Inhibitory Effect Of Zn Research Articles

Evaluating interactive toxic impact of heavy metals and variations of microbial community during fermentative hydrogen production

This study investigated the stimulating or inhibitory effect of Zn, Cu, Cd, Pb, and their mixtures on fermentative hydrogen production. Heavy metals inhibited the production process at all concentrations except 0.50 ppm of Zn. Consistent with the half of the maximum inhibitory concentration (IC50), Cu + Pb caused the greatest inhibition as it is more toxic than other heavy metal mixtures. As the concentration of heavy metal increased, the HBu/HAc ratio (butyrate/acetate) tended to decrease and the amount of hydrogen production decreased. Clostridium sp., Klebsiella sp., Dysgonomas sp., Enterobacter sp., and other hydrogen-producing bacteria were observed by polymerase chain reaction (PCR) – analysis by denaturing gradient gel electrophoresis (DGGE) during the fermentative hydrogen process. A significant relationship was observed between the richness index and the hydrogen production (p-value = 0.01 < 0.05) from the Pielou index. The Pielou index (E′) and the Shannon-Weaver index (H’) had no significant relationship (p-value = 0.12 > 0.05).

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations.

Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

PH-dependence in the inhibitory effects of Zn2+and Ni2+ on tolaasin-induced hemolytic activity

Pseudomonas tolaasii에 의해 분비되는 톨라신은 펩티드 독소로서, 버섯 자실체 구조와 세포를 파괴하여 갈반병을 일으킨다. 톨라신의 독성은 용혈활성을 측정함으로서 평가하며, 이는 톨라신 분자가 적혈구 막에 pore를 형성하여 세포 구조를 파괴하기 때문이다. 이전 연구에서, Zn2+ 뿐만 아니라 Ni2+이 톨라신의 세포독성에 억제효과를 가짐을 확인하였다. Ni2+은 농도가 증가함에 따라 톨라신에 의한 용혈작용을 저해하였으며, 이의 Ki 값은 1.8 mM이었다. 용혈활성은 10 mM 이상의 농도에서 완전히 저해되었다. Ni2+의 효과는 pH에 따라 크게 변하지 않았으나, Zn2+의 톨라신 세포독성 억제 효과는 염기성 pH에서 크게 증가하였다. 완충액의 pH를 7에서 9로 증가시키면, 50% 용혈작용이 일어나는 시간인 T50은 1 mM Ni2+에 의해 조금 증가하였으나 100 μM Zn2+에서는 크게 증가하였다. Zn2+와 Ni2+을 반응용액에 동시에 처리하였을 때, 두 양이온의 상승효과는 모든 pH에서 나타났다. 서로 다른 pH 의존성을 보이는 두 금속이온의 분자적 설명은 톨라신의 pore 형성과 세포 독성에 관한 기작의 이해에 기여할 것이다.

Speciation of nickel and zinc, its short-term inhibitory effect on anammox, and the associated microbial community composition

This study provides insight into the short-term effects of nickel and zinc on anammox. The impacts of these heavy metals are evaluated based on their potentially bioavailable fractions, including the intracellular, surface-bound, soluble, free-ion, and weak (labile) complexes of heavy metals, in the presence of certain inorganic/organic species. Results showed that the IC50 values for soluble, intracellular, cell-associated, surface-bound, and free-ion Ni concentrations are 5.99, 0.250, 0.930, 0.680, and 1.36 mg/L, respectively. The inhibitory effect of Zn is found to be lower with respect to Ni, with IC50 values of 6.76, 11.9, 15.1, and 2.71 mg/L for the soluble, intracellular, cell-associated, and free-ion Zn concentrations, respectively. This is the first detailed evaluation of anammox inhibition based on the fractionation of heavy metals. Metagenomic analysis reveals that Candidatus Kuenenia constitute approximately 89% of the entire Planctomycetes population, whereas Candidatus Brocadia are detected in relatively low fractions (3%).

Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn2+ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn2+ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the Ki value of 1.4μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn2+ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.

