Preparation and 113Cd NMR studies of homogeneous reconstituted metallothionein: reaffirmation of the two-cluster arrangement of metals

113Cd NMR analysis of rabbit liver metallothionein 2 reconstituted with 113Cd at all seven binding sites has previously indicated that the metals are arranged in two metal-thiolate clusters [Otvos, J.D., & Armitage, I.M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. Spectra of the protein always contained more than seven resonances, however, suggesting the samples were in some way heterogeneous. Results of a recent study of 113Cd metallothionein reconstituted in a different manner but also giving spectra with more than seven resonances have been interpreted as arguing against the two-cluster model of metal binding and in favor of a model in which structural flexibility of the protein allows many configurational substates of the cluster(s) to coexist [Vasak, M., Hawkes, G.E., Nicholson, J.K., & Sadler, P.J. (1985) Biochemistry 24, 740-747]. Data are presented here that indicate that dimers and larger oligomers of metallothionein formed as byproducts of metal reconstitution are the likely source of at least some of the 113Cd resonances attributed by these workers to configurational substrates. Removal of the contaminating oligomers by gel filtration yields a verifiably homogeneous protein whose 113Cd spectrum consists of seven resonances of comparable intensity. Unambiguous confirmation of the existence and structures of the two previously proposed metal-thiolate clusters was obtained by two-dimensional chemical shift correlation spectroscopy and spectral simulation of the 113Cd-113Cd splitting patterns of the individual resonances.     

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.     

Tissue-specific expression of two metallothionein genes in common carp during cadmium exposure and temperature shock

Two metallothionein cDNA isoforms (MT-1 and MT-2) were isolated from carp (Cyprinus carpio) by RT-PCR. Sequence analysis of the cDNAs revealed two amino acid differences between the coding regions and markedly different 3'-untranslated ends. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal MT-1 and MT-2 mRNA levels and to follow the inducer-specific expression of MT genes in different tissues during in vivo studies. In the brain and muscle, the uninduced levels of the two MT mRNAs were similar. In the kidney and liver, the MT-1 gene product predominated, while in the heart the relative expression levels of the two genes were opposite. Both the MT-1 and MT-2 mRNA levels increased with Cd concentration in a time- and dose-dependent manner. The expression of MT-2, however, was more responsive to a high Cd concentration. In parallel with the induction of the MTs by Cd, we followed the accumulation of this metal in the kidney and liver. Although the Cd level was always higher in the kidney during treatment, the rate of accumulation was higher in the liver. Cold stress resulted in a significantly higher induction of MT-1 than of MT-2, while heat shock had no effect on the expression of either gene.     

113Cd NMR studies on metal-thiolate cluster formation in rabbit Cd(II)-metallothionein: evidence for a pH dependence

The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

113Cd NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility

A reproducible method for the reconstitution of rabbit liver metallothionein (MT) containing seven cadmium atoms per mole of protein is described. This protein was studied in detail by 113Cd NMR at 88-, 55-, and 44-MHz frequencies, including the effects of pH, temperature, and ionic strength on the spectra. Our results differ significantly from previous reports of 113Cd NMR on similar samples. Thus, the spectra of both chromatographically distinguishable isoforms MT1 and MT2 were not identical, and neither could be interpreted in terms of a unique static model with the seven Cd ions forming two independent clusters of four and three Cd ions. Large differential shifts of 113Cd resonances were observed with changes in temperature over the range 277-320 K and ionic strength (0.02-0.5 M). At low temperature a slow structural change (half-life of several minutes) was detected. The structure was more rigid at high ionic strength. The frequency dependence and two-dimensional J-resolved spectra revealed that 113Cd resonances were composed of several overlapping peaks, complicating the interpretation of fine structure in one-dimensional spectra. A new flexible model of the Cd cluster in metallothionein is proposed. This model incorporates dynamic thiolate exchange reactions and involves several configurational substates of the protein. The possible relationship of such flexibility to the function of metallothionein is discussed.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

The effect of nitric oxide on metal release from metallothionein-3: gradual unfolding of the protein

