Characterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver

Weser, Ulrich; Rupp, Heinz; Donay, F; Linnemann, Frithjof; Voelter, Wolfgang; Voetsch, Walter; Jung, Günther

doi:10.1111/j.1432-1033.1973.tb03111.x

Cited by 201 publications

(48 citation statements)

References 38 publications

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“…All available data indicate that the metal ions are bound through mercaptide linkages (3,20,43,44) and that all cysteinyl residues of the protein participate in metal coordination. The ratio of cysteinyl residues to the sum of cadmium and zinc is close to S in all forms of metallothionein examined thus far (2-5, 16, 20, 45).…”

Section: ---4-4-4-4-4--4-4 N-acetyl-met-asp-pro-asn-cys-ser-cys-val-amentioning

confidence: 99%

Amino-acid sequence of equine renal metallothionein-1B.

Kojima¹,

Berger²,

Vallée³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

212

106

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Section: ---4-4-4-4-4--4-4 N-acetyl-met-asp-pro-asn-cys-ser-cys-val-amentioning

confidence: 99%

Amino-acid sequence of equine renal metallothionein-1B.

Kojima¹,

Berger²,

Vallée³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

212

106

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“…They are unique in their high cysteine content (30%) and their absence of aromatic amino acids [l -31. Metallothioneins are induced by heavy metals in the livers of many species, including rat [4,5], rabbit [6], chicken [7] and fish [8]. Metallothioneins are also induced by metals in crustacea [9], mollusks [9,10], yeast [I 11, and Neurospora [12].…”

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confidence: 99%

Induction of Metallothionein mRNA in HeLa Cells by Dexamethasone and by Heavy Metals

1981

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Synthesis of metallothionein, a cysteine-rich heavy metal binding protein, is induced in cultured HeLa cells both by the heavy metals CdZ+, Zn2+ and Cu2+, and by the glucocorticoid hormone dexamethasone. The accumulation of [35S]cysteine-labeled metallothionein and the amount of translatable metallothionein mRNA show identical concentration dependences in response to dexamethasone treatment and in response to zinc exposure. Induction of translatable metallothionein mRNA is rapid in response to both the metal and glucocorticoid inducers. Increased synthesis and accumulation of metallothionein in response to either metal or glucocorticoid exposure is regulated by the level of translatable metallothionein mRNA in HeLa cells.Metallothioneins are low-molecular-weight proteins which bind the heavy metals ZnZf, CdZ+, Hg", Ag2+ and Cu2+. [20].Hepatic metallothioneins are also induced by various types of stress in rats [21]. The stress-related induction of metallothioneins in vivo suggested that this protein might be regulated by hormonal mechanisms. We have recently demonstrated the induction of metallothionein by the synthetic glucocorticoid dexamethasone, both in HeLa cells [22] and in primary hepatocytes [I 51. The induction of metallothionein by glucocorticoids in HeLa cells is mediated through the cbassic glucocorticoid receptor [23]. The metallothioneins induced in HeLa cells by Zn2+ and glucocorticoids are interia [24], although Zn2+ and glucocorticoid hormones appear to induce metallothioneins by mechanisms independent of one another in these cells [25].In rat liver, metallothionein induction by heavy metals is mediated through an increase in translatable metallothionein mRNA [26,27]. Experiments using actinomycin D and cordycepin suggest that metal regulation of metallothionein synthesis in rat liver is probably at the level of available MRNA [28]. Enger et al. [29] MATERIALS AND METHODS ChemicalsTranslation grade [35S]cysteine (> 600 Ci/mmol) was obtained from New England Nuclear; [3H]cysteine (0.5-2 Ci/ mmol) and [3H]leucine (60 Ci/mmol) were from Amersham. Phenol was distilled under N2 and stored at -20°C. lodoacetic acid was recrystallized from light petroleum (b.p. 30-60 "C). Dexamethasone and dithiothreitol were from Sigma.All other chemicals were reagent grade. Guanidine . HCI (ultrapure) was from Schwartz-Mann. Nonidet P40 was from Bethesda Research Labs.Cell Culture in a serum-free hormone-supplemented medium.HeLa cells were grown as described previously [20.22,30] RNA Extraction HeLa cells from three confluent 100-mm plates were scraped into 1.4 ml of ice-cold phosphate-buffered saline and collected by centrifugation in a Beckman microfuge B for 45 s. The pellet was resuspended in 0.35 ml Tris/saline buffer (0.01 M Tris/HCl, pH 7.0; 0.15 M NaCI, 2 mM MgC12) containing 0.5 "/, Nonidet P40 and lysed for 5 min on ice. Nuclei were pelleted by centrifugation for 2 min in a Beckman microfuge and RNA was extracted from the supernatant by one of two different extraction procedures. In the case of the p...

