Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.     

Cloning, expression and characterization of metallothionein from the Antarctic clam Laternula elliptica

The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT.     

Antioxidant effect of zinc and zinc-metallothionein in the acute cytotoxicity of hydrogen peroxide in Ehrlich ascites tumour cells

This study was concerned with the role of zinc (Zn) and zinc-metallothionein (Zn-MT) in oxidative stress. Hydrogen peroxide-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from control host mice, mice pretreated with 10 mg/kg ZnSO4 (i.p.) to increase cellular Zn/Zn-MT levels, and mice exposed to Zn-deficient diet to reduce the cellular Zn/Zn-MT levels. The results of the present study showed that Ehrlich cells with seven-fold differences in Zn-MT concentrations could be obtained by manipulating the Zn status of host mice and that high Zn and Zn-MT levels can make Ehrlich cells more resistant to H2O2-induced oxidative injury (cell viability, lipid peroxidation, [Ca2+]i) while cells with reduced Zn/Zn-MT levels were more susceptible to this treatment. H2O2 treatment resulted in oxidation of MT thiolate groups and loss of its metal binding capacity with translocation of Zn released from oxidized MT to other cellular sites. Preincubation of Ehrlich cells with ZnSO4 in vitro also conferred some degree of resistance to H2O2 toxicity, suggesting the inherent antioxidative property of Zn ions. These data suggested that Zn-MT can be considered as an antioxidant by virtue of its thiolate groups and its Zn ions that are released in the presence of oxidative stress.     

蛹虫草金属硫蛋白分离纯化及性质研究

研究探讨锌离子胁迫下蛹虫草Cordyceps militaris金属硫蛋白的产生及性质。蛹虫草菌丝体以15g/L Zn2+在10L发酵罐中诱导培养56h后收集,产率为每升发酵液收集12.021g菌丝体（干重）,细胞破碎取上清液通过两次凝胶柱层析,冷冻干燥得到蛹虫草金属硫蛋白纯品。利用Bradford法进行蛋白质含量测定,用银饱和分析法结合原子吸收光谱（AAS）测定MT含量,发酵终点处金属硫蛋白含量为12.876mg/g菌丝体（湿重）。用电喷雾质谱法测得金属硫蛋白的分子量为7 390Da,用Ellman’s方法和火焰原子吸收法分别测得每分子蛋白质含有14个巯基、结合5个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含57个氨基酸,其中含有13个半胱氨酸,疏水氨基酸占29.8%,且含有组氨酸。以上表明,研究中的蛹虫草金属硫蛋白与哺乳动物金属硫蛋白结构差异较大,但与酵母菌金属硫蛋白结构组成类似。     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

Observations on the use of a cation exchange resin for the preparation of metal-depleted media for lymphocyte culture

A chelating resin specific for divalent cations (Chelex) was used to prepare metal-depleted media for lymphocyte culture. A batch procedure (resin in pH 7.4 phosphate buffer/specimen, 1:1) removed 70-80% of iron, 77-87% of copper and 88-98% of zinc, calcium and magnesium. At variance with other reports, when a resin/specimen ratio of 1:4 was used, iron chelation decreased to 40%, whereas other cation chelation remained unchanged. Best chelation for iron and calcium was obtained at pH 5-6.4; for copper, zinc and magnesium, at pH 7.4-8.0. During the procedure protein content decreased by 8-10%; arginine and lysine by 80%; asparagine, cystine, tyrosine and phenylalanine by 60%, other amino acids by 35%. These new data suggest that cation-depleted media prepared with Chelex may be used to study the effects of cations on lymphocytes in culture, provided that the most appropriate pH and resin/specimen ratio are selected and adequate amino acid replacement is performed. Results on normal human lymphocytes are reported.     

Degradation of zinc-metallothionein in monolayer cultures of rat hepatocytes

The degradation of zinc-metallothionein (MT) was studied in monolayer cultures of adult rat hepatocytes. Hepatocytes were incubated overnight in serum-free medium containing either [35S]cysteine or [3H]leucine and 100 microM zinc to induce MT synthesis. Total cellular 35S-MT was measured in the heat-stable extract of cell homogenate and quantified by fast protein liquid chromatography. When zinc was removed from the medium, 35S-MT turnover was almost 3-fold faster than that of [3H]Leu protein (t1/2 = 11 and 29 hr, respectively). The decrease in the cellular level of 35S-MT reflected degradation since less than 1% of total cellular 35S-MT was secreted into the medium. The rate of MT degradation was inversely proportional to cellular zinc content. In contrast, the degradation of [3H]Leu protein was not affected by changes in cellular zinc concentration. Chloroquine, a lysosomotrophic amine, and tosyl lysine chloromethyl ketone, an inhibitor of trypsin-like neutral protease activity, inhibited 35S-MT degradation by 65% and 50%, respectively, when cells were incubated in medium with 1 microM zinc. Turnover of [3H]Leu protein, but not 35S-MT, was enhanced by insulin deprivation. These data suggest that the degradation of hepatic MT (i) is primarily regulated by cellular zinc content and (ii) occurs in both lysosomal and nonlysosomal compartments.     

Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression.

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 microM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.     

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.     

Protective role of zinc-metallothionein (Zn-MT) in iron nitrilotriacetate (Fe-NTA)-induced renal oxidative damage

Several studies have shown the role of thiol-rich proteins especially metallothionein (MT) in the therapeutic interventions against oxidative damage. Previously, we have provided strong evidence for the involvement of ROS in iron nitrilotriacetate (Fe-NTA)-induced renal toxicity, which may have relevance to its carcinogenicity. The purpose of this study was to evaluate the role of zinc metallothionein (Zn-MT) on the protection against Fe-NTA-induced renal oxidative damage. The results demonstrate that Zn-MT pretreatment provided protection against Fe-NTA-induced mortality in mice (40% protection). Similarly, Zn-MT pretreatment also provided protection against Fe-NTA-induced lipid peroxidation (26% inhibition, P < 0.001). It is proposed that Zn-MT protects kidney tissue against the noxious effect of Fe-NTA primarily by interference with lipid peroxides. It is concluded that Zn-MT may serve as an excellent physiological antioxidant against Fe-NTA-mediated renal oxidative damage.     

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.     

Counterpoint,patronage and professionalism

Abstract

Several studies have shown the role of thiol-rich proteins especially metallothionein (MT) in the therapeutic interventions against oxidative damage. Previously, we have provided strong evidence for the involvement of ROS in iron nitrilotriacetate (Fe-NTA)-induced renal toxicity, which may have relevance to its carcinogenicity. The purpose of this study was to evaluate the role of zinc metallothionein (Zn-MT) on the protection against Fe-NTA-induced renal oxidative damage. The results demonstrate that Zn-MT pretreatment provided protection against Fe-NTA-induced mortality in mice (40% protection). Similarly, Zn-MT pretreatment also provided protection against Fe-NTA-induced lipid peroxidation (26% inhibition, P < 0.001). It is proposed that Zn-MT protects kidney tissue against the noxious effect of Fe-NTA primarily by interference with lipid peroxides. It is concluded that Zn-MT may serve as an excellent physiological antioxidant against Fe-NTA-mediated renal oxidative damage.     

Rates of metallothionein synthesis, degradation and accretion in a chicken macrophage cell line

To understand the regulation of metallothionein (MT) accretion in a chicken-macrophage cell line, fractional rates of MT synthesis (FRS) and degradation (FRD) were measured by following decay kinetics of [35S]cysteine in MT. To obtain valid measurements, we added various amounts of cysteine to medium to ensure that the isotope tracer was adequately diluted after MT was labeled in the presence of various levels of zinc. We also demonstrated that the measured fractional rate of MT accretion closely approximated the difference between FRS and FRD. All fractional rates were similar for the two MT isoforms isolated. FRD did not change during the 27-hr decay period, but FRS and fractional rate of MT accretion changed over time. FRS of MT was 0.097 and 0.012 hr-1 from 0 to 9 and 9 to 27 hr, respectively, after cells were incubated in medium supplemented with 50 microM zinc. FRD of MT was 0.020 hr-1. Addition of 1100 microM unlabeled cysteine to medium supplemented with 50 microM zinc increased FRD and decreased FRS and fractional rate of MT accretion, as compared with not adding cysteine. Overall, these results indicate that rates of MT synthesis and degradation can both regulate MT accretion. Further experiments with various amounts of zinc and cysteine added to medium suggested that the effect of added cysteine on MT fractional rates was due to chelation of unbound zinc. Elimination of the cysteine effect on MT fractional rates was accomplished by adding more zinc to cysteine-supplemented medium. Thus, the concentration of unbound zinc affects the rates of MT synthesis and degradation.     

