Separation and purification of (Cd, Cu, Zn)-metallothionein in carp hepato-pancreas

Kito, Hideaki; Ose, Youki; Mizuhira, Vinci; Sato, Takahiko; Ishikawa, Tetsuya; Tazawa, Teijiro

doi:10.1016/0306-4492(82)90178-2

Cited by 47 publications

(24 citation statements)

References 29 publications

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“…The precipitate was removed by centrifugation and the 109 Cd(II) bound to MTLP was measured with a 'Wizard 3 1480 Automatic gamma counter (PerkinElmer, Zaventem, Belgium). The MTLP concentrations were calculated on the basis of a molar ratio of Cd/MT of 7 (Kito et al, 1982).…”

Section: Metallothionein Quantificationmentioning

confidence: 99%

Differences in metal sequestration between zebra mussels from clean and polluted field locations

et al. 2009

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a b s t r a c tOrganisms are able to detoxify accumulated metals by, e.g. binding them to metallothionein (MT) and/or sequestering them in metal-rich granules (MRG). The different factors involved in determining the capacity or efficiency with which metals are detoxified are not yet known.In this work we studied how the sub-cellular distribution pattern of cadmium, copper and zinc in whole tissue of zebra mussels from clean and polluted surface waters is influenced by the total accumulated metal concentration and by its physiological condition. Additionally we measured the metallothionein concentration in the mussel tissue. Metal concentration increased gradually in the metal-sensitive and detoxified sub-cellular fractions with increasing whole tissue concentrations. However, metal concentrations in the sensitive fractions did not increase to the same extent as metal concentrations in whole tissues. In more polluted mussels the contribution of MRG and MT became more important. Nevertheless, metal detoxification was not sufficient to prevent metal binding to heat-sensitive low molecular weight proteins (HDP fraction). Finally we found an indication that metal detoxification was influenced by the condition of the zebra mussels. MT content could be explained for up to 83% by variations in Zn concentration and physiological condition of the mussels.

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Section: Metallothionein Quantificationmentioning

confidence: 99%

Differences in metal sequestration between zebra mussels from clean and polluted field locations

et al. 2009

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“…and since then the characteristics of fish MT separated from eel (5), plaice (6), staghorn sculpin (7), carp (8,9), skipjack (10), rainbow trout (11), and gibel (12) were elucidated.…”

Section: Introductionmentioning

confidence: 99%

“…We studied the induction of MT in carp (Cyprinus carpio) exposed to Cd (13), separated MT from carp receiving IP injections of Cd and clarified amino acid compositions and physicochemical characteristics of these proteins (8,9). In addition we examined immunohistological localization of MT in carp organs (14), and the detoxification effect of MT to Cd toxicity (15).…”

Section: Introductionmentioning

confidence: 99%

“…In Japan the environmental quality standard on water pollution of Cd has been established as <0.01 mg/mL. and since then the characteristics of fish MT separated from eel (5), plaice (6), staghorn sculpin (7), carp (8,9), skipjack (10), rainbow trout (11), and gibel (12) were elucidated.We studied the induction of MT in carp (Cyprinus carpio) exposed to Cd (13), separated MT from carp receiving IP injections of Cd and clarified amino acid compositions and physicochemical characteristics of these proteins (8,9). In addition we examined immunohistological localization of MT in carp organs (14), and the detoxification effect of MT to Cd toxicity (15).…”

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confidence: 99%

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Cadmium-binding protein (metallothionein) in carp.

