- Email: [email protected]
Oyster metallothioneins: Purification and properties
Abstracts
221
Subcellular Cadmium Sequestration by the Chioragocytes of Earthworms Living in Highly Contaminated Soil. A. J. MORGAN, J. E. MORGAN & C. WINTERS.School of Pure and Applied Biology, University of Wales College of Cardiff, PO Box 915, Cardiff CF1 3TL, UK. Litter consuming earthworm species (Dendrodrilus rubidus and Lumbricus rubellus) living on the calcareous spoil heaps of a disused Pb/Zn mine at Draethen, South Wales (OS Ref. No. = 217877) are exposed to very high 'total' (i.e. conc. H N O 3 extractable) concentrations of Pb = 5500 g/g dry wt, Zn = 29 000 #g/g, Cd = 450 #g/g. The earthworms accumulate the metals (L. rubellus: P b = 800/~g/g, concentration factor, C F = 0 . 1 5 ; Z n = 2700#g/g, CF = 0"09; Cd = 820/zg/g, CF = 1-8; D. rubidus: Pb = 2250 #g/g, CF = 0"40; Zn = 1900/~g/g, CF = 0"06; Cd = 1300 #g/g, CF = 2"9), with high proportions of the body burden located in the posterior alimentary canal. Electron probe X-ray microanalysis (EPXMA) ofchloragogenous smears indicates that Pb, Zn and Cd accumulate within this tissue, which separates the intestinal epithelium from the coelom; within these cells the metals are compartmentalized: Pb and Zn are preferentially accumulated within a Prich organelle (chloragosome granules) and Cd in S-rich vesicles, tentatively named 'cadmosomes'. Biochemical studies show that the Cd is sequestered in both worms by metallothioneins with 15-20% cysteine. In the present study we have: (a) performed qualitative EPXMA on unfixed freeze-substituted and cryosectioned/freeze-dried specimens to show that the 'cadmosomes' are not confined to a subpopulation of chloragocytes, thus invoking intracellular metal-recognition pathways; (b) quantitative X-ray microanalysis of the 'cadmosome' matrix, which shows that the S:Cd ratios (D. rubidus = 4"69 +_0"25, n = 1 9 ; L. rubellus= 2"55 + 0"28, n = 13) differ substantially from the SH:Cd values determined in the isolated proteins (D. rubidus isoform B = 2"2; L. rubellus, isoform B = 1'7); (c) used the Shikata orcein histochemical method on 10/~m cryostat sections to show that polymerized metallothionein is located within discrete vesicular structures. We hypothesize that the 'cadmosomes' are a lysosomal compartment containing Cd-metallothionein complex.
Oyster Metallothioneins: Purification and Properties. G. ROESIJADI, a S A N D R A K I E L L A N D b & P A U L KLERKS. a ~UniversiO' of Maryland, Chesapeake Biological Laboratory, Box 38, Solomons, Maryland 20688, USA & bDepartment of Biochemistry and Microbiology, University qf Victoria, Victoria, British Columbia, Canada. Two low-molecular-mass, cadmium-induced, metal-binding proteins were purified from the oyster Crassostrea virginica by acetone precipitation,
222
Abstracts
Sephadex gel chromatography, and anion-exchange and reverse-phase high performance liquid chromatography. Although clearly separate proteins by chromatography, the molecular weights, metal and amino acid compositions, and electrophoretic behaviour were similar. These proteins contained a large number of glycine residues, in addition to cysteine, and lacked methionine, histidine, arginine, and the aromatic amino acids phenylalanine and tyrosine. Determination of the NHz-terminal amino acid sequence of these molecules showed that they were identical in primary structure in this region and differed only in the presence of an NHz-terminal modification in one. This provided an explanation for the isolation of two proteins with otherwise identical characteristics. The sequence was most similar to that of mammalian metallothioneins when compared with other proteins, with all cysteines in the first 27 residues of the oyster metallothionein aligning with those in the mammalian forms. On this basis, these proteins were classified as class I metallothionein. Kinetic experiments on cadmium uptake and binding by gills showed that these proteins were induced between 1 and 4 days of exposure. Induction was associated with increased Cd-binding rates for metallothionein and reduced Cd-binding rates for other intracellular structures in the gills. The intracellular distribution of cadmium could be explained on the basis of competitive binding to ligands with various affinities for the metal, with metallothionein having the highest.
