Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein

Summary Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAF-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.Abbreviations MT Metallothionein - PBS Phosphate Buffered Saline - SDS Sodium Dodecyl Sulfat - PAGE Polyacrylamide Gel Electrophoresis Part of the work was reported in Proceedings of 54th Annual General Meeting of the Society of Biological Chemists, India, 1985.     

A single form of metallothionein is Present in both heavy metal induced and neonatal chicken liver

MultiPlicity of metallothionein and their genes in higher animals are documented extensively in recent literature. In contrast, chicken liver Produced aPParently a single form of metallothionein uPon heavy metal exPosure. This Protein was Purified by gel filtration and ion exchange chromatograPhy and another technique based on heat treatment and acetone fractionation, followed by ion exchange chromatograPhy. In adult uninduced chicken liver the Presence of metallothionein was below the detection limit. But, like mammalian system, chicken liver was found to contain high amount of metallothionein at neonatal stage. This naturally occurring neonatal chicken hePatic metallothionein was Purified and comPared with the heavy metal induced adult hePatic metallothionein. The biochemical and immunobiological comParative analysis of adult and neonatal hePatic metallothionein showed identical characteristics. The neonatal metaltothionein exPressed naturally was a zinc metallothionein and unlike few other mammalian neonatal metallothionein did not contain any coPPer. Metallothionein was undectable in unfertilized eggs, in early embryos, and in Postnatal chicken, from 4 weeks after birth. The highest level of this naturally occurring neonatal metallothionein was found in 1–4 day old neonatal liver, which was about 1.5% of the total cytosolic Protein. This is the first rePorted evidence for the Presence of ontogenically modulated exPression of metallothionein in avian system. Possible biological role of neonatal metallothionein and their cellular interactions has been discussed.     

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--I. Exposed to an artificial mixture of zinc, copper and cadmium

Rainbow trout developed elevated hepatic metallothionein concentrations after 4 weeks in a solution containing zinc, copper and cadmium in a fixed ratio of 400:20:1. Resistance to a combination of these metals increased in proportion to the concentration to which they were exposed for 4 weeks. The concentration of copper but not zinc or cadmium in the low molecular weight proteins separated by gel filtration was related to the concentrations of metallothionein present. The combined toxicity of the metals in the mixture was additive.     

Metal-binding proteins of the Syrian hamster ovaries: apparent deficiency of metallothionein

A deficiency of metallothionein, a high-affinity metal-binding protein thought to detoxify cadmium, has been observed in rat and mouse testes, tissues that are highly susceptible to the necrotizing and carcinogenic effects of cadmium. Like the testes, the ovaries undergo a hemorrhagic necrosis when exposed to cadmium, and female Syrian hamsters have recently been shown to be highly susceptible to cadmium. However, the nature of cadmium-binding proteins in the ovary is unknown; thus, this study was undertaken to define the nature of any such proteins in the Syrian hamster ovary. A low molecular weight (Mr) zinc- and cadmium-binding protein was detected in cytosol derived from the ovaries after gel filtration that eluted with a relative elution volume similar to authentic metallothionein. This protein was extractable by heat-treatment and sequential acetone precipitation. When such extracts were further purified with a reverse phase high performance liquid chromatography (HPLC) technique developed for the isolation of metallothionein isoforms, two forms were separated. However, neither of these could be classified as metallothionein on the basis of amino acid composition, since both were particularly low in cysteine, a very common amino acid in metallothionein. The ovarian protein also contained significant amounts of aromatic amino acids, unlike metallothionein--which is devoid of aromatics, and contained much more glutamate than metallothionein. Hamsters were also made resistant to cadmium-induced ovarian necrosis by zinc treatment. Such zinc treatment, however, did not alter levels of this protein, yet caused a marked induction of hepatic metallothionein. Likewise, cadmium treatment did not increase the levels of the ovarian metal-binding protein yet markedly induced hepatic metallothionein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cd-metallothionein and metal-enzymes interactions in the goldfish Carassius auratus

Goldfish injected with cadmium chloride synthesized metallothionein. Ten days after the first injection, cadmium reached a maximum in the metallothionein peak (2 micrograms/ml) obtained after gel filtration of liver cytosol. Pyruvate kinase activity was inhibited from the beginning of the experiment; after the fourth day, the enzyme activity again started to increase but did not reach the control level. Alkaline phosphatase and fructose biphosphatase did not show any apparent inhibition. From the results here reported, a detoxifying role of metallothionein could be suggested.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Is cadmium released from metallothionein in rejected human kidneys?

Summary Concentrations of metallothionein and metals, i.e. cadmium, copper and zinc, were determined in six rejected transplanted human kidneys and one kidney prepared for transplantation. Tissue samples separated by gel chromatography showed that almost all of cadmium in tissue was in the form of firmly bound cadmium-metallothionein.     

Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure

Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtail Orchesella cincta by determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0 μmol Cd/dry food. Cadmium is primarily located within the gut of O. cincta and isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115 μg metallothionein/g fresh gut. The O. cincta metallothionein gives the possibility of using this protein as a biomarker for heavy metal exposure in soil insects.     

Uptake of cadmium is diminished in transfected mouse NIH/3T3 cells enriched for metallothionein.