The effects of metal ions in Euphorbia cf. lactea latex on the fibrinogenolytic activity of a plant protease.

Two synchrotron-based techniques, synchrotron X-ray fluorescence (SXRF) and X-ray absorption spectroscopy (XAS), have demonstrated that Ca(2+) and Zn(2+) were the major metal ions distributed in the natural latex of Euphorbia cf. lactea. Both metal ions were found to affect the fibrinogenolytic activity of a homodimeric protease purified from the latex of this plant. The dimeric protein had an estimated molecular mass of about 82 kDa analyzed by SDS-PAGE. Therefore this protein was called as EuP-82. Based on the results of circular dichroism (CD) spectroscopy and the fibrinolytic activity measurement, it was found that Ca(2+) could activate the proteolytic activity of the enzyme by stabilizing its backbone structure. The intact conformation of EuP-82 was predicted from CD spectrum, which consisted of 51 % α-helix and 9 % β-sheet. Zn(2+) (10 mM) could decrease the fibrinolytic activity of EuP-82 to 30 ± 1 %. CD spectrum also supported that the inhibitory effect of Zn(2+) on the enzyme activity occurred by the drastic change of the enzyme structure with increasing the random coil conformation and by switching between α-helix and β-sheet structure. These results could be of first importance for further application to use EuP-82, the natural source protease as a potential drug for the thrombosis treatment. The fibrinolytic activity of EuP-82 may be enhanced by plasma Ca(2+) which generally involves in human hemostasis system.

Diethylpyrocarbonate modification reveals HisB5 as an important modulator of insulin amyloid formation.

More than 30 amyloid proteins are reported to be associated with amyloidosis diseases. Studies have implicated histidine may be critically involved in amyloid formation. Here, we used diethylpyrocarbonate (DEPC) modification to obtain a His(B5) mono-ethyloxyformylated insulin (DMI-B(5)). The secondary structure, amyloidogenicity, metal ion interaction, and cytotoxicity of DMI-B(5) and insulin were compared. DMI-B(5) was less prone to aggregation in acidic condition but easier to aggregate at neutral pH. DEPC modification resulted in attenuated inhibitory effect of Zn(2+) on aggregation, whereas DMI-B(5) fibrils induced more severe erythrocytes haemolysis compared to insulin fibrils. This study not only provides a fast new approach for studying the impact of imidazole ring in amyloid formation, but also reveals the critical modulating role of histidine imidazole ring on the amyloidogenicity of insulin.

Does zinc in livestock wastewater reduce nitrous oxide (N2O) emissions from mangrove soils?

Zinc (Zn) affects nitrogen cycling but the effect of Zn in wastewater on the emission of nitrous oxide (N2O) from the soil has not been reported. This study compared N2O emissions from mangrove soil receiving livestock wastewater containing various Zn2+ concentrations and evaluated how long the effects of Zn would last in these soil-wastewater microcosms. Significant increases in N2O flux were observed soon after the discharge of wastewater with a low Zn content. On the other hand, the flux was reduced significantly in the wastewater with high Zn levels but such inhibitory effect was not observed after tidal flushing. Continuous monitoring of the N2O fluxes also confirmed that the inhibitory effect of Zn was confined within a few hours and the fluxes recovered in 6–9 h after the wastewater was completely drained away. These results indicated that the inhibitory effect of Zn on N2O fluxes occurred immediately after wastewater discharge and disappeared gradually. In the surface soil, nitrate levels increased with the addition of wastewater but there was no significant accumulation of NH4+−N, irrespective of the Zn content in the wastewater. The study also showed that nitrification potential and immediate N2O emissions were inhibited by high Zn levels in the soil, but the total oxidation of ammonium to nitrate was not affected.

