Noncooperative cadmium(II) binding to human metallothionein 1a

The two-domain (βα) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain βα-rhMT and the isolated β-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd₃-β-rhMT and Cd₇-βα-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd₄-β- and Cd₈-βα-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.     

The fate of Cd,Cu, Ca,Zn, and Fe in rat during the recovery period following cessation of repeated exposure to Cd

Cadmium was administered subcutaneously to male Wistar rats, 0.1 mL/rat in 0.9% saline 3 times a wk for 4 wk at 3 mg Cd/kg. Saline was administered to control animals in an equivalent manner, without Cd. After the end of the dosing period, the distribution and excretion of Cd, Cu, Ca, Zn, and Fe were observed in some organs and excreta for 35 d (1, 7, 14, 21, 28, and 35 d). Cadmium dosing caused significant disturbances in the metabolism of Zn, Cu, Fe, and Ca, especially during the recovery period. Growth in Cd-dosed animals did not accelerate, even after 5 wk of recovery. There was evidence of mobilization of some elements among organs. Accumulation of Cd occurred in liver, kidney, and spleen during dosing, and during the recovery period it was retained in kidney and testes (for 2 wk) and cleared steadily in liver and RBC (for 5 wk), but increased in spleen (first 3 wk). The pattern of Cd excretion was closely associated with the binding of Cd with metallothioneins in kidney and liver for the first 21 and 7 d, respectively. This was associated with the excretion of Cd-metallothioneins (Cd-MT) in urine from d 1 to 21 during recovery. Cadmium caused higher Ca accumulations in testes and liver, which were probably associated with the lesions observed in these organs. Significant increases of Cu (in kidney d 7) and Fe (in liver) were observed during recovery. Furthermore, significant reductions of Cu and Fe were found in plasma, spleen, and RBC (after 5 wk) and kidney, spleen, and testes (on d 7), and blood (after 5 wk).     

Xenopus lipovitellin 1 is a Zn2+- and Cd2+-binding protein

This report discusses the identification of a Zn²⁺- and Cd²⁺-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with ⁶⁵Zn²⁺ or ¹⁰⁹Cd²⁺. The resulting autoradiograms showed binding of both radio-nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (～115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley-Liss, Inc.     

Genetic differences in zinc and copper induction of liver metallothionein in inbred strains of the mouse

Differences in Zn-induced levels of hepatic metallothionein (MT) in inbred strains of the mouse are described. Three low-producing strains, C57BL/6, C57BL/10, and NIH, are identified, while C3H and CBA display the highest levels of hepatic MT following Zn treatment. These interstrain differences affect not only the level of MT protein, but also the amount of MT-bound Zn and the total hepatic Zn concentration. Both MT isoforms are equally affected. A similar interstrain difference following Cu treatment is present in C3H and C57BL/6. The origin of these interstrain differences is discussed.     

金属硫蛋白在心肌细胞保护中与抗氧化酶的关系

目的：探讨金属硫蛋白（MT）在心肌细胞缺氧／复氧损伤（H／R）和预缺氧（HRC）保护中与抗氧化酶的关系。方法：建立培养的乳鼠心肌细胞缺氧／复氧模型,检测HPC、锌诱导、外源MT和MT抗体等处理下,心肌细胞MT含量变化及抗氧化酶（SOD,CAT,GSHpx）活力的相应变化。结果：HPC和锌诱导组MT含量及抗氧化酶的活力均显著高于H／R组P＜0. 05、P＜0.01）：外源MT处理后SOD活力显著高于对照组（P＜0.01）,CAT和GSHpx活力虽然显著低于对照组但显著高于H／R组（P＜0.01）;使用MT抗体后,酶活力最低。结论：MT参与HPC的心肌细胞保护作用,并通过保护抗氧化酶的活力的机制减轻H／R所致的心肌损伤。     

Analogous copper(I) coordination in metallothionein from yeast and the separate domains of the mammalian protein

The three-dimensional structures of both vertebrate Cu12-metallothionein (class 1) and yeast Cu8-thionein (class 2) are still unknown. The different copper:protein stoichiometry compared with that of the (ZnCd)7-metallothioneins was expected to alter the metal-thiolate cluster structure considerably. In order to avoid possible domain interactions in the hepatic rat metallothionein, separate chemically synthesized alpha- and beta-domains were used rather than the apoprotein. Apo yeast thionein, and the alpha- and beta-domains of rat liver metallothionein-2 were reconstituted by Cu(I) titration. Reconstitution steps were monitored using spectroscopic methods including luminescence emission and circular dichroism. Upon UV irradiation a linear increase in intensity of the orange-red luminescence was observed near 600 nm up to 6 Cu eq using either compound regardless of the different cysteine sulfer content (yeast thionein 12S, alpha-domain 11S, beta-domain 9S). The characteristic dichroic properties of the yeast copper-protein between 240 and 400 nm were in good agreement with those of the respective class 1 metallothionein domains. All observed Cotton bands were of similar shape and appeared in the same wavelength regions. However, the molar ellipticities were less pronounced in the alpha- and beta-fragments employed. There appears to be a striking similarity between the oligonuclear Cu(I) binding centers in all metallothionein species.     

