Isolation and chemical characterization of the heavy metal-binding protein metallothionein from marine invertebrates

Olafson, Robert W.; Sim, Rob; Boto, Kevin

doi:10.1016/0305-0491(79)90111-1

Cited by 57 publications

(29 citation statements)

References 28 publications

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“…Throughout the feeding period the zinc concentrations in the digestive glands of lobsters tended to increase ( The results of this investigation have shown that the concentrations of cadmium and zinc in the individual digestive glands of fed lobsters increased throughout the experiment but were extremely variable, and that the induction of a cadmium-containing metallothionein was dependent upon the tissue concentration of cadmium. In other investigations with crustaceans, it was assumed that the induction of cadmium metallothionein was immediate (4,15,16), as was shown to occur in the gills of the blue crab (11). In many of these other investigations, however, injection was used as the mode of exposure rather than having the metal incorporated into food or dissolved in water.…”

Section: Resultsmentioning

confidence: 99%

“…Crustaceans (1)(2)(3)(4)(5)(6) and particularly the American lobster, Hornarus americanus (7,8), concentrate metals, such as cadmium, copper, and zinc in their digestive glands (i.e., hepatopancreas). In addition to being a possible direct hazard to the organism, these metals also may be hazardous to organisms that consume digestive glands of crustaceans, including man.…”

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confidence: 99%

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Cadmium and copper metallothioneins in the American lobster, Homarus americanus.

Engel¹,

Brouwer²

1986

Environ Health Perspect

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Lobsters were fed cadmium-rich oysters for 28 days, and the induction of cadmium metallothionein and its relation to concentrations of cadmium, copper, and zinc in the digestive gland and gills was determined.A portion of the tissues also was retained for determining the cytosolic distribution of these metals by gel filtration and ion-exchange chromatography. The digestive gland contained a majority of the cadmium, copper, and zinc, and both cadmium and zinc were actively accumulated from the oysters. Gel chromatography of the digestive gland cytosol showed that initially cadmium and zinc were bound to macromolecules with molecular weights of > 70,000, -45,000 and < 5000, and for copper > 70,000, 10,000-7,000, and < 5000. Therefore, only copper was bound to a protein with a molecular weight in the range of metallothionein (i.e., 10,000-7,000). However, after feeding on cadmium-laden oysters for 28 days, both cadmium and copper were bound to the metallothioneinlike protein. Further purification ofthe cadmium/copper protein by ion-exchange chromatography showed that a large portion of the copper and all of the cadmium did not bind to DEAE-Sephacel. The induction of cadmium metallothionein in the digestive gland is correlated with tissue cadmium concentration. There is, however, a tissue threshold concentration of cadmium of 80 to 100 1Lg Cd/g wet weight required for induction. Coincident with the induction of the cadmium metallothionein was a cytosolic redistribution of copper. The distribution of zinc was not affected.Crustaceans (1-6) and particularly the American lobster, Hornarus americanus (7,8), concentrate metals, such as cadmium, copper, and zinc in their digestive glands (i.e., hepatopancreas). In addition to being a possible direct hazard to the organism, these metals also may be hazardous to organisms that consume digestive glands of crustaceans, including man. In the case of the lobster, such a situation already has occurred near Belledune Harbor, New Brunswick, Canada (7,9). Here, digestive glands from local animals were shown to contain up to 203.7 ,ug/g wet weight of cadmium (9). These levels were considered a health hazard to the local population, who consumed the digestive gland. A similar situation also has occurred for blue crabs, Callinectes sapidus, (1,2) from the Hudson River, where animals from Foundry Cove had cadmium concentrations in the digestive glands of up to 20 ,ug/g wet weight.Because lobsters can accumulate large quantities of metals in the digestive gland, a physiologically active detoxification system must be present to sequester the accumulated metals, such as cadmium, copper, and zinc. It is, therefore, of interest to determine the chemical forms of these metals in the tissue. In our earlier research, we demonstrated that lobsters accumulate cadmium more efficiently from food than from water (8,10). Also the lobster has m etal-binding proteins in the diges-

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Cadmium and copper metallothioneins in the American lobster, Homarus americanus.