Hypoxia Limits Inhibitory Effects of Zn2+ on Spreading Depolarizations

Spreading depolarizations (SDs) are coordinated depolarizations of brain tissue that have been well-characterized in animal models and more recently implicated in the progression of stroke injury. We previously showed that extracellular Zn2+ accumulation can inhibit the propagation of SD events. In that prior work, Zn2+ was tested in normoxic conditions, where SD was generated by localized KCl pulses in oxygenated tissue. The current study examined the extent to which Zn2+ effects are modified by hypoxia, to assess potential implications for stroke studies. The present studies examined SD generated in brain slices acutely prepared from mice, and recordings were made from the hippocampal CA1 region. SDs were generated by either local potassium injection (K-SD), exposure to the Na+/K+-ATPase inhibitor ouabain (ouabain-SD) or superfusion with modified ACSF with reduced oxygen and glucose concentrations (oxygen glucose deprivation: OGD-SD). Extracellular Zn2+ exposures (100 µM ZnCl2) effectively decreased SD propagation rates and significantly increased the initiation threshold for K-SD generated in oxygenated ACSF (95% O2). In contrast, ZnCl2 did not inhibit propagation of OGD-SD or ouabain-SD generated in hypoxic conditions. Zn2+ sensitivity in 0% O2 was restored by exposure to the protein oxidizer DTNB, suggesting that redox modulation may contribute to resistance to Zn2+ in hypoxic conditions. DTNB pretreatment also significantly potentiated the inhibitory effects of competitive (D-AP5) or allosteric (Ro25-6981) NMDA receptor antagonists on OGD-SD. Finally, Zn2+ inhibition of isolated NMDAR currents was potentiated by DTNB. Together, these results suggest that hypoxia-induced redox modulation can influence the sensitivity of SD to Zn2+ as well as to other NMDAR antagonists. Such a mechanism may limit inhibitory effects of endogenous Zn2+ accumulation in hypoxic regions close to ischemic infarcts.

The GLRA1 Missense Mutation W170S Associates Lack of Zn2+Potentiation with Human Hyperekplexia

Hyperekplexia is a neurological disorder associated primarily with mutations in the α1 subunit of glycine receptors (GlyRs) that lead to dysfunction of glycinergic inhibitory transmission. To date, most of the identified mutations result in disruption of surface expression or altered channel properties of α1-containing GlyRs. Little evidence has emerged to support an involvement of allosteric GlyR modulation in human hyperekplexia. Here, we report that recombinant human GlyRs containing α1 or α1β subunits with a missense mutation in the α1 subunit (W170S), previously identified from familial hyperekplexia, caused remarkably reduced potentiation and enhanced inhibition by Zn(2+). Interestingly, mutant α1(W170S)β GlyRs displayed no significant changes in potency or maximum response to glycine, taurine, or β-alanine. By temporally separating the potentiating and the inhibitory effects of Zn(2+), we found that the enhancement of Zn(2+) inhibition resulted from a loss of Zn(2+)-mediated potentiation. The W170S mutation on the background of H107N, which was previously reported to selectively disrupt Zn(2+) inhibition, showed remarkable attenuation of Zn(2+)-mediated potentiation and thus indicated that W170 is an important residue for the Zn(2+)-mediated GlyR potentiation. Moreover, overexpressing the α1(W170S) subunit in cultured rat neurons confirmed the results from heterologous expression. Together, our results reveal a new zinc potentiation site on α1 GlyRs and a strong link between Zn(2+) modulation and human disease.

Removal of nickel and zinc from single and binary metal solutions by Sargassum angustifolium.

The capability of Sargassum angustifolium in removing nickel (Ni) (II) and zinc (Zn) (II) from single and binary metal solution was determined. In binary solution the presence of the secondary metal inhibited the sorption of the primary metal by S. angustifolium but the inhibitory effect of Ni during sorption of Zn is stronger than the inhibitory effect of Zn in absorption of Ni. The total metal (Ni + Zn) sorbed from the binary metal solution by S. angustifolium cells always remained lower than the total sorption of individual metals from their respective single metal solutions, thereby suggesting competition between Ni(II) and Zn(II) for the common binding sites on S. angustifolium. The maximum uptake capacities of the S. angustifolium, which was collected near Bushehr, Persian Gulf, Iran in the natural form, at the optimal conditions for Ni(II) and Zn(II) ions in single metal solutions were approximately 0.71 and 0.93 mmol/g dry S. angustifolium, respectively. Under the binary system Ni(II) and Zn(II) uptake capacities were 0.41 mmol Ni/g and 0.36 mmol Zn/g, respectively. Better fitness of equilibrium metal sorption data to the Langmuir than the Freundlich model suggests multilayer adsorption of test metals onto the cell surface.