Cd Uptake in Fish: Metabolic Effects and Cd Complexes. L. TALLANDIN I, M. T U R C H E T T O , O. COPPELLOTTI & C. MARCASSA. lnstituto de
Biologia Animale, Universi O' ~[' Padua, Italy. Cd mechanisms have been widely studied in many organisms, including fish. However, the metabolic routes and effects of the metal have not yet been extensively clarified. While the induction of metallothionein (MT) as an -SH polidentate Cd ligand seems to be the most general detoxification response to Cd, there is not, up to now, a general description of the -SH level regulation inside the cell. In this study we follow the response of fish (Zosterisessor ophiocephalus Pall.) to Cd administration by intraperitoneal injections. After Cd exposure for a maximum total amount of 0.66 ppm (by three 0.22ppm distinct injections, the highest non-lethal dose, for this fish), fish were killed and Cd levels checked in the different tissues. The liver accumulated up to 74% of the total administered metal, which was mainly found in the cytosol fraction (70%), linked (up to 80%) to a protein fraction with an apparent molecular weight of 10000, which was isolated and is now under
221
Subcellular Cadmium Sequestration by the Chioragocytes of Earthworms Living in Highly Contaminated Soil. A. J. MORGAN, J. E. MORGAN & C. WINTERS.School of Pure and Applied Biology, University of Wales College of Cardiff, PO Box 915, Cardiff CF1 3TL, UK. Litter consuming earthworm species (Dendrodrilus rubidus and Lumbricus rubellus) living on the calcareous spoil heaps of a disused Pb/Zn mine at Draethen, South Wales (OS Ref. No. = 217877) are exposed to very high 'total' (i.e. conc. H N O 3 extractable) concentrations of Pb = 5500 g/g dry wt, Zn = 29 000 #g/g, Cd = 450 #g/g. The earthworms accumulate the metals (L. rubellus: P b = 800/~g/g, concentration factor, C F = 0 . 1 5 ; Z n = 2700#g/g, CF = 0"09; Cd = 820/zg/g, CF = 1-8; D. rubidus: Pb = 2250 #g/g, CF = 0"40; Zn = 1900/~g/g, CF = 0"06; Cd = 1300 #g/g, CF = 2"9), with high proportions of the body burden located in the posterior alimentary canal. Electron probe X-ray microanalysis (EPXMA) ofchloragogenous smears indicates that Pb, Zn and Cd accumulate within this tissue, which separates the intestinal epithelium from the coelom; within these cells the metals are compartmentalized: Pb and Zn are preferentially accumulated within a Prich organelle (chloragosome granules) and Cd in S-rich vesicles, tentatively named 'cadmosomes'. Biochemical studies show that the Cd is sequestered in both worms by metallothioneins with 15-20% cysteine. In the present study we have: (a) performed qualitative EPXMA on unfixed freeze-substituted and cryosectioned/freeze-dried specimens to show that the 'cadmosomes' are not confined to a subpopulation of chloragocytes, thus invoking intracellular metal-recognition pathways; (b) quantitative X-ray microanalysis of the 'cadmosome' matrix, which shows that the S:Cd ratios (D. rubidus = 4"69 +_0"25, n = 1 9 ; L. rubellus= 2"55 + 0"28, n = 13) differ substantially from the SH:Cd values determined in the isolated proteins (D. rubidus isoform B = 2"2; L. rubellus, isoform B = 1'7); (c) used the Shikata orcein histochemical method on 10/~m cryostat sections to show that polymerized metallothionein is located within discrete vesicular structures. We hypothesize that the 'cadmosomes' are a lysosomal compartment containing Cd-metallothionein complex.
Oyster Metallothioneins: Purification and Properties. G. ROESIJADI, a S A N D R A K I E L L A N D b & P A U L KLERKS. a ~UniversiO' of Maryland, Chesapeake Biological Laboratory, Box 38, Solomons, Maryland 20688, USA & bDepartment of Biochemistry and Microbiology, University qf Victoria, Victoria, British Columbia, Canada. Two low-molecular-mass, cadmium-induced, metal-binding proteins were purified from the oyster Crassostrea virginica by acetone precipitation,
222
Abstracts
Sephadex gel chromatography, and anion-exchange and reverse-phase high performance liquid chromatography. Although clearly separate proteins by chromatography, the molecular weights, metal and amino acid compositions, and electrophoretic behaviour were similar. These proteins contained a large number of glycine residues, in addition to cysteine, and lacked methionine, histidine, arginine, and the aromatic amino acids phenylalanine and tyrosine. Determination of the NHz-terminal amino acid sequence of these molecules showed that they were identical in primary structure in this region and differed only in the presence of an NHz-terminal modification in one. This provided an explanation for the isolation of two proteins with otherwise identical characteristics. The sequence was most similar to that of mammalian metallothioneins when compared with other proteins, with all cysteines in the first 27 residues of the oyster metallothionein aligning with those in the mammalian forms. On this basis, these proteins were classified as class I metallothionein. Kinetic experiments on cadmium uptake and binding by gills showed that these proteins were induced between 1 and 4 days of exposure. Induction was associated with increased Cd-binding rates for metallothionein and reduced Cd-binding rates for other intracellular structures in the gills. The intracellular distribution of cadmium could be explained on the basis of competitive binding to ligands with various affinities for the metal, with metallothionein having the highest.
Cd Uptake in Fish: Metabolic Effects and Cd Complexes. L. TALLANDIN I, M. T U R C H E T T O , O. COPPELLOTTI & C. MARCASSA. lnstituto de
Biologia Animale, Universi O' ~[' Padua, Italy. Cd mechanisms have been widely studied in many organisms, including fish. However, the metabolic routes and effects of the metal have not yet been extensively clarified. While the induction of metallothionein (MT) as an -SH polidentate Cd ligand seems to be the most general detoxification response to Cd, there is not, up to now, a general description of the -SH level regulation inside the cell. In this study we follow the response of fish (Zosterisessor ophiocephalus Pall.) to Cd administration by intraperitoneal injections. After Cd exposure for a maximum total amount of 0.66 ppm (by three 0.22ppm distinct injections, the highest non-lethal dose, for this fish), fish were killed and Cd levels checked in the different tissues. The liver accumulated up to 74% of the total administered metal, which was mainly found in the cytosol fraction (70%), linked (up to 80%) to a protein fraction with an apparent molecular weight of 10000, which was isolated and is now under