3D solution structure of copper and silver-substituted yeast metallothioneins.

3D solution structural calculations for yeast silver(I)-substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein volume conserved to within 6.5%.

Detection of carboxymethylmetallothionein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A sensitive method for detecting metallothioneins (MTs) by using silver staining and autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs is described. Carboxymethylation of MTs is indispensable because it prevents their aggregation, thereby allowing each of them to appear as a single band using SDS-PAGE. Metallothioneins can be detected with a limit of nanogram levels per lane. This method can be applied to MTs induced in in vitro cultured cells and in in vivo tissues.

Metabolism of orally administered cadmium-metallothionein in mice.

The main source of cadmium in the diet is cereal or meat, especially in liver and kidney. Since the cadmium in both liver and kidney is bound to metallothionein, a heat-stable protein, the gastrointestinal absorption and metabolism of cadmium metallothionein (CdMt) was studied in detail. The selective renal cadmium deposition after oral CdMt was analogous to the studies on injected CdMt. Metallothionein with 109Cd or 35S-cysteine radioactive label was isolated from rat liver and administered orally (60 microgram Cd) through a gastric tube to mice (C57 BL/6J). After 4 hr, a major portion of the ingested CdMt was isolated intact from intestinal mucosal cells. However, only a small amount of cadmium bound metallothionein was present in the kidney supernatant. The protein moeity was also degraded completely in kidney. The absorption and tissue distribution of cadmium from oral-cysteine and cadmium-glutathione complexes were similar to that after oral CdCl2 in mice. These results that oral CdMt may be absorbed intact from the gastrointestinal tract and the protein is degraded during renal deposition.

Identification of metallothionein receptors in human astrocytes.

Metallothionein (MT) isoforms are low molecular weight (6000-7000 Da) zinc binding proteins containing 60-68 amino acid residues, 25-30% cysteine, no aromatic amino acids, and binding between 5-7 g zinc/mol of protein. Since the synthesis of MT is induced by endotoxin, cytokines, and glucocorticoids, MT is now considered to be an acute phase protein protecting against oxygen radicals and oxidative damages caused by inflammation, tissue injury, and stress to the central nervous system. By postulating that a specific mechanism must exist to foster the induction of MTs I and II by numerous and diversified factors, we searched for and identified for the first time, MT receptors on U373MG cell membrane preparations, by using fluoresceinated MT I isoform probe; and by employing cysteine, glutathione, and four MT isoforms to determine high affinity and specific binding. MT receptors revealed a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein. Moreover, MT receptors were found in greater density on the surface of aggregated astrocytes. We postulate that conditions or agents generating reactive oxygen species may influence the expression of MT receptors.

Metallothionein-like metal-binding protein in the biomonitor Chaoborus: occurrence and relationship to ambient metal concentrations in lakes.

Larvae of the insect Chaoborus are used to monitor cadmium (Cd) in lakes. We set out to determine if this animal possesses a metallothionein-like protein to which its Cd could be bound and if the concentrations of such a protein are correlated with those of Cd in the insect and in lakewater. To achieve our goals, we collected water and larvae of several Chaoborus species from 10 lakes situated along an environmental Cd gradient. We found that all of the Chaoborus species possess a metallothionein-like protein and that concentrations of the protein and of Cd differed among species. Concentrations of the metallothionein-like protein were directly related to those in Chaoborus and in lakewater. These direct relationships support the use of Chaoborus larvae as a Cd biomonitor.

Cadmium-induced production of a metallothioneinlike protein in Tubifex tubifex (Oligochaeta) and Chironomus riparius (Diptera): correlation with reproduction and growth.

Laboratory-cultured Chironomus riparius and Tubifex tubifex were exposed to sediments artificially enriched with a range of cadmium (Cd) concentrations. Both species accumulated Cd in a concentration-dependent manner. The concentration of a metallothioneinlike protein (MTLP), as measured by a mercury saturation assay, increased with increasing Cd exposure. After reaching a threshold of Cd exposure, the whole-body endpoints of reproductive output in T. tubifex and growth in C. riparius declined significantly. The threshold effect concentrations for T. tubifex and C. riparius were 2.68 and 0.134 micromol Cd/g dry sediment, respectively. Metallothioneinlike protein and Cd tissue concentrations were more sensitive indicators of exposure than the whole-body endpoints. For T. tubifex, the concentrations of MTLP and tissue Cd were significantly elevated above control levels after exposure to the 0.67 micromol Cd/g dry sediment treatment. In C. riparius, MTLP concentration and tissue Cd concentration were both significantly elevated above control levels after exposure to the 3.8 x 10(-3) micromol Cd/g dry sediment treatment. Analysis of these data suggests that MTLP and tissue Cd concentrations are sensitive subcellular endpoints, which can be used to predict exposure to and the effects of metals at the individual or population level.

