Metal binding feature of copper‒induced metallothionein from freshwater crab Sinopotamon henanense reveals its Cu‒thionein character.

Sinopotamon Henanense expresses two metal‒induced metallothioneins (MTs), Cd‒induced MT and Cu‒induced MT (ShCuMT). The Cd‒induced MT has been characterized as a Cd‒thiolate MT. However, it is unknown whether ShCuMT is a Cu‒thiolate MT. In the present study, ShCuMT was expressed heterologously in Escherichia coli and purified by Ni‒NTA column and superdex‒75 column. And its metal‒binding feature was evaluated by DTNB reaction, circular dichroism spectroscopy (CD), isothermal microtitration (ITC), electrospray flight mass spectrometry (ESI‒TOF‒MS), and matrix‒assisted laser desorption ionization flight mass spectrometry (MALDI‒TOF‒MS). Bioinformatics analysis demonstrated that ShCuMT possessed the cysteine‒triplet motif of a Cu‒specific MT. Expression and purification of ShCuMT illustrated that SUMO tag used as the production system for ShCuMT resulted in a high production yield. The stability order of ShCuMT binding metal ions were Cu (Ⅰ) > Cd (Ⅱ) > Zn (Ⅱ). The CD spectrum indicated that ShCuMT binding with Cu (I) exhibited a compact thiol metal clusters structure. Besides, there emerged no a visible nickel‒thiol absorption after Ni‒NTA column affinity chromatography. The ITC results implied that Cu‒ShCuMT possessed the optimal thermodynamic conformation and the highest stoichiometric number of Cu (Ⅰ). Overall, the results suggested that SUMO fusion system is a robust and inexpensive approach for ShCuMT expression and Ni‒NTA column had no influence on metal binding of ShCuMT and Cu(Ⅰ) was considered its cognate metal ion, and ShCuMT possessed canonical Cu‒thiolate characteristics. The metal binding feature of ShCuMT reported here contributes to elucidating the structure‒function relationship of ShCuMT in S. Henanense.

Purification, secondary structure and antioxidant activity of metallothionein zinc-binding proteins from Arca subcrenata.

Zinc-binding proteins named MT-M-I and MT-M-II were obtained after purification from metal-exposed hairy clams (Arca subcrenata) using gel permeation and ion-exchange chromatography. MT-M-I and MT-M-II were resolved by ion-exchange chromatography, and they were found to have similar molecular weights. MT-M-I and MT-M-II can bind 6 and 7 equivalents of Zn2+ in vitro, and they showed unusual migration behaviors in Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Such migration behaviors may be due to themetal thiolate clusters in these proteins. In terms of amino acid composition, the proportion of cysteine in MT-M-I and MT-M-II was approximately 30%, and glycine accounted for approximately 15%, where as aromatic amino acids were absent. Considering the performance in Tricine-SDS-PAGE and the amino acid compositions, MT-M-I and MT-M-II conform to the molecular characteristics of the metallothionein proteins. The structures were explored using circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR). Also determined the antioxidant activities in terms of DPPH radical scavenging ability, hydroxyl radical (·OH) scavenging ability, and ferric-reducing/antioxidant power. The antioxidant activities of MT-M-I were found to be stronger than those of MT-M-II.

The Enigmatic Metallothioneins: A Case of Upward-Looking Research.

In the mid-1950s, Bert Lester Vallee and his colleague Marvin Margoshes discovered a molecule referred to today as metallothionein (MT). Meanwhile, MTs have been shown to be common in many biological organisms. Despite their prevalence, however, it remains unclear to date what exactly MTs do and how they contribute to the biological function of an organism or organ. We investigate why biochemical research has not yet been able to pinpoint the function(s) of MTs. We shall systematically examine both the discovery of and recent research on Dr. Vallee's beloved family of MT proteins utilizing tools from philosophy of science. Our analysis highlights that Vallee's initial work exhibited features prototypical of a developing research tradition: it was upward-looking, exploratory, and utilized mere interactions. Since the 1960s, MT research has increasingly become intervention- and hypothesis-based while it remained largely upward-looking in character. Whilst there is no reason to think that upward-looking research cannot successfully yield structure-function mappings, it has not yet been successful in the case of MTs. Thus, we suggest it might be time to change track and consider other research strategies looking into the evolution of MTs. Recent studies in mollusks render research in this direction worthy of pursuit.

