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.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.     

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.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Organization and assembly of metal-thiolate clusters in epithelium-specific metallothionein-4

Mammalian metallothionein-4 (MT-4) was found to be specifically expressed in stratified squamous epithelia where it plays an essential but poorly defined role in regulating zinc or copper metabolism. Here we report on the organization, stability, and the pathway of metal-thiolate cluster assembly in MT-4 reconstituted with Cd(2+) and Co(2+) ions. Both the (113)Cd NMR studies of (113)Cd(7)MT-4 and the spectroscopic characterization of Co(7)MT-4 showed that, similar to the classical MT-1 and MT-2 proteins, metal ions are organized in two independent Cd(4)Cys(11) and Cd(3)Cys(9) clusters with each metal ion tetrahedrally coordinated by terminal and bridging cysteine ligands. Moreover, we have demonstrated that the cluster formation in Cd(7)MT-4 is cooperative and sequential, with the Cd(4)Cys(11) cluster being formed first, and that a distinct single-metal nucleation intermediate Cd(1)MT-4 is required in the cluster formation process. Conversely, the absorption and circular dichroism features of metal-thiolate clusters in Cd(7)MT-4 indicate that marked differences in the cluster geometry exist when compared with those in Cd(7)MT-1/2. The biological implication of our studies as to the role of MT-4 in zinc metabolism of stratified epithelia is discussed.     

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.     

Silver binding to rabbit liver metallothionein. Circular dichroism and emission study of silver-thiolate cluster formation with apometallothionein and the alpha and beta fragments

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

锌_7与镉_7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白制备去金属金属硫蛋白、锌７与镉７金属硫蛋白．在不同ｐＨ条件下,比较后二者清除羟自由基能力;在ｐＨ６条件下,比较锌７－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理ｐＨ条件下锌７－金属硫蛋白清除羟自由基能力远强于镉７－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., K?gi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.     

Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+.

Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing experiments were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, respectively. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Co(n)-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric analysis of the titration suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titration of free sulfhydryl groups (S) in Co(n)-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Co(n)-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.     

Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia.

The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.     

Influence of chloride ligands on the structure of Zn- and Cd-metallothionein species

It is now commonly accepted that non-proteic ligands contribute to the structure and stability of metal-metallothionein (M-MT) species, although this contribution may differ substantially depending on the MT and the metal ions involved. Conversely, literature data are unconnected, lacking correlation studies between the contribution of inorganic ligands to the M-MT complexes and the corresponding CD and UV-vis fingerprints. To contribute towards filling this gap, we have analyzed the influence of chloride anions in the Zn- and Cd-MT complexes of mammalian MT1 and MT4 isoforms. Starting from the initial hypothesis that the shoulders appearing at 240nm in the UV-vis difference spectra during the Cd(II) titrations of Zn-MTs would be indicative of chloride participation in these metal-MT complexes, we can now propose that, while their absence definitely rules out these ligands being involved in metal coordination, their presence should not necessarily be attributed to the formation of metal-Cl bonds. Instead, we identified a global blue shift for the UV-vis difference spectral envelope as the most liable indication of chloride participation in the binding sites of the M-MT species. Following this criterion, we determined that chloride anions are bound to the Cd(7)-MT1 and Cd(4)-alphaMT1 complexes but not in the isostoichiometric Zn complexes, nor in the Zn- or Cd-complexes of the homologous MT4 peptides.     

M?ssbauer studies on the metal-thiolate cluster formation in Fe(II)-metallothionein

The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using M?ssbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting delta EQ and isomer shift delta as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vasák (1986) Biochemistry 25,8353--8356]. The M?ssbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in M?ssbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the M?ssbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)4-MT form appear to be magnetically isolated, whereas in the fully metal-saturated 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of M?ssbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.