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Comparative luminescence of rat liver Cu-thionein and its chemically synthesized alpha-domain.

A peptide corresponding to the alpha-domain of rat liver metallothionein-2 was chemically synthesized employing the solid phase peptide synthesis technique. Its luminescence properties that depend on the coordinated Cu(I) have been studied using luminescence spectrometric titration in the presence of Cu(I). Unlike the intact metallothionein which has been converted into the Cu species, the emission and excitation spectra of the Cu-alpha-fragment showed a red shift by 20 nm and 65 nm, respectively, suggesting a more compact and stable luminophore in the alpha-domain. Saturation of Cu(I) coordination was reached in the presence of 6.5 mol eq Cu(I) when the alpha-fragment was used and 12 mol eq Cu(I) were specifically bound by the intact metallothionein. The emission bands were homogeneous and no decline of the cluster structure was observed when excessive Cu(I) was added after saturation. A rearrangement of the Cu-cluster in metallothionein during its formation seems to be plausible.

Amino Acid Sequence↗
PubMed1978

Degradation of rat liver metallothioneins in vitro.

The degradation of zinc and cadmium-induced hepatic metallothionineins was investigated in vitro. Both zinc-thionein and cadmium-thionein were labeled in vivo with [35S]cystine. The labeled proteins were isolated and purified by gel filtration and DEAE-ion exchange chromatography. Purified zinc[35S]thionein and cadmium-[35S]thionein were incubated with trypsin, chymotrypsin and pronase for varying times up to 24 h. The rate of degradation of zinc-thionein was twice that of cadmium-thionein when the proteins were incubated with trypsin. Virtually no digestion occurred when the proteins were incubated with chymotrypsin, whereas the rates of degradation were about equal when they were incubated with pronase. In contrast, degradation of zinc-thionein was twice that observed with cadmium-thionein when the proteins were incubated at pH 5.0 with a purified lysosomal extract. Degradation of these proteins by the lysosomal proteases was 77 and 46% within 3 h for zinc-thionein and cadmium-thionein, respectively. Thionein, the metal-free form of metallothionein, was degraded extremely rapidly by both neutral and lysosomal proteases. Chromatography of the digestion products on Sephadex G-25 demonstrated that all three forms of metallothionein were degraded to species of approximately 100-300 daltons. These data indicate that metals stabilize thionein polypeptides and suggest that the degradation of metallothionein in vivo is regulated in part by the species of metal bound.

Animals↗
PubMed1987

Metal constitution of metallothionein influences inhibition of delta-aminolaevulinic acid dehydratase (porphobilinogen synthase) by lead.

This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd,Zn-thionein, although minimal binding of 203Pb to kidney Cd,Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd,Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd,Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd,Zn-thionein much greater than kidney Cd,Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd,Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.

Animals↗
PubMed1981

Metabolism of zinc and copper in the neonate: accumulation and function of (Zn, Cu)-metallothionein in the liver of the newborn rat.

1. Measurements were made of the hepatic concentrations and contents of total and thionein-bound zinc and copper in late foetal and newborn rats and of the distribution of these metals between the particulate and soluble components of the liver. 2. The decrease in the hepatic concentration of thionein-bound Zn, which occurred with age after birth, was proportional to the increase in liver weight until the 16th day post partum; thereafter it was greater. 3. Throughout the period from birth to 25 d of age the Zn concentration remained constant in the cytosolic non-thionein fraction (i.e. total cytoplasmic Zn--thionein-bound Zn), but decreased in other compartments of the liver. 4. The same constant concentration of cytoplasmic non-thionein-bound Zn also was observed in animals with reduced total hepatic Zn contents, but normal body-weights, and in 20-d-old Zn-deficient pups. 5. The concentration of thionein-bound Cu increased significantly only between the 6th and 14th day post partum. The age-related variations in Cu contents of the particulate components closely paralleled those in the whole liver, whereas the Cu contents of the cytosolic thionein and non-thionein fractions did not increase appreciably until after the 10th day. 7. It is concluded that the cytosolic non-thionein fraction of newborn rat liver may contain particularly important metabolic sites that require Zn and a major function of Zn-thionein is to regulate the supply of the metal to these sites. As, from birth to 26 d of age, the sum of the concentrations of thionein-bound Zn and Cu was correlated with whole liver Zn, the accumulation of Cu as a soluble metallothionein seems to be a secondary event, dependent on the hepatic Zn concentration.

Aging↗
PubMed1978

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil

Cadmium↗
PubMed1989

Do cadmium and dexamethasone induce different types of metallothioneins?

1. Dexamethasone induced thionein in rat liver cross reacts with antibody made towards Cd-thionein from rat liver. 2. Dex-thionein binds Cd. 3. Compared to Cd-thionein, dex-thionein behaves differently on SDS polyacrylamide gels. 4. On molecular filtration on Sephadex G-75 columns, dex-thionein elutes slightly more towards the low molecular weight region than Cd-thionein. 5. Dex-thionein elutes in one peak with two shoulders after DEAE-Sephadex A-50 ion exchange chromatography, while Cd-thionein elutes in two peaks.