To determine the relationship between cellular uptake of cadmium and content of metallothionein, we measured uptake of 109Cd in cells that differed in content of metallothionein (MT). MT cells were derived from NIH/3T3 cells by transfection with a plasmid containing the genome of bovine papilloma virus and the mouse metallothionein-I gene, driven by the promotor for the glucose-regulated protein of 78 kDa. Control cells were similarly transfected with bovine papilloma virus-based plasmids with the gene for metallothionein inverted and thus separated from the promoter (TM), or deleted, along with the promoter (BPA). The number of copies of bovine papilloma virus-based plasmids was similar in MT, TM, and BPA cells, approximately 100 per cell. MT cells were more than 10 times more resistant to the lethal effect of cadmium than were the control cells. Synthesis of metallothionein was 15-fold greater in the MT cells than in the TM or BPA cells. The uptake of 109Cd by the cells enriched in metallothionein was 4-fold less than by the control cells. These data suggest that an increased content of metallothionein may protect some cells from the toxic effects of cadmium, in part, by diminishing uptake of the metal.     

Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium

Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.     

Cadmium speciation studies in the intestine of Lumbricus terrestris by electrophoresis of metal proteins complexes

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO₃)₂ supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (¹⁰⁹Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.     

Cadmium-copper interaction in intestinal mucosal cell cytosol of mice

In vivo as well as in vitro protein-metal interaction was studied in cytosolic fractions from intestinal mucosal cells. Female Swiss-Webster mice wre pretreated with cadmium (25 ppm) or copper (100 ppm) in drinking water for 3 weeks. Treatment groups were divided into subgroups receiving Cd or Cd+Cu for an additional 6 weeks. In the in vitro study, mucosal cytosol obtained from pretreated animals was incubated with Cd-109 or Cd-109+Cu. Proteins were separated by gel filtration chromatography and metals determined by furnace AAS or gamma-spectrometry. Cadmium-induced synthesis of metallothionein-like proteins (MTP) in cytosol was indicated by increased Cd in those eluted fractions corresponding to the molecular weight of purified equine renal metallothionein. This cadmium level reached a plateau after 3 weeks of cadmium treatment. In addition, an increased amount of cadmium bound to MTP was noted when copper was added to cadmium in drinking water of mice pretreated with copper. This was not the case for Cd-pretreated animals. The in vitro experiments produced similar results, in that MTP fractions retained a greater percentage of Cd when animals were pretreated with copper compared to controls. Cadmium pretreatment resulted in even higher amounts of cadmium bound to MTP. The existence of a Cd as well as a separate Cu MTP, each with specific metal-binding properties, is suggested.     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 microM cadmium chloride (Cdr) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [35S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cdr lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [35S]Cysteine-labelled protein eluted at a Ve/Vo = 2.1 on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7-17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound 109Cd2+.     

Application of a modified 203Hg binding assay for metallothionein

A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of ²⁰³Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound ²⁰³Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by ²⁰³Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl₂. Similar separation methods demonstrated [³⁵S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples.     

Circular dichroism and thermal denaturation studies of chromatin and DNA from BrdU-treated mouse fibroblasts

A simple procedure for the isolation and pruification of metallothionein from rat liver is described. This method involves only four steps and is especially useful for large scale isolation of this protein. The final isolated preparation was homogeneous both in Sephadex gel filteration and in polyacrylamide gel electrophoresis. Isoelectric focussing shows the presence of two cadmium binding proteins with isoelectric points of 4.2 and 4.7. Metallothionein is isolated from dog liver using this method.     

Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes.

An isolated rat hepatocyte preparation was used to study the cellular toxicity of cadmium and the protective effects of metallothionein on cadmium-induced toxicity. Exposure of primary suspension cultures of isolated rat hepatocytes to Cd2+ (0-35.7 microM) for 15 min resulted in a dose-dependent reduction in the synthesis of cellular proteins during a subsequent 6 h incubation. Such inhibition could not be correlated with cellular lethality or gross membrane damage. Pre-induction of metallothionein in hepatocytes by zinc treatment in vivo of donor rats protected hepatocytes in vitro from cadmium-induced inhibition of protein synthesis. The protective effects in zinc-pre-induced hepatocytes are not due to alterations in the level of total cellular cadmium, but could be accounted for by the redistribution of intracellular cadmium in the presence of high levels of zinc-metallothionein. The data suggest that metallothionein exerts its protective effect by a kinetic detoxification mechanism, i.e. a decrease in reactive intracellular cadmium.     

Induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells is controlled by the level of intracellular copper

A study was carried out on the uptake of copper, zinc, or cadmium ions and their induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells. The main difference between Menkes' and normal cells in the uptake of these metal ions was an increased uptake of copper ions in Menkes' cells at a low concentration of CuCl2 (2.1 microM). The CuCl2 concentration necessary to induce metallothionein synthesis in Menkes' cells was 50 microM, whereas that in normal cells was about 200 microM. The levels of zinc or cadmium ions needed to induce metallothionein in Menkes' cells were similar to those in normal cells. At least four isomers of metallothionein were induced by copper, zinc, and cadmium ions in both types of cells. Metallothionein synthesis in Menkes' and normal cells was induced when the amounts of intracellular copper reached a threshold level of approximately 0.2 nmol/10(6) cells, and the rate of metallothionein synthesis in these cells was increased as a function of the amounts of intracellular copper (0.2-1.7 nmol/10(6) cells). These results indicate that the induction of metallothionein synthesis in lymphoblastoid cells is controlled by the level of intracellular copper, suggesting that the major defect in Menkes' cells is not due to the abnormal regulation of metallothionein synthesis but to an alteration of the copper metabolism in cells by which the levels of intracellular copper become larger than those in normal cells and just lower than the threshold level for induction of metallothionein synthesis.