Metallothionein-3 (MT-3), or neuronal growth inhibitory factor, which exhibits growth inhibitory activity, is a brain-specific metallothionein. In this study, the effect of nitric oxide (NO) on metal release (using Cd²⁺ as a probe) from MT-3 was examined by ¹¹³Cd and 2D [¹H–¹⁵N] heteronuclear single-quantum coherence NMR spectroscopy. The exposure of human MT-3 to NO leads to a nonselective release of the three metals from the β-domain. In contrast to metallothionein-1 and metallothionein-2, two of the bound metals in the α-domain were also partially released, with the domain structure remaining almost unchanged. Further addition of NO resulted in the complete release of metals and concomitant unfolding of the protein. The preference of release of the two metals in the α-domain was attributed to the presence of two slightly different coordination environments for the four cadmium/zinc atoms.     

Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal-thiolate clusters.

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.     

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [113Cd7]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [113Cd, Zn]MT2 were obtained from rat liver following injection of 113Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of 113Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [1H, 113Cd]-correlated spectra that are indistinguishable from those of [113Cd7]MT2, thereby implying identity of cluster coordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.     

Properties of metal-ion coordinated imidazoles: nmr and C-2 H Exchange in Co(III) Complexes

The ¹H and ¹³C nmr spectra of Co(NH₃)₅ImH³⁺ and the ¹H nmr spectra of αCotrien(ImH)₂³⁺ and βCotrien(ImH)₂³⁺ are reported. The pK_a values determined from the dependence of the chemical shift on pH are 10.0, 9.6, and 10.1, respectively. The range of the chemical shift between the acid and base forms is unusually small in the ¹H nmr, 0.5–0.7 ppm for the C-2 H and about 0.25 ppm for the C-4 H and C-5 H. In the ¹³C nmr, C-2 and C-4 have large shifts to low field and C-5 a small shift to high field on deprotonation. The C-2 proton is not exchanged with solvent ²H under acidic or basic conditions, in marked contrast to the corresponding proton in both imidazole and 1-methylimidazole. These spectroscopic and chemical properties should be useful for the direct identification of metal-ion coordinated histidines in proteins.     

Immunological properties of low molecular-weight proteins binding heavy metals in rat kidney and liver

Two homogenous fractions of hepatic metallothioneins ((Cd,Zn) MT-1 and (Cd,Zn) MT-2) and renal metal binding proteins ((Bi,Cu) BP-1 and (Bi,Cu) BP-2) were isolated from rats exposed to heavy metals and specific antisera to them were produced in rabbits.These antisera were tested by immunodiffusion and immunoelectrophoresis for their ability to bind different fractions of hepatic Cd,Zn -metallothionein and renal (Bi,Cu)-, (Hg,Cu)- and (Cd,Cu)-binding proteins. It was found that anti (Bi,Cu) BP antisera did not cross-react with hepatic (Cd,Zn) MT-1 and (Cd,Zn) MT-2. Strong immunological cross-reactions were detected between anti (Bi,Cu) BP antisera and individual forms of (Cd,Cu)-, (Hg,Cu)- and (Bi,Cu)-binding proteins isolated from rat kidneys.     

113Cd NMR study of bovine prothrombin fragment 1 and factor X

The interaction of Cd2+ with bovine prothrombin fragment 1, prothrombin intermediate 1, factor X, and a modified (Gla-domainless) factor X has been studied with 113Cd NMR. All the 113Cd resonances observed in this study were in the chemical shift range expected for oxygen ligands, suggesting that cadmium is binding at the same sites where calcium binds. Both fragment 1 and factor X displayed two major resonances, one near 10 ppm from 113Cd2+ that did not exchange rapidly with unbound 113Cd2+ (the high-affinity, or H, resonance) and one near -15 ppm from 113Cd2+ that exchanged rapidly with unbound 113Cd2+ (the low-affinity, or L, resonance). The difference between the chemical shift of the H resonance and the chemical shift range of -90 to -125 ppm that has been reported for three other small calcium-binding proteins is postulated to be due to different coordination geometries for monocarboxylate and dicarboxylate ligands; Cd2+ binds to fragment 1 and factor X through the dicarboxylate side chains of gamma-carboxyglutamate (Gla) residues. This allows contribution of only one oxygen per carboxyl group. At least one of the first few 113Cd2+ ions bound to fragment 1 did not appear in the 113Cd NMR spectrum until a total of five 113Cd2+ had been added. This could be due to exchange broadening of initial 113Cd2+ resonances due to sharing of ligands among several sites. Filling all sites would then restrict ligand exchange. Addition of Zn2+ displaced 113Cd2+ from the H resonance sites. Factor X did not display the interactions among ion binding sites proposed for fragment 1.     

Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.

Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a metal-sensitive yeast strain in which the endogenous metallothionein gene has been deleted. The mutant proteins have single or double cysteine to tyrosine replacements (C13Y, C50Y, and C13,50Y), single cysteine to serine replacements (C13S and C50S), or a single cysteine to alanine replacement (C50A). These proteins function in their yeast host in cadmium detoxification to differing extents. Metallothioneins which contain a cysteine mutation at position 50 (C50Y, C50S, C50A, and C13,50Y) conferred markedly less cadmium resistance than wild type metallothionein, or metallothionein with a single cysteine mutation at position 13 (C13Y and C13S). Wild type and three of the mutant Chinese hamster metallothioneins (C13Y, C50Y, and C13,50Y) were purified from yeast grown in subtoxic levels of either CdCl2 or 113CdCl2. All three of the mutant proteins bound less cadmium than the wild type protein when metal-binding stoichiometries were determined. The one-dimensional 113Cd NMR spectrum of the recombinant wild type Chinese hamster metallothionein was compared to the spectra of native rat and rabbit liver metallothioneins. The close correspondence between the 113Cd chemical shifts in these metallothioneins is consistent with the presence of two separate metal clusters, A and B, corresponding, respectively, to the alpha- and beta-domains, in the recombinant metallothionein. The one-dimensional 113Cd NMR spectra recorded on each of the three mutant metallothioneins, on the other hand, provide some indication as to the structural basis for the reduced, by one, metal stoichiometry of each of the mutant metallothioneins. For the C13Y mutant, it appears that the beta-domain now binds a total of two metal ions whereas with the C50Y mutant, the alpha-domain appears metal-deficient. For the double mutant, C13,50Y, the 113Cd resonances are indicative of major structural reorganizations in both domains.     

Radioimmunoassay of rat liver metallothionein

A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of ¹⁰⁹Cd(II) to a specific activity of 1–2 × 10⁶ cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Mercury,cadmium, zinc and copper concentrations and stable isotope ratios of carbon and nitrogen in tiger sharks (Galeocerdo cuvier) culled off Ishigaki Island,Japan

We analyzed Hg, Cd, Zn and Cu in the liver as well as Hg and stable isotope ratios of carbon and nitrogen (δ¹³C and δ¹⁵N) in muscle from tiger sharks (Galeocerdo cuvier) in Japan. The Hg concentration in the muscle increased slightly and proportionally with increases in body length, but the Hg concentration in the liver increased markedly after maturation (exceeding 2.7 m precaudal length). The Hg concentration in the liver of mature shark was higher than that in the muscle. The Cd concentration in the liver increased with increases in body length. On the other hand, the Zn and Cu concentrations in the liver decreased during the growth stage, but thereafter increased with increased Cd burden due to growth. The marked increase in hepatic Hg in mature sharks may be explained by the continuous intake of Hg via food, slower growth and Hg–Se complex formation. High concentrations of Zn and Cu in the liver of immature sharks and concomitant increases in Zn and Cu with the Cd burden in the liver of mature sharks may be explained by the physiological requirements of Zn and Cu during the growth stage, the induction of metallothionein synthesis due to the Cd burden and the subsequent binding of these metals to metallothionein. The δ¹⁵N and δ¹³C values decreased with increases in body length, suggesting a shift from coastal feeding to pelagic feeding with shark growth. The Hg and Cd concentrations tended to be negatively correlated with the δ¹⁵N and δ¹³C values as a result of the increase in Hg and Cd accumulation due to the growth and the decreases in δ¹⁵N and δ¹³C values due to the sift of feeding area.     

Purification and characterization of recombinant Caenorhabditis elegans metallothionein

The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.