Arsenic induces and enhances rat hepatic metallothionein production in vivo.

Metallothionein genes (MT) are inducible by a variety of agents, including heavy metals. We report the induction of MT expression by arsenite (As3+) in rat liver in vivo. As3+ (but not arsenate [As5+]) injection increased MT protein and MT-1 and MT-2 mRNA accumulation in liver only, but not in kidney or pancreas. In addition, As3+ enhanced zinc-induced MT protein accumulation in liver without any increase in MT mRNA levels. These data indicate that arsenic may increase MT expression either directly (by inducing MT mRNA accumulation), or indirectly by altering post-transcriptional events. This constitutes an unusual mechanism of enhancement of MT gene expression and appears to be mediated by processes not specifically associated with binding of arsenite to MT in vivo.

A luminescence probe for metallothionein in liver tissue: emission intensity measured directly from copper metallothionein induced in rat liver.

We report the first use of an emission probe based on the Cu(I)-thiolate chromophore, for the direct observation of copper metallothionein located in samples of rat liver. Elevated synthesis of Cu-MT in the rat liver was induced by subcutaneous injections of a series of aqueous CuCl2 solutions containing increasing amounts of Cu(II). Luminescence intensity in the 600 nm region, detected from frozen solutions of Cu-MT and from slices of the liver frozen at 77 K, following excitation in the 300 nm region, was dependent on the concentration of the Cu(II) used in the inducing solution. No such luminescence intensity was found for control samples obtained from the livers of rats not exposed to copper salts. It is suggested that this new method will allow direct visualization of Cu-MT in tissue where genetic disorders impare copper metabolism.

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.

Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein alpha-fragment.

The yield of the alpha-fragment of rabbit liver metallothionein 2 was used to test the domain-specificity and mobility of Cd2+ and Zn2+ when bound to metallothionein. Increasing molar ratios of Cd2+ were added to either Zn7-metallothionein or the metal-ion-free apo-metallothionein. The enzyme subtilisin was used to digest those parts of the peptide chain that were not bound to Cd2+. Analysis of the digestion products was carried out by separation by polyacrylamide-gel electrophoresis. The chelation agent EDTA was used as a competitive chelator. It was found that the presence of excess EDTA greatly enhances the formation of the Cd4-metallothionein alpha-fragment, and catalyses the complete digestion of all other the metal-ion-containing peptides, so that even Cd7-metallothionein, formed when 7 molar equivalents of Cd2+ are added to Zn7-metallothionein, is digested to the alpha-fragment. These results suggest that the Cd2+ bound in the beta-sites is very labile, much more labile than the kinetics of the off-reaction would suggest. The observation of significant amounts of alpha-fragment on the gels, even when the stoichiometry of the metal ions initially present in the protein should not have resulted in much concentration of Cd4-alpha-fragment clusters, indicates that as the digestion proceeds the metal ions move to sites that form complete clusters and therefore selectively protect that part of the peptide chain from digestion. We also find that rabbit Cd4-metallothionein 2 alpha-fragment stains near to the top of the gel, in complete contrast with the location of rat Cd4-metallothionein 2 alpha-fragment. This difference in the mobilities suggests that the alpha-fragment prepared from rabbit metallothionein 2 is much less negatively charged than the analogous protein fragment prepared from rat liver metallothionein 2.

Luminescent Ag12-metallothionein: dependence of emission intensity on silver-thiolate cluster formation.

We report the observation of emission intensity at 77 K that is a function of Ag(I)-thiolate bonds formation within the protein metallothionein. The emission characteristics (a large, 250 nm, Stokes shift and long emission lifetime) suggests that the transition occurs from the excited triplet state. The emission intensity and circular dichroism both indicate that silver(I) clusters form with stoichiometric ratios of 12 Ag(I) to the 20 thiolate sulfur groups that are present in the protein. These data are the first to show that Ag(I)-metallothionein complexes are luminescent and that a specific Ag12-MT species forms.

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes.

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.

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.

Information on metal binding properties of metallothioneins from optical spectroscopy.

Absorption, circular dichroism and emission measurements made during titrations of rabbit liver Zn-MT and calf liver Cu, Zn-MT with Cd2+ and Cu+ are reported. There are systematic changes in the CD and emission spectra that can be associated with the formation of several species during these titrations. Addition of Cu+ to Zn-MT results in the formation of distinct species that form at specific stoichiometries, these are: Cu6-MT, Cu12-MT and Cu20-MT. The emission intensity due to Cu+ provides a sensitive indication of the presence of Cu-S clusters for the Cu6-MT and Cu12-MT species, suggesting that Cu6-Sx clusters form in both the alpha and beta domains of the protein. The data also demonstrate that Cd7-MT will bind 12 Cu+ to form a species with the stoichiometry of 12 Cu: 4 Cd, and that, surprisingly, Cu12-MT will also bind Cd2+ to form this same new species. It is suggested that the new species incorporates a Cu6 cluster in the beta domain and a mixed-metal Cu6, Cd4 cluster in the alpha domain.

