Relative cadmium-binding capacity of metallothionein and other cytosolic fractions in various tissues of the rat.

The Cd-binding capacity of soluble proteins in 10 tissues of normal rats not excessively exposed to heavy metals was measured by saturation of freshly isolated cytosol with 109CdCl2 in vitro followed by Sephadex G-75 chromatography. The Cd-binding capacity of a 10,000 molecular weight Cd-binding peak (10,000 MW Cd-BP), which had a high affinity for Cd and was probably metallothionein, was the highest in kidney (78nmol Cd/g fresh tissue), followed by testis (63 nmol/g), liver (38 nmol/g) and then by brain (14 nmol/g). The amount of the Cd-BP in these tissues (assuming that it was metallothionein and bound 9 mol Cd/10,000g) was calculated to be 87, 70, 42 and 16 mg/kg fresh tissue in kidney, testis, liver and brain, respectively, or in the order of 10(-5) to 10(-6) mol/kg tissue. A significant amount of the 10,000 MW Cd-BP was also found in small intestine. It was present in rather small amounts in heart and lung, and possibly in spleen and skeletal muscle as well. In contrast, the protein was not detectable by this technique in plasma. The results suggest that metallothionein is a rather ubiquitous, intracellular protein in tissues of normal animals and may have other biological functions, besides its possible fortuitous role in heavy metal detoxification. A 30,000 molecular weight Cd-binding peak (30,000 MW Cd-BP) having a very high affinity Cd, apparently higher than that of the 10,000 MW Cd-BP, was found only in testes, among the 10 tissues examined. Its estimated Cd-binding capacity was 51 nmol Cd/g of testis, slightly less than that of metallothionein in testis. These findings support the hypothesis that the 30,000 MW Cd-BP is a plausible target of Cd in Cd-induced testicular injury, and suggest a basis for the peculiar sensitivity of the rat testis to Cd.     

Interactions between cadmium,selenium and glutathione peroxidase in rat testis

In rats given a minimal damaging dose of ¹⁰⁹CdCl₂ (0.011 mmole/kg, s.c.), a visible hemorrhagic response was evident after 48 h when testicular Cd uptake exceeded a level of approx. 150 ng/g. Glutathione peroxidase (GSH-Px) activity was elevated in homogenates of these damaged testes. In rats whose testes were not damaged, the Cd levels were below 150 ng/g and the GSH-Px activity was similar to that of control animals injected with sodium acetate. Rat testis cytosol was found to contain two different GSH-Px activities when assayed with cumene hydroperoxide. These could be separated by gel filtration chromatography. The larger species (GSH-Px A) was eluted in the void volume on Sephadex G-150 and incorporated ⁷⁵Se from Na₂⁷⁵SeO₃ given 4 weeks earlier. The smaller species, of approx. 42 000 molecular weight (MW) (GSH-Px B), did not incorporate ⁷⁵Se and could be distinguished from GSH-Px A by its insensitivity to cyanide (10 mM). CdCl₂ (1 mM) did not inhibit GSH-Px activity when added in vitro to GSH-Px A or B from testicular cytosol, or to purified GSH-Px isolated from ovine erythrocytes. When ¹⁰⁹CdCl₂ was given in vivo to rats injected 4 weeks previously with a tracer dose of Na₂⁷⁵SeO₃ or added in vitro to cytosol prepared from similarly labeled rats, Sephadex G-150 chromatography of cytosol showed that most of the ¹⁰⁹Cd was eluted in a major peak of 34 000 MW. Little or no ¹⁰⁹Cd was found in association with ⁷⁵Se (major peak 140 000 MW) or GSH-Px activity. When ¹⁰⁹CdCl₂ was injected into rats given an equimolar dose of Na₂⁷⁵SeO₃ 30 min previously, ¹⁰⁹Cd uptake in cytosol was increased and both ¹⁰⁹Cd and ⁷⁵Se was shifted into a peak of 110 000 MW.The ¹⁰⁹Cd-binding peak of approx. 30 000–34 000 MW was the major Cd-binding fraction in cytosol of 7-week-old rats but was not detectable in 4-week-old rats. Susceptibility of the testes to Cd did not correlate with the presence of this peak, however, since 4-week-old rats were occassionally damaged by CdCl₂.     

Variation in cadmium accumulation potential and tissue distribution of cadmium in tobacco

Variation in Cd accumulation between Nicotiana species but not varieties has been observed in seedlings grown in solution culture with moderate-to-low levels of Cd. Nicotiana tabacum has been characterized as a leaf and root accumulator while Nicotiana rustica is shown to be primarily a root accumulator, having about half the leaf Cd per gram dry weight of N. tabacum. This phenotype is retained in the mature N. rustica plant. To characterize these two species which differ in their modes of Cd accumulation, tissue Cd distribution, partitioning of metal in soluble and insoluble fractions and the contribution of soluble Cd-binding proteins (peptides) to total plant Cd was assessed using mature solution cultured plants. Metal accumulation was highest in the most mature leaves and in young roots. The preponderance of young roots in N. rustica may, in part, account for low leaf/high root Cd accumulation in this species. While Cd-binding peptides appear to be a principal form of Cd in leaves and roots of seedlings and these also occur in mature leaves, Cd is equally distributed between soluble (about 80% as Cd-binding peptide) and uncharacterized insoluble forms in mature plant roots.     