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“…yielded either one (20,24) or two species (12,25) behind the front. Inasmuch as it moved with the electrophoretic front at pH 8.9 and required high ionic strength for elution from DEAE-cellulose, tomato root Cd-binding protein must be a very anionic molecule.…”

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confidence: 99%

Partial Characterization of a Cadmium-binding Protein from the Roots of Cadmium-treated Tomato

1980

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A Cd-binding protein has been isolated from the roots of Cd-treated tomato plants cv. Rutgers. Almost all the Cd from a high-speed supernatant fraction was recovered in a 10,000-dalton fraction from a gel filtration column coincident with 250-nanometer absorbing material. DEAE-cellulose chromatography of this 10,000-dalton material yielded one major component, which eluted at 0.34 molar NaCI, had an absorption spectrum characteristic of metaDlothioein, and showed absorption changes upon acidification typical of metallothionein. Although the Cd-binding protein did not behave like metallothioneins from animal sources during gel electrophoresis at pH 8.9, a single band containing Cd and staining with Coomassie brilliant blue could be detected following electrophoresis at pH 6.9. Synthesis of the Cd-binding protein appeared to be "induced" by treatment of the plants with Cd2". Recent concern over Cd as an environmental contaminant (23) has stimulated research on the response of plants to Cd (7,15,16). Although dose-response data have been obtained for many plant species, little is known concerning the biochemical mechanisms of Cd toxicity or tolerance (19,22,23 Preparation of Cd-Thionein. The high-speed supernatant was fractionated via gel filtration using Sephadex G-50 fine (Pharmacia Fine Chemicals). The column was equilibrated and the sample eluted with 50 mM Tris (pH 7.8). The appropriate fractions were combined and then subjected to DEAE-cellulose chromatography (Whatman DE52). The DEAE-cellulose column was equilibrated with 50 mM Tris (pH 7.8) and, after sample application, washed with the same buffer. The sample was eluted with 200 ml of a linear gradient of 0.20 to 0.40 M NaCl in 50 mm Tris (pH 7.8). The appropriate fractions were combined and then desalted with coarse Sephadex G-25 using deionized H20 as eluant. The sample then was concentrated by ultrafiltration using an Amicon UM-2 filter. Column dimensions are indicated in the figure legends.Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis was via tube gels, 10 cm long and 0.5 cm in diameter. The running gel was 12% acrylamide (0.4% bisacrylamide) and contained 0.03% (v/v) TEMED5, 0.0 175% (w/v) ammonium persulfate, and 0.38 M bis-Tris adjusted to pH 6.9 with HC1. The stacking gel was 2.5% acrylamide (0.625% bisacrylamide) and contained 23% (w/v) sucrose, 0.0625% (v/v) TEMED, 0.07% (w/v) ammonium persulfate, and 62.5 mM bis-Tris adjusted to pH 5.9 with HC1. The reservoir buffer was 20 mm bis-Tris adjusted to pH 6.5 with glutamic acid. Electrophoresis was at room temperature with DNP-aspartate as "tracking dye." Prior to electrophoresis, samples were mixed with an equal volume of sample buffer consisting of 32% (w/v) sucrose and 100 mm bis-Tris adjusted to pH 5.9 with HC1. Duplicate gels were run. One was stained at 5 Abbreviations: bis-Tris: bis(2-Hydroxyethyl)imino-tris(Hydroxymethyl) methane; DNP-aspartate: 2,4-dinitrophenyl aspartate;

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Characterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver

Cited by 201 publications

References 38 publications

Amino-acid sequence of equine renal metallothionein-1B.

Amino-acid sequence of equine renal metallothionein-1B.

Induction of Metallothionein mRNA in HeLa Cells by Dexamethasone and by Heavy Metals

Partial Characterization of a Cadmium-binding Protein from the Roots of Cadmium-treated Tomato

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