Inhibitory effect of Zn2+ on α-glucosidase: Inhibition kinetics and molecular dynamics simulation

α-Glucosidase (EC 3.2.1.20) is a critical enzyme with clinical relevance to type 2 diabetes mellitus. Therefore, research on this enzyme's inhibition is important. In the present study, we investigated Zn2+-induced inhibition and the structural changes of α-glucosidase. α-Glucosidase activity was significantly inhibited by Zn2+ in a dose-dependent manner. The inhibition followed a multi-phase kinetic process with a first-order reaction. Zn2+ inhibited α-glucosidase in a parabolic mixed-type reaction (Ki=0.102±0.001mM) and directly induced the unfolding of α-glucosidase, resulting in a slight hydrophobic exposure. We also performed 10ns molecular dynamics simulations on α-glucosidase and Zn2+. The simulations suggest that ten Zn2+ ions possibly interact with 57 α-glucosidase residues. The molecular dynamics simulations also confirmed the binding mechanism of Zn2+ to α-glucosidase and suggest that the Zn2+ binding sites are not located in the glucose binding pocket of α-glucosidase. Our study provides insights into the mechanism of Zn2+-induced unfolding of α-glucosidase and inhibition of ligand binding and suggests that Zn2+ could act as a potent inhibitor of α-glucosidase for the treatment of type 2 diabetes mellitus.

Steric Hindrance Mutagenesis in the Conserved Extracellular Vestibule Impedes Allosteric Binding of Antidepressants to the Serotonin Transporter

The serotonin transporter (SERT) controls synaptic serotonin levels and is the primary target for antidepressants, including selective serotonin reuptake inhibitors (e.g. (S)-citalopram) and tricyclic antidepressants (e.g. clomipramine). In addition to a high affinity binding site, SERT possesses a low affinity allosteric site for antidepressants. Binding to the allosteric site impedes dissociation of antidepressants from the high affinity site, which may enhance antidepressant efficacy. Here we employ an induced fit docking/molecular dynamics protocol to identify the residues that may be involved in the allosteric binding in the extracellular vestibule located above the central substrate binding (S1) site. Indeed, mutagenesis of selected residues in the vestibule reduces the allosteric potency of (S)-citalopram and clomipramine. The identified site is further supported by the inhibitory effects of Zn(2+) binding in an engineered site and the covalent attachment of benzocaine-methanethiosulfonate to a cysteine introduced in the extracellular vestibule. The data provide a mechanistic explanation for the allosteric action of antidepressants at SERT and suggest that the role of the vestibule is evolutionarily conserved among neurotransmitter:sodium symporter proteins as a binding pocket for small molecule ligands.

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC50 of 278.57μM, 0.35μM, 41.56μM and 198.86μM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn2+) in a concentration-dependent manner with an IC50 value of 275μM, and the inhibitory effect of Zn2+ was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by ∼34%; thus, the vaccination trial was ∼73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Decreased Dissolution of ZnO by Iron Doping Yields Nanoparticles with Reduced Toxicity in the Rodent Lung and Zebrafish Embryos

We have recently shown that the dissolution of ZnO nanoparticles and Zn(2+) shedding leads to a series of sublethal and lethal toxicological responses at the cellular level that can be alleviated by iron doping. Iron doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1-10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium, and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung, and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release, and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn(2+) on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe doping was associated with decreased polymorphonuclear cell counts, LDH, and albumin levels. All considered, the above data show that Fe doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment.