Effect of ethanol on troponin calcium binding

The Chelex resin method was found to be suitable for studying drug effects on Ca2+ binding of proteins. In comparison to conventional dialysis techniques, the Chelex method has the following advantages: Ca2+-EGTA buffer is not necessary, free Ca2+ concentration as low as 10(-9) M can be determined directly, and the reaction is complete in 30 min, thus creating fewer problems with protein denaturation at elevated temperatures. Methods to cope with problems inherent to this assay, such as the excluded volume effect of the resin and protein adsorption by the resin are described. The validity of the method was confirmed by the measurements of Ca2+ binding of troponin in the presence and absence of Mg2+. Using this method, it was demonstrated that ethanol concentration as high as 25% does not influence the Ca2+ binding of troponin.     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

The search for an 111In labeled agent for the solid component of gastric emptying

¹¹¹In-labeled solid meal was prepared by chelation of ¹¹¹In with Chelex resin bead. The effect of grinding of normal Chelex bead on ¹¹¹In chelation and retention in solid meal was evaluated in an in vitro system. The Chelex resin beads were ground in a mortar-pestle to form ground Chelex resin beads. Fine particles were removed by resuspension in distilled water and centrifugation (1000 g). One hundred to 150 μCi of ¹¹¹In chloride was diluted with 0.1 N HC1 and mixed with 1 g of Chelex resin beads. Unbound ¹¹¹In was removed by centrifugation (1000 g). The ¹¹¹In-labeled Chelex resin beads were mixed with fresh egg and ¹¹¹In-labeled solid meal was prepared by heating until solid. The meals were digested with HCl-pepsin (1.2mg/mL of pepsin in 0.1 N HC1) for 4 h in a stirrer-bath (37 °C). Aliquots were collected at intervals for determination of ¹¹¹In loss from ¹¹¹In-labeled solid meal.These results suggest that ¹¹¹In Chelex resin beads were retained in solid meals at a higher level than normal Chelex resin beads and other ¹¹¹In-tracers.     

Gold sodium thiomalate toxicity in cadmium-sensitive and cadmium-resistant Chinese hamster ovary cells

Gold sodium thiomalate was incubated with one cadmium-sensitive cell line and two cadmium-resistant variants. The resistant lines have been reported to synthesize metallothionein (MT) in response to both cadmium and zinc, whereas the sensitive line does not. All cell lines showed a dose-dependent inhibition of growth as a result of gold sodium thiomalate treatment. However, daily comparisons of cell numbers indicate that the cadmium-resistant lines actually increase in number at the highest gold concentrations, whereas numbers of cells in the nonresistant line decrease. MT biosynthesis was measured by monitoring the incorporation of [35S )cysteine into low molecular weight protein. None of the cells synthesized MT in response to gold. When incubated with both zinc and gold, MT was synthesized by both of the cadmium resistant lines; however, the amount of MT synthesized was reduced in the presence of gold which appears to inhibit the uptake of [35S]cysteine by all the cell lines. Although MT is synthesized in the presence of zinc and gold sodium thiomalate, the MT does not have a significant effect on the ability of these cells to withstand high concentrations of gold.     

Metallothionein 2A induction by zinc protects HEPG2 cells against CYP2E1-dependent toxicity

Zinc has been shown to have antioxidant actions, which may be due, in part, to induction of metallothionein (MT). Such induction can protect tissues against various forms of oxidative injury because MT can function as an antioxidant. The objective of this study was to investigate if zinc or MT induction by zinc could afford protection against CYP2E1-dependent toxicity. HepG2 cells overexpressing CYP2E1 (E47cells) were treated with 60 microM arachidonic acid (AA), which is known to be toxic to these cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. E47 cells were preincubated overnight in the absence or presence of metals such as zinc or cadmium that can induce MT. The culture medium containing the metals was removed, AA was added, and cell viability determined after 24 h incubation. Preincubation overnight with 150 microM zinc sulfate or 5 microM cadmium chloride induced a 20- to 30-fold increase of MT2A mRNA; high levels of MT2A mRNA were maintained during the subsequent challenge period with AA, even after the zinc was removed. MT protein levels were increased about 4- to 5-fold during the overnight preincubation with zinc and a 20- to 30-fold increase was observed 24 h after zinc removal during the AA challenge. The treatment with zinc was associated with significant protection against the loss of cell viability caused by AA in E47 cells. The zinc pretreatment protected about 50% against the DNA fragmentation, cell necrosis, the enhanced lipid peroxidation and increased generation of reactive oxygen species, and the loss of mitochondrial membrane potential induced by AA treatment in E47 cells. CYP2E1 catalytic activity and components of the cell antioxidant defense system such as glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxidase (GPX), catalase, Cu,Zn superoxide dismutase (SOD), and MnSOD were not altered under these conditions. Zinc preincubation also protected the E47 cells against BSO-dependent toxicity. When E47 cells were coincubated with zinc plus AA for 24 h (i.e., zinc was not removed, nor was there a preincubation period prior to challenge with AA), AA toxicity was increased. Thus, zinc had a direct pro-oxidant effect in this model and an indirect antioxidant effect, perhaps via induction of MT. MT may have potential clinical utility for the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.