Animals↗
PubMed1985

Cd2+-induced synthesis of metallothionein in HeLa cells.

HeLa cells synthesize metallothioneins in response to Cd2+. The kinetics of thionein (apoprotein of metallothionein) synthesis was studied by pulse-labelling the cells with [35S]cysteine and measuring relative amounts of the labelled thioneins separated by electrophoresis. Thionein synthesis rapidly increases in the first 6-8 h after exposure to 0.5 micrograms of Cd2+/ml, and begins to decrease in a few hours after reaching the maximum. However, the rate of synthesis never returns to the basal value at least in 30 h after Cd2+ exposure; instead, the second increase in thionein synthesis occurs at 16-18 h. A possible regulatory mechanism of thionein synthesis is discussed from these results, together with the data on intracellular accumulation and subcellular distribution of 109Cd2+. The initial increase of thionein synthesis is accompanied by an increase of mRNA coding for thioneins (MT-mRNA). The induction of MT-mRNA is sensitive to Actinomycin D, but not to cycloheximide, suggesting transcriptional regulation without any mediating protein synthesis. Two thionein isomers are coded for by mRNA molecules of almost the same size, which is similar to that of hepatic MT-mRNA of mouse and rat.

Cadmium↗
PubMed1981

Long-term turnover of cadmium metallothionein in liver and kidney following a single low dose of cadmium in rats.

Rats were injected subcutaneously on two consecutive days with CdCl2, and sampled animals, killed at monthly intervals from 1 to 6 months thereafter, exhibited the presence of Cd,Zn-thionein in both the liver and kidney. At 6 months, hepatic thionein was present as the two major polymorphic forms previously demonstrated in short term Cd-injection studies. [35S]cysteine incorporation studies showed that both polymorphic forms of thionein underwent continual turnover at similar rates throughout th study. The slow hepatic and renal turnover of Cd, therefore, was not due to a highly stable form of Cd-thionein, but apparently due to an inefficient mechanism for excretion of Cd from these tissues. The Cd/Zn ratio of hepatic thionein remained relatively constant, suggesting that continual thionein induction results in a long-term hepatic trapping of Zn by thionein, but the ratio of renal thionein showed a marked increase during the course of the study.

Animals↗
PubMed1981

Complete amino acid sequence of mouse liver metallothionein-II.

The complete amino acid sequence of thionein-II, one of the two major mouse liver thionein components, was determined. The main fragmentation of thionein-II, which consists of 61 amino acid residues, was accomplished by digesting the S-[14C]-carboxymethylated protein and the cyanogen bromide-treated oxidized protein with trypsin. The peptides obtained by papain digestion of S-[14C]carboxymethylated thionein-II were used to align the major tryptic peptides. The sequence was determined by a combination of automated and manual Edman degradation techniques. Remarkable structural homology is observed in mouse thionein-I, mouse thionein-II, and thioneins from man and horse.

Amino Acid Sequence↗
PubMed1978

Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid.

Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing [3H]glycine, [3H]lysine and [3H]serine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.

Animals↗
PubMed1993

Copper-dependent antioxidase defenses in inflammatory and autoimmune rheumatic diseases.

Gel-filtered sera of patients with various inflammatory and autoimmune rheumatic diseases (N = 354) were screened for the presence of the inflammation marker Cu-thionein. The concentrations of Cu-thionein were significantly diminished in patients with connective tissue diseases (P < 0.001). Sera of patients suffering from inflammatory rheumatic diseases were almost totally depleted of this low-molecular-weight copper protein that exerts pronounced superoxide dismutase activity and scavenges effectively hydroxyl radicals and singlet oxygen. Cortisone treatment of patients with rheumatoid arthritis, systemic lupus erythematosus, and polymyalgia rheumatica replenished impressively the serum concentration of Cu-thionein. The partial oxidation of the EPR-silent Cu(I)-chromophore to Cu(II)/Cu(I)-thionein, which is essential for the catalytic dismutation of superoxide, was monitored by electron paramagnetic resonance in the presence of activated neutrophils and monocytes. Release of Cu-thionein during the oxidative burst of peripheral blood monocytes was demonstrated in vitro. The role of prooxidant-antioxidant imbalances in the pathogenesis of rheumatic diseases is discussed.

Acquired Immunodeficiency Syndrome↗
PubMed2006

The Saccharomyces cerevisiae Crs5 Metallothionein metal-binding abilities and its role in the response to zinc overload.