Zn(II) ions co-secreted with insulin suppress inherent amyloidogenic properties of monomeric insulin

Insulin, a 51-residue peptide hormone, is an intrinsically amyloidogenic peptide, forming amyloid fibrils in vitro. In the secretory granules, insulin is densely packed together with Zn(II) into crystals of Zn(2)Insulin(6) hexamer, which assures osmotic stability of vesicles and prevents fibrillation of the peptide. However, after release from the pancreatic beta-cells, insulin dissociates into active monomers, which tend to fibrillize not only at acidic, but also at physiological, pH values. The effect of co-secreted Zn(II) ions on the fibrillation of monomeric insulin is unknown, however, it might prevent insulin fibrillation. We showed that Zn(II) inhibits fibrillation of monomeric insulin at physiological pH values by forming a soluble Zn(II)-insulin complex. The inhibitory effect of Zn(II) ions is very strong at pH 7.3 (IC(50)=3.5 microM), whereas at pH 5.5 it progressively weakens, pointing towards participation of the histidine residue(s) in complex formation. The results obtained indicate that Zn(II) ions might suppress fibrillation of insulin at its release sites and in circulation. It is hypothesized that misfolded oligomeric intermediates occurring in the insulin fibrillation pathway, especially in zinc-deficient conditions, might induce autoantibodies against insulin, which leads to beta-cell damage and autoimmune Type 1 diabetes.

Zinc ions bind to and inhibit activated protein C

Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible approximately 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 +/- 1.5 microM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

Effects of Tolaasin Inhibitory Factors on Tolaasin Peptide Channel Evaluated by Competition with Zn2+

Tolaasin is a 1.9 kDa bacterial lipodepsipeptide toxin and forms membrane pores causing brown blotch disease on the cultivated mushrooms. Molecular multimerization of tolaasin is required to form membrane channel. Previously, we showed that various chemicals inhibit the multimerization of tolaasins and thus prevent from brown blotch disease. These chemicals named tolaasin-inhibitory factor (TIF) were applicable to control the disease. Various candidates were chosen from different food additives and they were successful for disease control at 1-100 μM. Interestingly, after tolaasin molecules formed channels on the erythrocyte membrane and hemolysis started, further hemolyses were stopped as soon as TIF's were added. The added TIF was not washed by centrifugation and addition of fresh HBS solution. These result imply that TIF's are able to inhibit the preformed tolaasin channels. TIF may inhibit tolaasin-induced hemolysis either by plugging the channel pores or by the dissociation of tolaasin multimers. Zn2+ is a potent tolaasin inhibitor known to bind to tolaasin channel. In order to characterize the TIF-induced inhibition, competition effect of Zn2+ and TIF on tolaasin channel activity was investigated. When Zn2+ and TIF-9 were added simultaneously, no additive effects were observed at various concentration combinations. However, in the combinations of Zn2+ plus either TIF-11 or TIF-16, no additive effect was measured and the inhibitory effect of Zn2+ was reduced in the presence of low concentration of Zn2+. In these experiments, the inhibition of hemolysis was dominated by Zn2+concentration. Based on these results, we suggest that TIF has high affinity to tolaasin channel than Zn2+ although Zn2+ is a strong inhibitor.

Zinc Binding to Protein C and Activated Protein C Modulates Their Interaction with Endothelial Cell Protein C Receptor.