Metalation of a rice type 1 metallothionein isoform (OsMTI-1b).

The simultaneously functions of Metallothioneins (MTs) are relied on their metalation mechanisms that can be divided into non-cooperative, weakly cooperative and strongly cooperative mechanisms. In this study, we recombinantly synthesized OsMTI-1b, N- and C-terminal Cys-rich regions as glutathione-S-transferase (GST)-fusion proteins in E. coli. In comparison with control strains (The E. coli cells containing pET41a without gene), transgenic E. coli cells showed more tolerance against Cd2+ and Zn2+. The recombinant GST-proteins were purified using affinity chromatography. According to in vitro assays, the recombinant proteins showed a higher binding ability to Cd2+ and Zn2+. However, the affinity of apo-proteins to Cu2+ ions were very low. The coordination of Cd2+ ions in OsMTI-1b demonstrates a strongly cooperative mechanism with a priority for the C-terminal Cys-rich region that indicates the detoxifying of heavy metals as main role of P1 subfamily of MTs. While the metalation with Zn2+ conformed to a weakly cooperative mechanism with a specificity to N-terminal Cys-rich region. It implies the specific function of OsMTI-1b is involved in zinc homeostasis. Nevertheless, a non-cooperative metalation mechanism was perceived for Cu2+ that suggests the fully metalation does not occur and OsMTI-1b cannot play a significant role in dealing with Cu2+ ions.

Cloning and functional characterization of a novel metallothionein gene in Antarctic sea-ice yeast (Rhodotorula mucilaginosa).

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu2+ , Pb2+ , Zn2+ , Cd2+ , and Ag+ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.

Binding of Cd(II), Pb(II), and Zn(II) to a type 1 metallothionein from maize (Zea mays).

Metallothioneins (MTs) are a family of ubiquitous, low-molecular-mass, cysteine-rich proteins that play a significant role in maintaining intracellular metal homeostasis, eliminating metal toxification, and protecting cells against oxidative damages. Research activity on plant MTs, although known for 30 years, has only moderately increased in the past few years. In this study, a type 1 MT from maize (Zea mays) (ZmMT1) was successfully expressed in Escherichia coli strain BL21 (DE3). The UV absorption spectra recorded after the reconstitution of apo-ZmMT1 with different metals demonstrated that ZmMT1 can coordinate up to six Zn(II) ions, six Cd(II) ions, and even higher amounts of Pb(II). In addition, the general metal ion coordination abilities of ZmMT1 characterized by pH-dependent zinc-, lead- and cadmium-binding stability and by the competitive reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were evaluated. Results showed that the affinity of metal ions for the recombinant form of ZmMT1 can be arranged as follows: Cd(II) > Pb(II) > Zn(II). The observation revealed that chelating agents, such as ethylene diamine tetraacetic acid (EDTA) and ATP, accelerate the oxidation of ZmMT1 in the following order: EDTA ≫ L-histidine > ATP ≈ citrate. Meanwhile, commonly used buffers increase the reactivity of ZmMT1 with DTNB in the following order: PBS > Tris-HCl > HEPES.

Metallothionein is elevated in liver and duodenum of Atp7b(-/-) mice.

Different mutations in the copper transporter gene Atp7b are identified as the primary cause of Wilson's disease. These changes result in high copper concentrations especially in the liver and brain, and consequently lead to a dysfunction of these organs. The Atp7(-/-) mouse is an established animal model for Wilson's disease and characterized by an abnormal copper accumulation, a low serum oxidase activity and an increased copper excretion in urine. Metallothionein (MT) proteins are low molecular weight metal-binding proteins and essential for the zinc homeostasis but also play a role for the regulation of other metals, e.g. copper. However the molecular mechanisms of MT in regard to Atp7b remain still elusive. In this study we investigate the expression of MT in the liver and duodenum of Atp7b(-/-) mice and wildtype mice. Hepatic and duodenal expression of MT was measured by real-time reverse transcription-polymerase chain reaction and post-translational expression was analyzed by immunoblot and immunofluorescence. Expression of MT in liver und duodenum was significantly higher in Atp7b(-/-) mice than in controls. Hepatic and duodenal copper, iron and zinc content were also studied. Compared to control hepatic copper and iron content was significantly higher while hepatic zinc content was significantly lower in Atp7b(-/-) mice. In the duodenum copper and zinc content of Atp7b(-/-) mice was significantly lower than in controls. Duodenal iron content was also lower in Atp7b(-/-) mice, but did not reach statistical significance. The results of our study suggest that metallothionein is elevated in the liver and duodenum of Atp7b(-/-) mice.

Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum plumbizincicola.

Mechanistic insight into metal hyperaccumulation is largely restricted to Brassicaceae plants; therefore, it is of great importance to obtain corresponding knowledge from system outside the Brassicaceae. Here, we constructed and screened a cDNA library of the Cd/Zn hyperaccumulator Sedum plumbizincicola and identified a novel metallothionein-like protein encoding gene SpMTL. SpMTL showed functional similarity to other known MT proteins and also to its orthologues from non-hyperaccumulators. However, three additional cysteine residues were observed in SpMTL and appeared to be hyperaccumulator specific. Removal of these three residues significantly decreased its ability to tolerate Cd and the stoichiometry of Cd against SpMTL (molar ratio of Cd/SpMTL) to a level comparable to those of Cd/SaMTL and Cd/SeMTL in the corresponding non-hyperaccumulating relatives. SpMTL expressed in S. plumbizincicola roots at a much higher level than those of its orthologues in the non-hyperaccumulator roots. Interestingly, a positive correlation was observed between transcript levels of SpMTL in roots and Cd accumulation in leaves. Taking these results together, we propose that elevated transcript levels and heterotypic variation in protein sequences of SpMTL might contribute to the trait of Cd hyperaccumulation and hypertolerance in S. plumbizincicola.

Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis.

A new copper metallothionein (TfCuMT) gene has been identified from a locally isolated ciliate Tetrahymena farahensis. It contains 327 nucleotides encoding a peptide chain of 108 amino acids and belongs to class MTT2 and subfamily 7b. Amplification from both gDNA and mRNA confirmed the intronless nature of this gene. Like most of the metallohtioneins, cysteine residues contribute nearly 30% content with the specific CKC motifs. Structural repeats present in peptide sequence of TfCuMT indicate internal duplication of gene at some stage of gene evolution. The predicted model of copper metallothionein protein showed that copper ions are mainly chelated by thiol sulfur of cysteine residues and are embedded in the folds of polypeptide chain. For in vivo expression of TfCuMT in Escherichia coli host cells the classical stop codons, which coded for glutamine in the ciliate were mutated to CAA and CAG through site directed mutagenesis. The mutated gene showed higher expression in pET28a expression vector compared with pET21a. Optimum expression was obtained after 6-8 h of 0.1 mM IPTG induction. Stability of His tagged TfCuMT in 5% SDS was low, with half-life of about 104 min. Presence of 1.0 µM copper increased the expression level by 1.65-fold. Presence of 100 µM Cysteine in culture medium caused 2.4-fold increase in expression level. His tagged TfCuMT was purified through affinity chromatography using NTN-His binding resin in the presence of 0.1 M imidazole and NaCl. The modeled structure of the TfCuMT showed a cleft for Cu binding with correct orientation of Cys residues in the motif CKC. J. Cell. Biochem. 117: 1843-1854, 2016. © 2016 Wiley Periodicals, Inc.

Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential.

Metallothioneins are essential in plants for metal detoxification in addition to their other roles in plant life cycle. This study reports the characterization of an olive (Olea europaea L. cv. Ayvalik) metallothionein with respect to molecular and functional properties. A cDNA encoding a type 2 metallothionein from olive was isolated from a leaf cDNA library, characterized and named OeMT2 after its molecular and functional properties. OeMT2 was expressed in Escherichia coli, and a single protein band was confirmed by protein gel blot analysis. Metal tolerance ability of bacterial cells expressing OeMT2 was determined against 0.2 mM CdCl2, 0.4 mM CdCl2 and 1 mM CuSO4 in the growth medium. Metal ion contents of bacterial cells expressing OeMT2 were measured by ICP. Metal tolerance assays and ICP measurements suggested that OeMT2 effectively binds Cu and Cd. Molecular analysis of OeMT2 revealed two introns, three exons, a short 3' UTR and a long 5' UTR. Comparing the genomic sequences from 14 olive cultivars revealed OeMT2 had both intron and exon polymorphisms dividing the cultivars into three groups. Real-time PCR analysis demonstrated that OeMT2 expresses more or less the same amounts in all tissues of the olive tree examined. The genomic copy number of OeMT2 was also determined employing real-time PCR which suggested a single copy gene in the olive genome while three other MT2 members were determined from the draft olive genome sequences of Ayvalik cultivar and that of wild olive. This is the first report on molecular and functional characterization of an olive metallothionein and shows that OeMT2 expressed in E. coli has the capability of effectively binding toxic heavy metals. This may suggest that OeMT2 plays an important role in metal homeostasis in addition to a good potential for environmental and industrial usage.