Influence of thyroparathyroidectomy and thyroxine replacement on CU and ZN cellular distribution and on the metallothionein level and induction in rats

Thyroid hormones are involved in copper and zinc distribution in rat tissues. We examined the influence of thyroparathyroidectomy (TPTY) and of a replacement therapy by T4 on Cu and Zn organ distribution. MT levels were also measured both in basal conditions and after induction by cadmium. The results confirm that a lack of T4 modified Cu and Zn in serum and tissues. In serum, TPTY increased Cu (+15%) and ceruloplasmin (+18%), and decreased Zn (−18%). In tissues, Cu was altered in liver (+13%), kidney (−24%), heart (−16%) duodenum (−18%), and Zn in liver (+25%) and kidney (−10%). The soluble fractions (100,000 g supernatant) were mainly affected in liver and kidney, and the subcellular fractions in heart and duodenum. MT levels were modified in basal conditions only in liver (+57%) and kidney (−36%). T4 administration partially prevented the effect of TPTY on both elements and MT concentrations. Therefore, no evidence is provided for a direct role of T4 in the metabolism of MT in a way comparable to the effects of glucocorticoids. However, MT could mediate the consequences of TPTY on metal distribution in certain organs, such as liver and kidney.     

Heavy metal intracellular balance and relationship with metallothionein induction in the gills of carp

Determination of metal levels (Cu, Zn, Cd, Ag, Hg) in soluble and insoluble fractions of gill homogenates has been performed after 7 d exposure of carp (Cyprinus carpio) to moderate concentrations of Cd, Ag, and Hg in water. Metallothionein levels have been quantified by polarographic method before and after contamination and a subsequent decontamination phase (7 d). The influence of pretreatment by zinc (7 d) has also been evaluated. Metallothionein level variations have been interpreted as having regard to interrelated flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in gill. Regardless of pretreatment, mercury gave the highest increase of gill MT, and after the decontamination MT level remained high compared to control. Cadmium and silver gave similar increases, but a significant difference with control appeared only after the decontamination step with silver, whereas 1 week of contamination was enough for cadmium. Our experimental conditions gave the following order of potency for MT induction in gill: Hg≫Cd>Ag>Zn.     

Role of thiocyanate ion in metallothionein induction and in endogenous distribution of essential elements in the rat liver

Thiocyanate is the major toxic metabolite of hydrogen cyanide, a toxic substance the organism may be exposed to as a result of cigarette smoking or industrial pollution. The complex interactions existing between metals and metallothionein induction are well known. However, the possible role of thiocyanate, which is also an anion, has not been established yet. Considering the interactions between metals and the metallothioneins, in this study the relationship between thiocyanate and the in vivo distribution of hepatic metallothionein and zinc, copper, iron, calcium, magnesium, and manganese are investigated in rats. This study implies that thiocyanate has, to some extent, an effect on the in vivo expression of metallothionein and endogenous distribution of essential elements in rat liver. Elevated levels of metallothionein and changes in hepatic concentrations of essential elements have suggested a role for thiocyanate in cellular metabolism and it might reflect a direct role of thiocyanate on alteration of cellular functional activities.     

Testicular toxicity induced by dietary cadmium is associated with decreased testicular zinc and increased hepatic and renal metallothionein and zinc in the bank vole <Emphasis Type="Italic">(Clethrionomys glareolus)</Emphasis>

Mechanism of testicular toxicity induced by dietary cadmium (Cd) has been less investigated than that following acute Cd injection. In the present study we characterized testicular injury in a small rodent, the bank vole, exposed subchronically to dietary Cd in a quantity of 0.9 mol/g, and determined the importance of some factors (Cd accumulation, metallothionein (MT), oxidative stress, and zinc (Zn)) in the injury. Dietary Cd induced moderate histopathological changes (hemorrhage in interstitium, necrosis and apoptosis in seminiferous tubule epithelium) in young (1 month old) bank voles fed, for 6 weeks, Fe-adequate (1.1–1.4 mol/g) and Fe-enriched (4.5–4.8 mol/g) diets. In contrast, adult (5 months old) bank voles appeared to be resistant to the toxic effects of dietary Cd, despite the fact that testicular Cd contents were higher and MT levels lower than those in the young animals. The Cd-induced histopathological changes and apoptosis were accompanied by increased testicular lipid peroxidation, decreased testicular Zn concentration and elevated levels of hepatic and renal MT and Zn. Supplemental dietary Zn (1.7–1.8 mol/g) prevented the Cd-induced testicular Zn depletion and injury. The data indicate that dietary Cd produces testicular lesions indirectly, through decreasing testicular Zn, which seems to be due to the sequestration of this element by the Cd-induced hepatic and renal MT.     