Crs5 is a Saccharomyces cerevisiae Metallothionein (MT), non-homologous to the paradigmatic Cu-thionein Cup1. Although considered a secondary copper-resistance agent, we show here that it determines survival under zinc overload in a CUP1-null background. Its overexpression prevents the deleterious effects exhibited by CUP1-CRS5-null cells when exposed to combined Zn/Cu, as it does the mouse MT1 Zn-thionein, but not Cup1. The detailed characterization of Crs5 in vivo and in vitro Zn(II)-, Cd(II)- and Cu(I)-binding abilities fully supports its resemblance to mammalian MTs. Hence, Crs5 exhibits a good divalent metal-binding ability, yielding homometallic, highly chiral and stable Zn and Cd complexes when expressed in media enriched with these metal ions. In Cu-supplemented cultures, heterometallic Zn,Cu complexes are recovered, unless aeration is kept to a minimum. These features define a Crs5 dual metal-binding behaviour that is significantly closer to Zn-thioneins than to Cu-thioneins. Protein sequence similarities fully support these findings. Overall, a Crs5 function in global metal cell homeostasis, based on its Zn-binding features, is glimpsed. The comparative evaluation of Crs5 in the framework of MT functional differentiation and evolution allows its consideration as a representative of the primeval eukaryotic forms that progressively evolved to give rise to the Zn-thionein lineage.

Copper↗
PubMed1977

Mouse liver metallothioneins. Complete amino acid sequence of metallothionein-I.

The amino acid sequence of thionein-I, a major mouse liver thionein, is presented. Thionein-I is a single chain polypeptide which consists of 61 amino acid residues and has the amino acid composition Cys20, Asp3, Asn1, Thr5, Ser9, Gln1, Pro2, Gly5, Ala5, Val2, Met1, Lys7, with N-acetylmethionine and alanine as NH2- and COOH-terminal amino acids, respectively. Automated sequence analysis of a major cyanogen bromide peptide consisting of 60 amino acid residues, as well as characterization of the peptides obtained from trypsin and papain digestion, led to the elucidation of the complete primary structure of this protein. Remarkable structural homology is observed between the mouse thionein-I and the equine renal thionein-1B (Kojima, Y., Berger, C., Vallee, B.L., and Kägi, J.H.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3413-3417).

Amino Acid Sequence↗
PubMed1982

The effect of zinc and copper pretreatment on the binding of gold (I) to hepatic and renal metallothioneins.

In the first part of this study, single s.c. injections of gold (I) sodium thiomalate were given to male Wistar rats at various dose levels and sacrificed either at 48 hr or 4 days later. The results indicate that though metallothioneins play a significant role in the sequestration of renal gold, the ability of gold itself to induce synthesis of the metalloprotein was limited in the kidneys and practically insignificant in the liver. Pre-injection with zinc however, (4 mg/kg body wt), significantly (P less than 0.01) enhanced the uptake of gold into the metallothioneins both, in the liver as well as the kidneys. The pretreatment increased the proportion of cytosolic gold incorporated into the thioneins from 29% to 48% in the kidneys and from 22% to 47% in the liver. Concurrent increases in the uptake of zinc and copper into the thioneins was also observed. Pre-injection with copper induced a similar effect in the kidneys only. There was no significant difference in the overall uptake of gold into the cytosol itself between the control animals (injected with gold only) and those pretreated with either zinc or copper. This indicated that the increased uptake of gold into the thioneins in the pretreated animals were due to enhanced synthesis of metallothioneins and subsequent increased binding of gold to thioneins rather than increased levels of intracellular gold itself. It is suggested that increased protection against the deposition and possible toxicity of gold in the cell organelles of the liver and kidney tissues may be provided by an increased binding of gold to metallothioneins through pretreatment with zinc, and to a lesser extent copper.

Animals↗
PubMed1990

Extracellular metallothionein effects on lymphocyte activities.

Metal cation influences on the immune response have been reported in a wide variety of experimental systems. These influences can either result in the augmentation or suppression of immunological activities. In order to investigate possible mechanisms of these influences, we examined the role that a metal cation-induced protein, metallothionein (MT), might play. Our findings suggest that thioneins, either as apoproteins or when complexed as Cd,Zn-MT, Zn-MT, or Cd-MT, are capable of inducing lymphocyte proliferation. This level of induction is substantially reduced when Zn,Cd-MT is added to lymphocyte cultures in the presence of 50 microM 2-mercaptoethanol. Apoprotein, Zn,Cd-MT, Zn-MT and Cd-MT also augment LPS-induced proliferation of splenic lymphocytes. Only the Zn,Cd-MT preparation significantly augmented ConA-induced proliferation. Hg-MT and Cu-MT were inhibitory as additions in either LPS or ConA mitogen proliferation assays, and did not stimulate proliferation when added alone to lymphocyte cultures. The capacity to induce proliferation correlates with the measurable thiol level of the particular thionein. Interestingly, Zn,Cd-MT and apothionein had an equivalent number of accessible thiols. Although Zn, Pb, Hg and Cu lowered the number of these sites, the immunoreactivity of these MTs was not altered substantially except by Pb. These results suggest that some metal influences on lymphocytes might be through a thionein intermediary. Our results also demonstrate that thioneins complexed with certain metal cations are detrimental to the normal cellular activities of lymphocytes. At least in these circumstances, MT does not play a role as a protective agent.

Animals↗