio fertilizer (F₀), application of mycorrhiza (F₁), plant-growth-promoting rhizobacteria (PGPR) (F₂) and both application PGPR and mycorrhiza (F₃)] and four nano zinc oxide levels [(without nano zinc oxide (Zn₀) as control, application of 0.3 (Zn₁), 0.6 (Zn₂) and 0.9 (Zn₃) g?L^?1)]. Results showed that water limitation decreased chlorophyll content, relative water content, stomatal conductance, quantum yield and grain yield of triticale, whereas electrical conductivity and the activity of catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and polyphenol oxidase (PPO, EC 1.14.18.1) enzymes were increased. Inoculation of plants with bio fertilizers and zinc application improved these traits (except electrical conductivity) under water-limitation condition as well as normal irrigation. Based on the results, it was concluded that bio fertilizers and nano zinc oxide application can be recommended for profitable triticale production under water-limitation condition.