Some properties of a unique cadmium-binding moiety in the soluble fraction of rat testes.

A 30,000 molecular weight testicular Cd-binding peak (30,000 MW Cd-BP) previously implicated in Cd-induced testicular injury was unstable during storage with respect to apparent molecular weight determined by Sephadex G-75 chromatography. Storage of testicular cytosol labeled with 109Cd in vivo or in vitro for several days at 4 degrees C under nitrogen resulted in disappearance of the 30,000 MW Cd-BP and increased 109Cd uptake in other protein fractions. Rechromatography of the previously isolated 30,000 MW Cd-BP after storage gave rise to a 109Cd peak eluting in the higher molecular weight region. The latter effect was prevented by 1 mM dithiothreitol, suggesting that sulfhydryl groups were involved in the apparent aggregation. The 30,000 MW Cd-BP found in testes of rats was not present in testes of roosters, nor in liver and kidney of either species, providing further evidence of a correlation between the occurrence of 30,000 MW Cd-BP protein in the tissue and susceptibility to Cd-injury. The inability of parenterally administered HgCl2 to induce testicular injury compared to the same dose of CdCl2(0.011 mmol/kg) is apparently related to the poor uptake of Hg in the testes (one-eighteenth that of Cd) rather than to an inability of Hg to bind to the 30,000 MW Cd-BP. Our studies indicate that binding of Cd to this unique 30,000 MW testicular component, as yet unidentified, is a possible basis for the unique sensitivity of the testis to Cd injury.     

Toxicity of Cadmium to Amoeba Proteus: A Biochemical Approach

SYNOPSIS The cadmium ion (Cd²⁺) was accumulated by Amoeba proteus in all cellular fractions, the highest level being associated with the cytosol fraction. On gel separation of the cytosol fraction, Cd-binding protein appeared in 2 peaks: one >45,000 MW (peak I) and the other 12,000 MW (peak II). Added cysteine increased the total Cd²⁺ taken up by the cells and resulted in disproportionate increase of Cd incorporated into the Cd-binding protein of peak II. the Cd-binding protein of peak II is analogous to the low-MW, Cdbinding proteins in Anacystis nidulans, Mytilus edulis , and to the metalloprotein of some vertebrates.     

Selenium-containing proteins of rat kidney

Rat kidney selenium (Se)-containing proteins were studied by isotopic labeling with [75Se]selenite or [75Se]selenomethionine via three routes: oral, intraperitoneal injection, and incubation of kidney slices with the isotope. The two major Se-containing proteins in kidney were fractionated and partially characterized. 75Se elution profiles from Sephadex G-150 chromatography were similar for each labeling protocol, except for the profile obtained following incubation of slices with [75Se]selenomethionine. Of the two major 75Se-containing proteins, the one eluting at the void volume during Sephadex G-150 fractionation had a subunit of 23,000 Mr. The 75Se-labeled tryptic peptide from this protein and a 75Se-containing tryptic peptide from glutathione peroxidase had the same elution time from an HPLC column. A 75,000 Mr 75Se-containing protein had a 65,000 Mr subunit, and the 75Se-labeled tryptic peptide from this protein eluted from the HPLC column before that of glutathione peroxidase. Glutathione peroxidase is the most abundant kidney selenoprotein. Injection of animals with 75Se is the method of choice for isotopic labeling of rat kidney Se-containing proteins. Appropriate methods were developed that can be used in future studies of kidney Se-containing proteins.     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

芦苇抗镉污染机理研究

研究了芦苇幼苗体内 Cd的积累、亚细胞微区分布、存在形态和其诱导蛋白以及植物络合素合成抑制剂 (BSO)对芦苇光合作用和生长的影响。在 Cd污染条件下 ,芦苇幼苗植株和根皮层细胞中可积累大量的Cd,但 Cd在芦苇各器官和根皮层细胞亚细胞结构中的分布显著不均 ;Cd在芦苇幼苗体内的分配为 :根 >叶片 >茎 >地下茎 ,在根皮层细胞中的分布为 :细胞间隙 >细胞壁 >液泡 >细胞质。受 Cd污染的芦苇幼苗体内的 Cd以不同化学形态存在 ,其中 Na Cl提取态的 Cd在根和叶片中占的比例均为最大 ,其次为根内的醋酸提取态 ;在叶片中以水提取态为主 ,其它形态的含量相对较低。层析结果表明 ,根和叶片中各存在一种Cd结合蛋白 ,其中根内的 Cd结合蛋白可能是一种植物络合素聚合体。受 Cd诱导 ,芦苇幼苗根中还新合成了一种小分子蛋白或多肽 ,但另有一种蛋白因 Cd影响而消失。此外 ,BSO实验证明了植物络合素对 Cd的解毒作用。可见 ,芦苇的抗 Cd机理与以下几个方面有关 :根部截留 ,细胞间隙积累 ,细胞壁沉淀 ,液泡区域化 ,形成活性较低的难溶化合物 ,形成 Cd结合蛋白     