Metalloselenonein, the selenium analogue of metallothionein: synthesis and characterization of its complex with copper ions.

We used an automated peptide synthesizer to produce a peptide, metalloselenonein, that contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein. Metalloselenonein binds 3 mol of Cu(I) per mol. This adduct shows a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the copper-metalloselenonein complex shows a positive band around 245 nm attributable to asymmetry in metal coordination.

Natural variation in a metallothionein-like protein in Tubifex tubifex in the absence of metal exposure.

Concentrations of a metallothionein-like protein (MTLP) in Tubifex tubifex were consistent throughout the various life stages with no significant variation associated with reproductive stage. The mean concentration from cocoon through 10 weeks of age was 2.5 (+/-0.6) nmol MTLP/g. T. tubifex was subjected to a number of potential environmental stressors to determine whether MTLP production could be induced in the absence of metal exposure. There were no significant differences in the concentrations of MTLP between the controls and worms subjected to decreased food supply, increased handling, or reduced dissolved oxygen at a given exposure temperature. However, at 12 degrees C T. tubifex had significantly (P < 0.01) higher MTLP than T. tubifex held at 23 degrees C. There were also significant differences in wet weight and reproduction at the two temperatures, which may have implications for the levels of MTLP.

Metal-binding and detoxification effect of synthetic oligopeptides containing three cysteinyl residues.

Metallothionein is a naturally occurring metal-binding protein with high cysteine content. Oligopeptides containing three cysteinyl residues and having amino acid sequences analogous to portions of this protein were synthesized by the solid-phase method. Strong affinity of the synthetic peptides to Cd2+ and Zn2+ was observed, and the dissociation constants of the peptide-metal complexes were 2-4 orders of magnitude lower than those of cysteine-metal and dithioerythritol-metal complexes. Effectiveness of detoxification of the peptides against Cd toxicity was demonstrated by the higher survival rates of mice treated with the peptides and by the neutralization of Cd toxicity by the peptides in tissue cultures.

Identification of a structural gene encoding a metallothionein-like domain that includes a putative regulator protein for Streptomyces protease gene expression.

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation analysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.

Correlation of increased levels of Ha-ras T24 protein with extent of loss of gap junction function in rat liver epithelial cells.

Although it is known that cells transformed by ras and other oncogenes show reduced gap junction function, to date there has been no investigation of the quantitative relationship between intracellular levels of ras oncoprotein and loss of cell-cell communication. Using the rat liver epithelial cell line MTR6, which carries a zinc-inducible metallothionein ras T24 (MTrasT24) fusion gene, we showed a direct correlation between the accumulation of ras T24 protein and the loss of dye transfer as measured by interactive laser cytometry. After stimulation with zinc sulfate, changes in both parameters were rapid and measurable by 24 h. Similarly, there was a dose-response relationship between loss of gap junction function and increase in ras T24 protein. Northern analysis of two gap junction proteins (connexins 43 and 32) showed no differences between cells that expressed high levels of ras and control cells. These data demonstrate that the degree of loss of gap junction function is dependent on the amount of increase in ras T24 protein levels, but the mechanism by which these changes are effected remains unclear.

Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy).

Selected toxic and essential metals (mercury, Hg; cadmium, Cd; and zinc, Zn) were determined in the liver and in the kidney collected from 13 dolphins (Stenella coeruleoalba and Tursiops truncatus) stranded along the southern coasts of Italy from 1991 to 1999. Liver samples were also analysed for their methyl mercury (MeHg) and selenium (Se) contents. For subcellular fractionation, liver and kidney samples were homogenized in Tris-HCl buffer; after centrifugation, the supernatant (cytosol) was separated from pellets (insoluble fraction), heated at 80 degrees C for 10 min and centrifuged in order to separate the precipitate containing the thermolabile high molecular weight proteins. The cytosol heat-stable fraction, including metallothionein-like proteins (MTLPs), was then purified by gel filtration chromatography on Sephadex G-75 column. The three subcellular fractions collected (insoluble fraction, thermolabile fraction and purified heat-stable fraction) were analysed for their Hg, Cd and Zn contents. The analytical results confirm previous similar studies on toothed whales in showing that: (1) in the liver, as well as in the kidney, Hg was mainly found in the insoluble fraction, therefore, metallothioneins seem to have no role in the Hg detoxification; (2) in the liver, the molar ratio between Se and inorganic Hg was very close to 1; this suggests that the final compound of MeHg detoxification could be HgSe (tiemannite); (3) in almost all the samples, Cd and Zn were detected in the purified heat-stable fraction (including MTLPs). The mechanism of Cd detoxification and Zn homeostasis is also discussed.