Engel¹,

Brouwer²

1986

Environ Health Perspect

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“…1) demonstrate that all the cadmium was associated with a CdMT fraction with an average apparent molecular weight of 13 700 Da, which was different from the molecular weight of 7328 Da obtained on calibrated gel SDS-PAGE electrophoresis. This discrepancy is largely reported in well-characterised MT proteins from other organisms (Mackay et al, 1993;Olafson et al, 1979;Roesijadi et al, 1989), and is attributed to their flexible (Vasak et al, 1984), nonglobular, prolate ellipsoid shape with a calculated axial ratio of 6 (Kagi et al, 1974).…”

Section: Discussionmentioning

confidence: 99%

“…Suggestions included different specificity of the promoter region of the gene (Schmidt et al, 1985), different metal-binding affinities (Winge and Miklossy, 1982) or simply the need for a large number of copies to facilitate a rapid response when necessary (Olafson et al, 1979). Considering the NH 2 -terminal sequence results (Table 2), the absence of methionine is consistent with other aquatic invertebrate MTs (Roesijadi et al, 1989;Mackay et al, 1993) indicating that MTs in this types of organisms are subject to NH 2 -terminal modifications following initial synthesis that does not occur in the mammalian MTs.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

2003

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Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus ) using gelpermeation and ion-exchange chromatography. Four cadmium Á/metallothioneins (CdMTs) were resolved by ionexchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd Ã/S aggregates. The NH 2 -terminal sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH 2 -terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure. #

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“…This invertebrate MT, like the mammalian MTs, can be induced by the administration of cadmium (36). Native crab MT isolated from l3Cd-injected crabs is homogeneous in its metal composition (36,37) and is therefore particularly amenable to study by 113Cd-NMR (31). Figure 2A shows the 'H-decoupled 113Cd-NMR spectrum of native crab 113Cd-MT-1.…”

Section: '3cd-nmr Of Invertebrate Metallothioneinmentioning

confidence: 99%

NMR analysis of the structure and metal sequestering properties of metallothioneins.

Hunt

Boulanger

Fesik

et al. 1984

Environ Health Perspect

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113Cd-NMR studies have been used to elucidate the structure of the metal-binding sites in mammalian and invertebrate (Scylla serrata) metaliothioneins (MTs). Chemical shift data have shown that all Cd ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. Homonuclear decoupling experiments have shown that the 7 g-atoms of metal bound per mole of mammalian protein are located in a three-and a four-metal cluster while the 6 g-atoms of metal in the invertebrate MT are located in two three-metal clusters. The different metal binding affinities of the two mammalian clusters have been determined by 113Cd-NMR. The threemetal cluster prefers Cu > Zn > Cd whereas exactly the reverse order applies in the fourmetal cluster. Proteolytic cleavage of the protein produced a 32-residue fragment which contained the four-metal cluster and demonstrated the presence of two separate domains in the protein. 500 MHz 'H-NMR has been employed to elucidate the arrangement of these metal clusters in the tertiary structure of the protein. The 1H resonances were assigned from their scalar and dipolar connectivities obtained from extensive one and two-dimensional NMR experiments. A specific application of 2D correlation spectroscopy (COSY) to the assignment of the 1H resonances in crab MT-1 is discussed. A molecular model, representing the three-dimensional solution structure of this protein, has been constructed based on an analysis of all thepe data. Detailed structural features of this model are discussed, with particular emphasis on their relationship to the function and evolution of the protein.

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Isolation and chemical characterization of the heavy metal-binding protein metallothionein from marine invertebrates

Cited by 57 publications

References 28 publications

Cadmium and copper metallothioneins in the American lobster, Homarus americanus.

Cadmium and copper metallothioneins in the American lobster, Homarus americanus.

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

NMR analysis of the structure and metal sequestering properties of metallothioneins.

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