Abstract 331▪▪This icon denotes an abstract that is clinically relevant. Introduction/background:Zinc is a multi-functional element that is essential for life and the second most abundant metal ion, after iron in eukaryotic organisms. Zinc deficiency has been associated with bleeding disorders and with defective platelet aggregation, suggesting it may play an important role in hemostasis. Zinc ions have been shown to enhance activation of the intrinsic pathway of coagulation but to down-regulate the extrinsic pathway of coagulation. All vitamin K-dependent coagulation proteins have calcium binding sites and may therefore to some extent, interact with other divalent metal ions, including zinc, through these sites. Recent crystallography studies identified a pair of Zn2+ binding sites in FVIIa protease domain, and with the exception of Glu220, all the side chains involved in both the Zn1 and Zn2 coordination in FVIIa are unique to FVIIa and are not present in other vitamin K-dependent clotting factors (Bajaj et al., J Biol Chem 2006; 281:24873-88). Nonetheless, Zn2+ may bind to other vitamin K-dependent clotting factors at sites different from those identified in FVIIa. Objective:The aim of the present study is to investigate the effect of zinc ions on the protein C pathway, particularly on protein C/APC binding to EPCR, protein C activation and APC catalytic activity. Methods:Protein C and APC binding to EPCR on endothelial cells was examined by radioligand binding studies. Protein C activation and APC catalytic activity were evaluated in chromogenic assays. Equilibrium dialysis was used to measure zinc binding to protein C/APC. Conformational changes in protein C/APC were monitored by intrinsic fluorescence quenching. Results:Zn2+ does not replace the Ca2+ as a mandatory cofactor for protein C/APC binding to EPCR but Zn2+ at physiologically relevant concentrations (10 to 25 μM) markedly increased Ca2+-dependent protein C and APC binding to EPCR (∼2 to 5-fold). The kinetic analysis of protein C and APC binding to EPCR suggested that Zn2+ enhanced protein C/APC binding to EPCR by increasing the binding affinity of protein C/APC to its receptor (Kd for APC: – Zn2+, 117 ± 27 nM; + Zn2+, 9.3 ± 3.3 nM; Kd, for protein C: – Zn2+, 96 ± 26 nM; + Zn2+, 21.4 ± 6.6 nM). The enhancing effect of Zn2+ on APC binding to EPCR was also observed in the presence of physiological concentrations of Mg2+, which itself increased the APC binding to EPCR, two-fold. Zn2+-mediated increased protein C binding to EPCR resulted in increased APC generation. The effect of Zn2+ was not limited to enhancing protein C and APC binding to EPCR but also affected the catalytic activity of APC. Zn2+ inhibited the amidolytic activity of APC half-maximally at 50 to 100 μM. Zn2+ also inhibited the amidolytic activity of Gla domain deleted (GD)-APC in a similar fashion. The inhibitory effect of Zn2+ was partially reversed by physiological concentrations of calcium. Addition of Zn2+ to protein C or APC quenched the intrinsic fluorescence of both APC and GD-APC. Data from the equilibrium binding studies performed with 65Zn2+ revealed that Zn2+ binds to both GD-APC and APC, but that the amount of Zn2+ bound to APC was 3 to 4-fold higher than the amount bound to GD-APC. Kinetic analysis of equilibrium binding studies suggested that two Zn2+ atoms bind to APC outside the Gla domain with relatively high affinity (∼70 μM). At least one of the Zn2+ sites may overlap with the Ca2+ binding site as the Zn2+ binding to GD-APC was inhibited by approximately 50% by saturating concentrations of Ca2+. The substantially increased Zn2+ binding to the APC compared to GD-APC suggested that the N-terminus of the Gla domain of protein C contains multiple Zn2+ binding sites. Interestingly, Zn2+ bound to APC and GD-APC with a similar high affinity suggesting that the Gla domain, as well as the protease domain, may contain high affinity binding sites for Zn2+. A majority of the Zn2+ binding sites in the Gla domain appear to be distinct from the Ca2+ binding sites as less than 40% of the maximal Zn2+ binding could be blocked by Ca2+. The putative zinc binding sites in protein C/APC appeared to be unique as no consensus canonical zinc binding sequences homologous to other known zinc binding proteins were found in protein C. Conclusions:Our present data show that Zn2+ binds to protein C/APC and induces a conformational change in these proteins, which in turn leads to higher affinity binding to their cellular receptor EPCR. Overall our results suggest that zinc ions may play an important regulatory role in the protein C pathway. Disclosures:No relevant conflicts of interest to declare.

Zn(II)‐ and Cu(II)‐induced non‐fibrillar aggregates of amyloid‐β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.