Kito¹,

Ose²,

Sato³

1986

Environ Health Perspect

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When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with the duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl2), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein.By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them.By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. In the nontreated group, metallothionein was present in the acinar cells of hepatopancreas and renal convoluted tubules. In the Cd-treated group (2 mg/kg IP daily for 3 days), metallothionein was present in the nuclei, sinusoids, and extracellular space of hepatopancreas, in addition to the acinar cells. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups. The metallothionein contents increased in hepatopancreas and kidney in the order: Zn>Cd>control group. IntroductionIt is well known that cadmium-induced renal dysfunction may be the causal substance of Itai-Itai disease. During the 1960s fish kills occurred several times following an increase in water pollution of rivers, lakes and seas in Japan. Some of those were caused by Cd. In Japan the environmental quality standard on water pollution of Cd has been established as <0.01 mg/mL. and since then the characteristics of fish MT separated from eel (5), plaice (6), staghorn sculpin (7), carp (8,9), skipjack (10), rainbow trout (11), and gibel (12) were elucidated.We studied the induction of MT in carp (Cyprinus carpio) exposed to Cd (13), separated MT from carp receiving IP injections of Cd and clarified amino acid compositions and physicochemical characteristics of these proteins (8,9). In addition we examined immunohistological localization of MT in carp organs (14), and the detoxification effect of MT to Cd toxicity (15). This paper summarizes some of the main points. Experimental and Results Induction of Cd-Binding ProteinCarp were exposed to 5.30 ppm Cd of in a total volume of 100 L in a polypropylene water tank. Two carp were killed by destruction of the medulla oblongata after 1/3, 1, 4, and 24 hr and 4, 15, and 31 days, and each organ (hepatopancreas, kidney, gill, gastrointestine, spleen, bile, and muscle) were removed. All org...

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“…In addition, MTs function in the homeostasis of essential trace metals such as Cu and Zn (6,7). MTs have been quite well characterized in a number of fish such as rainbow trout (8)(9)(10), salmon (11), plaice (12), carp (13), and eels (14), among others.…”

Section: Introductionmentioning

confidence: 99%

Heavy metal-induced gene expression in fish and fish cell lines.

Price-Haughey¹,

Bonham²,

Gedamu³

1986

Environ Health Perspect

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Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl2 and ZnCl2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 1"Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium. We suggest that both MT and MIP may function in detoxification of heavy metals. IntroductionThe prevalence of toxic heavy metals in the environment is of increasing concern to both the environmental and medical communities. Since aquatic animals are often the first life forms to come into contact with these poisons, the detrimental effects, as well as the mechanisms by which these animals cope with such poisons is ofgreat interest. By such studies it may be possible to establish a biological index for heavy metal toxicity in the aquatic environment.One apparently universal method by which eukaryotes cope with heavy metals is by synthesizing a family of proteins known as metallothioneins (MTs) (1). These low molecular weight, thiol-rich proteins are induced at the transcriptional level in response to heavy metals as well as a variety of other agents and environmental stresses (2-5).It is widely believed that MTs function by binding to toxic metals such as Cd and Hg thus detoxifying the system in question (1). In addition, MTs function in the homeostasis of essential trace metals such as Cu and Zn (6,7). MTs have been quite well characterized in a number of fish such as rainbow trout (8-10), salmon (11), plaice (12), carp (13), and eels (14), among others.We report here the induction of MT and MT mRNA ResultsWe have previously shown (8) that when rainbow trout are subjected to a series of intraperitoneal injections of CdCl2, they respond by producing two isoforms of the low molecular weight metal binding protein metallothionein. These two isoforms, MT-I and MT-II, were purified by standard techniques involving heat denaturation, G-75 column chromatography and ion-exchange chromatography. Figure 1 shows an SDS-polyacrylamide gel demonstrating the MT purification scheme, and Table 1 shows the amino acid analysis of purified MT-I and MT-II. We have also shown (8) that RNA isolated from...

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Separation and purification of (Cd, Cu, Zn)-metallothionein in carp hepato-pancreas

Cited by 47 publications

References 29 publications

Differences in metal sequestration between zebra mussels from clean and polluted field locations

Differences in metal sequestration between zebra mussels from clean and polluted field locations

Cadmium-binding protein (metallothionein) in carp.

Heavy metal-induced gene expression in fish and fish cell lines.

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