Tissue-specific copper accumulation, zinc levels, induction, and purification of metallothionein in freshwater crab Sinopotamon henanense exposed to subacute waterborne copper.

Copper (Cu) is one of the most important essential metals for crustaceans, buttoxic in excess. Metallothioneins (MT) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis, detoxification, and cytoprotection. In the present study, Sinopotamon henanense were exposed to 0 (controls), 2.86, and 14.3 mg L(-1) waterborne Cu, Cu accumulation, zinc (Zn) levels and MT induction in gills and hepatopancreas were determined with Cd/Hemoglobin saturation assay and atomic absorption spectrophotometry method. Results showed that Cu accumulation and MT levels were both tissue-specific and revealed some time-dependent and dose-dependent, respectively. The highest Cu accumulations of 82.10 ± 16.38 µg g(-1) w wt were observed in the gill after 15 days of 14.3 mg L(-1) Cu exposure, the peak MT induction of 136.16 ± 19.39 µg g(-1) w wt were observed in the hepatopancreas after 3 day of 14.3 mg L(-1) Cu exposure.In addition, the essential metal homeostasis of Zn was disturbed in some ways by subacute Cu exposure. The calculated ratios of actual Cu to theoretical maximum metal bound by MT indicating that the hepatopancreas had much greater Cu-binding potentials than the gills. Positive correlation were shown between MT induction and Cu accumulation both in hepatopancreas and gills, indicating that MT induction in S. henanense can be considered as a biomarker for subacute waterborne Cu pollution. Furthermore, the Cu induced MT (CuMT) from S. henanense was purified using acetone precipitation (50-80%), followed by gel filtration chromatography and anion exchange chromatography. SDS-PAGE and time-of-flight mass spectrometry analysis showed that S. henanense CuMT possess two isoforms and both mainly existed as monomer and dimmer forms. These present studies will be helpful to increase the database information of heavy metal-induced MT in terms of crustaceans.

A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

Metal stoichiometry of isolated and arsenic substituted metallothionein: PIXE and ESI-MS study.

The stoichiometric analysis of the metal induced Metallothionein (MT) is pertinent for understanding the metal-MT interactions. Despite innumerable publications on MT, the literature addressing these aspects is limited. To bridge this gap, PIXE and ESI-MS analysis of the commercial rabbit liver MT1 (an isoform of MT), zinc induced isolated rat liver MT1, apo and Arsenic substituted rabbit liver MT1 have been carried out. These techniques in combination provide information about number and the signature of all the metal ions bound to MT. By using ESI-MS in the rabbit MT1, ions of Zn n MT1 (n = 0, 1, 4, 5, 6, 7) whereas, in rat MT1, the Zn1MT1 and Zn5MT1 ions are observed. PIXE analysis shows that some copper along with zinc is also present in the rabbit as well as rat MT1 which could not be assessed with ESI-MS. During As metallation reaction with rabbit MT1, with increase in arsenic concentration, the amount of arsenic bound to MT1 also increases, though not proportionally. The presence of both Zn and Cu in MT1 on Zn supplementation can be related to the role of MT in Zn and Cu homeostasis. Further, the presence of partially metallated MT1 suggests that MT1 may donate fractional amount of metal from it's fully metallated form to other proteins where Zn acts as a cofactor.

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening.

This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use.

A novel metallothionein gene from a mangrove plant Kandelia candel.