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

Enhancing Safety,Security, and Environmental Protection of the Straits of Malacca and Singapore: The Cooperative Mechanism

In November 2004, the International Maritime Organization, as part of its Protection of Vital Shipping Lanes Initiative, decided to convene a high-level conference to address the security of ships plying the Straits of Malacca and Singapore in collaboration with the three littoral states of Indonesia, Malaysia, and Singapore. Three meetings were held over the period 2005 to 2007. The outcome was the creation of a framework for cooperation between the users of the straits and the littoral states known as the Cooperative Mechanism, a historic breakthrough as Article 43 of the Law of the Sea Convention was implemented for the first time. This article outlines the decisions made at the three meetings, analyzes the responses of the various stakeholders during the meetings, explains the reasons for the success of the Cooperative Mechanism, and concludes by proposing ways in which the momentum of cooperation between the users and the littoral states could be sustained.     

Partitioning of Zn,Cd, Pb,and Cu in organic-rich soil profiles in the vicinity of a zinc smelter

Abstract

A five-step sequential extraction procedure was applied to organic-rich soil samples from five soil profiles situated 1–8 km from a zinc smelter. The partitioning of Zn, Cd, Pb, and Cu into five operationally defined fractions (exchangeable, “carbonate’’-bound, reducible, oxidizable, and residual) was studied at different soil depths down to 35cm. In the surface soil (0–1 cm) a major part of Pb and Cu was extracted in the oxidizable fraction, whereas for Zn and Cd slightly more was extracted in the ‘‘carbonate”-fraction than in the other four fractions. Extracted metal proportions in the oxidizable fraction were respectively of the order of 30%, 20%, 50%, and 80% for Zn, Cd, Pb, and Cu in the surface soil for all sites, but these proportions decreased with soil depth. In the surface soil less than 20% of all the elements were extracted in the residual fraction, but the proportions associated with this fraction generally increased with soil depth. In the C-horizon, differences in extracted proportions of Pb and Cu in the residual fraction were probably due to geochemical factors, whereas for Zn the low extracted proportion at a highly contaminated site (20%) may be due to Zn migration to the C-horizon at this site. For Cd the extracted proportions in the C-horizon were lower than for the other elements, generally below 20%, presumably because Cd is weaker in terms of its adsorption to the soil than the other elements studied. Total concentrations of the metals decreased strongly with increasing distance from the smelter, but less systematic differences were observed for their distributions among fractions. Potentially bioavailable metal proportions (exchangeable + “carbonate”-bound fraction) in the surface soil were about 50%, 60%, 20%, and 10% for Zn, Cd, Pb, and Cu, respectively. In C-horizon soil the mobility sequence Cd>Zn>Pb = Cu was generally observed. The present results indicate that the concentrations and chemical fractionation of Zn, Pb, and Cd in these soils represent a considerable risk to natural terrestrial food chains.     

Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.     

Effects of interaction between65Zn,cadmium, and copper in rats

Distribution and retention of zinc in the presence of cadmium and copper was studied in rats exposed repeatedly to these metals. The experiment was performed on white rats of the Wistar strain. The animals were divided into four groups/five rats each: 1)⁶⁵ZnCl₂; 2)⁶⁵ZnCl₂+CdCl₂; 3)⁶⁵ZnCl₂+CuCl₂; and 4) control group. Rats were administered sc every other day for two weeks:⁶⁵ZnCl₂−5 mg Zn/kg; CdCl₂−0,3 Cd/kg; and CuCl₂−2 mg Cu/kg. The zinc content was measured in rat tissues by γ-counting. Effect of Cd and Cu on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein were also examined. Whole body retention of zinc under the influence of cadmium was lower than that observed in animals treated with zinc alone. However, copper increased twofold the whole body retention of zinc. Cadmium elevated the accumulation of zinc only in the kidneys nuclear fraction and liver soluble fraction. In the kidneys and liver, copper elevated the accumulation of zinc, in the nuclear, mitochondrial, and soluble fractions. The level of metallothionein-like proteins (MT) in the kidneys after a combined supply of zinc and copper was significantly increased with respect to the group of animals treated with zinc alone. These results indicated complex interactions between cadmium, copper, and zinc that can affect the metabolism of each of the metals.