Spectroscopic properties of the alpha fragment of metallothionein.

Absorption, CD, and magnetic circular dichroism spectra are reported for the alpha fragment of rat liver Cd,Zn-metallothionein (MT) 2. The CD and magnetic circular dichroism spectra of the Cd4 cluster unit are particularly well-resolved and are remarkably similar to data of the complete Cd,Zn-MT. It is suggested that the high signal intensity in the 225 nm CD band may be attributed to an interaction between a terminal amino acid residue and the Cd4 cluster. Titration experiments with CdCl2 and [Cu(CH3CN)4]+ show that while no additional Cd2+ can be bound in the presence of excess Cd2+, Cu+ does replace the bound Cd2+ in a complex reaction to form at least two species. One of these species requires the presence of both Cu+ and Cd2+, with a stoichiometry of Cu 3.0, Cd 2.5. Further, Cu+ displaces all the remaining Cd2+, and the spectra recorded now closely resemble Cu-MT formed by titration of Cd,Zn-MT with greater than 8 mol eq of Cu+.

In vivo and in vitro binding of platinum to metallothionein.

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammineplatinum(II]. Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl2 significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl2 pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.

Spectroscopic characterization of rat kidney Hg,Cu-metallothionein.

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra are reported for rat kidney Hg,Cu-metallothionein isoform 3 isolated following induction of the metallothionein with HgCl2. While the absorption spectrum is featureless, both the CD and MCD spectra show resolved bands that arise from the Cu-thiolate and Hg-thiolate groups. The emission spectrum at 77 K is much more complicated than would be expected for a copper (I)-containing metallothionein. It is suggested the emission only arises from the copper-thiolate groups but that the presence of the mercury results in copper ions in several different environments depending on the nature of the nearest neighbour.

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.

Mercury-binding, copper-zinc proteins from rat kidney. Amino acid composition, molecular wieght and metal content.

Three isoforms of mercury-binding, copper-zinc protein were isolated from kidney of rats exposed to mercuric chloride. They contained 56-67 micrograms Hg/mg protein, 23.7-43.9 micrograms Cu/mg and 1.8-5.4 micrograms Zn/mg protein. Amino acid composition of isoforms 1, 2 and 3 was very similar, and also close to that of hepatic [Hg,Zn]- and [Cd,Zn]-metallothionein. Isoforms 1, 2 and 3 contained 29.7, 27.2 and 29.3 mol% cysteine, respectively. The molecular weight of isoforms 1, 2 and 3 was 6580, 6880 and 7000, respectively.

Purification of low molecular weight metal-binding proteins by preparative polyacrylamide gel electrophoresis: properties of electrophoretically purified rat liver (Cd, Zn) - metallothioneins.

A method is proposed for purification of metallothionein by preparative polyacrylamide gel electrophoresis. The method enables purification of 100-700 mg of a preparation containing (Cd, Zn) - metallothionein yielding preparations of considerably higher purity as compared with those obtained by ion-exchange chromatography.

Low molecular weight cadmium-and copper-binding proteins from rat kidneys.

Three isoforms of rat kidney cadmium- and copper-binding proteins [(Cd, Cu)-BP 1, 2, and 3] were isolated. They contained from 75.0 up to 89.0 µg Cd/mg protein, from 7.5 up to 28.0 µg Cu/mg, and from 1.5 up to 12.0 µg Zn/mg protein. Apparent molecular weights of all three isoforms were of about 10,000. Their amino acid compositions were similar to that of rat metallothionein, with cysteine amounting to 25.8-32.7% of all amino acids.

Induced synthesis of chromochelatin, the low molecular weight bismuth-binding protein in rat kidneys.

Bismuth administered subcutaneously to rats as BiCl3 is deposited in the kidneys, where it is bound to two classes of proteins: one of high molecular weight and a fraction of molecular weight approx. 7500 (chromochelatin). The latter fraction prevails on repeated exposure to bismuth. The bismuth-binding protein is heterogenous and using polyacrylamide gel may be divided into three fractions of which all contain bismuth and copper. In parallel with increasing concentration of chromochelatin due to bismuth administration, the incorporation of L-[35S]cysteine is elevated in all three fractions. The incorporation is augmented especially if repeated administration of bismuth is applied. Cycloheximide (CH) completely abolishes the inducing effect of bismuth on the incorporation of L-[35S]cysteine into chromochelatin both following single and repeated administration of bismuth. Actinomycin D (AcD) eliminates the incorporation only in the case of single dose of bismuth. The obtained results suggest that the elevation of chromochelatin levels in the kidney following administration of bismuth is due to the induction of the de novo protein synthesis.