A new metallothionein (MT) gene was cloned from Kandelia candel, a mangrove plant with constitutional tolerance to heavy metals, by rapid amplification of cDNA ends and named KMT, which is composed of two exons and one intron. The full length of KMT cDNA was 728 bp including 121 bp 5' noncoding domain, 240 bp open reading frame and 384 bp 3' termination. The coding region of KMT represented a putative 79 amino acid protein with a molecular weight of 7.75 kDa. At each of the amino- and carboxy-terminal of the putative protein, cysteine residues were arranged in Cys-Cys, Cys-X-Cys and Cys-X-X-Cys, indicating that the putative protein was a novel type 2 MT. Sequence and homology analysis showed the KMT protein sequence shared more than 60 % homology with other plant type 2 MT-like protein genes. At amino acid level, the KMT was shown homology with the MT of Quercus suber (83 %), of Ricinus communis (81 %) and of Arabidopsis thaliana (64 %). Function studies using protease-deficient Escherichia coli strain BL21 Star ™(DE3) confirmed the functional nature of this KMT gene in sequestering both essential (Zn) and non-essential metals (Cd and Hg) and the E. coli BL21 with KMT can live in 1,000 µmol/L Zn, 120 µmol/L Hg, and 2,000 µmol/L Cd. The information could provide more details of the causative molecular and biochemical mechanisms (including heavy metal sequestration) of the KMT in K. candel or a scientific basis for marine heavy-metal environment remediation with K. candel. This study also provides a great significance of protecting mangrove species and mangrove ecosystem.

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis.

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis.

Functional comparison of metallothioneins MTT1 and MTT2 from Tetrahymena thermophila.

Metallothioneins MTT1 and MTT2 from Tetrahymena thermophila have been characterized. The MTT1 contains mainly characteristic Cys-Cys-Cys and Cys-Cys clusters, but MTT2 contains mainly Cys-X-Cys cluster. Cd(16)-MTT1 mainly consists of α-helix and ß-turns, in contrast, Cd(11)-MTT2 mainly consists of random coils. Reaction of Cd(16)-MTT1 and Cd(11)-MTT2 with nitric oxide leads to intramolecular disulfide bond formation, respectively. Binding stabilities of Cd(2+), Hg(2+) and Zn(2+) to MTT1 are stronger than those to MTT2. Cu(2+) can not replace Cd(2+) from Cd(16)-MTT1 complex, but can replace Cd(2+) from Cd(11)-MTT2 complex. The analysis of qRT-PCR revealed MTT2 mRNA levels were 31-fold higher than those of MTT1 under basal conditions. These results further suggest MTT1 possibly play a role in the detoxification of heavy metal ions, and MTT2 may be involved in the homeostasis of copper ions.

Paramagnetic antibody-modified microparticles coupled with voltammetry as a tool for isolation and detection of metallothionen as a bioindicator of metal pollution.

Low-molecular mass proteins rich in cysteines called metallothioneins (MT) can be considered as markers for the pollution of the environment by metals. Here, we report on suggestion for an automated procedure for the isolation of MT followed by voltammetric analysis. Primarily, we optimized the automated detection of MT using an electrochemical analyser. It was found that the most sensitive and repeatable analyses are obtained at a temperature of 4 °C for the supporting electrolyte. Further, we optimized experimental conditions for the isolation of MT by using antibody-linked paramagnetic microparticles. Under the optimal conditions (4 h long interaction between the microparticles and MT), the microparticles were tested on isolation of various amounts of MT. The lowest isolated amount of MT by antibody-linked paramagnetic microparticles was 5 µg ml(-1) of MT (50 ng). The automated procedure of MT isolation was further tested on isolation of MT from guppy fish (Poecilia reticulata) treated with silver(i) ions (50 µM AgNO(3)). The whole process lasted less than five hours and was fully automated. We attempted to correlate these results with the standard method for MT isolation. The correlation coefficient is 0.9901, which confirms that results are in good agreement. Moreover, the concentration of silver ions in tissues of fish treated with Ag(i) ions was determined by high performance liquid chromatography with electrochemical detection.

Identification and functional analysis of cadmium-binding protein in the visceral mass of Crassostrea gigas.

The Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets.

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M(8) (II)-MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn(6)-MTC and Cd(7)-MTC species from M(8) (II)-MTC after treatment with Chelex 100. The NMR characterization of Cd(7)-MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal-Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1-31) reveal a Cd(3)Cys(9) cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32-64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu(8-9)-MTC). The subsequent generation of Cu(12)-MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.