Interactions of selenium and cadmium with metallothionein-like and other cytosolic proteins of rat kidney and liver

Following injection of rats with CdCl2 and [75Se]selenite using five different protocols, the metallothionein-like proteins (MTLPs) of kidney and liver cytosols were fractionated by Sephadex G-75 gel filtration and DEAE Sephacel ion-exchange chromatography. Cd and 75Se distribution in gel-filtration elution profiles was influenced mainly by the time that elapsed between administration of these elements and by the sequence of their administration. There was no Cd redistribution to high molecular weight proteins after long-term Cd injection when rats were killed 48 hr after 75Se injection. Cd was redistributed from MTLP to high molecular-weight proteins in the liver when Cd and 75Se were injected within 1-3 hr of each other. Incorporation of 75Se into MTLP of kidney and liver was independent of Cd injection. The strength of 75Se binding of MTLP was comparable to the covalent binding of 75Se to glutathione peroxidase. Cd and 75Se did not share binding sites on MTLP. In ligand-exchange studies, 1000 ppm Cd did not displace 75Se from MTLP, but 2% 2-mercaptoethanol displaced 10% of the presumably nonspecifically bound 75Se from kidney and liver MTLP. This study provides new information regarding the apparent covalent binding of Se to low molecular-weight, Cd-containing proteins in kidney and liver.     

Sulphate uptake and metabolism in water hyacinth and salvinia during cadmium stress

Water hyacinth (Eichhornia crassipes (Mart.) Solms) and salvinia (Salvinia auriculata Aubl.) were exposed to toxic levels of Cd with the objective of evaluating its effect on sulphate uptake and metabolism. Plants were treated with 0 and 5 μmol L⁻¹ Cd for 3 days and, then sulphate uptake, ATP sulfurylase activity, soluble thiol content and Cd-binding complexes were determined. Water hyacinth showed a lower sulphate uptake, but its kinetic parameters were not affected by Cd. In salvinia, however, both V_max and affinity to sulphate (1/K_m) decreased with Cd treatment. The ATP sulfurylase activity increased in Cd-treated plant of both species, except in the roots of salvinia. In the presence of Cd water hyacinth always exhibited higher activity of this enzyme. The total soluble thiol content was always higher in water hyacinth. In Cd treated plants it increased in the leaves of water hyacinth, but decreased in salvinia. Cysteine content increased only in water hyacinth leaves, while γ-glutamylcysteine content increased in the two parts of the plants of both species after Cd treatment, especially in water hyacinth. Glutathione contents, on the contrary, after Cd treatment, reduced in both parts of the plants of water hyacinth but only in the leaves of salvinia. The unidentified thiol fraction content increased with Cd treatment in both species, especially in water hyacinth. Root and leaf extracts of both species showed peaks with maxima at A₂₆₅/A₂₈₀. In treated plants these peaks coincided with Cd content peaks indicating the formation of Cd-binding peptides. It was estimated that in the presence of Cd about 97% of Cd was associated with these complexes and water hyacinth had 28% more Cd-binding peptides than salvinia. Despite its lower sulphate uptake, water hyacinth showed higher rates of sulfur reduction and assimilation into soluble thiols. Possibly, glutathione is used in water hyacinth roots to synthesize hitherto unidentified Cd-binding peptides.     

Effects of dietary zinc depletion and food restriction on intestinal transport of cadmium in the rat

The effects of Zn depletion and short-term fasting on intestinal transport of Cd were examined in perfused rat small intestines. The small intestine was isolated with its vascular network intact, then simultaneously perfused from the luminal and vascular sides. A Zn-depleted state that results in marked hypozincemia was produced in some rats by feeding a Zn-deficient diet for 4 days. Uptake of Cd from the luminal perfusate was greater in the Zn-depleted rats, whereas transport of Cd to the vascular perfusate was not affected. Fasting overnight prior to perfusion did not influence Cd transport nor alter the effect of Zn depletion on Cd uptake. The Cd concentration in the soluble fraction of intestinal mucosa from perfused intestines was not different between Zn-depleted and Zn-adequate rats. Gel filtration chromatography of the soluble fraction showed a shift in the distribution of Cd from metallothionein to high molecular weight ligands in intestines from Zn-depleted rats. The decrease in amount of metallothionein-associated Cd corresponded to a decrease of total intestinal metallothionein as measured by the Cd-binding assay. The results suggest association of Cd with intestinal metallothionein did not influence the absorption of Cd under these conditions.     

In vitro assessment of target cell specificity in cadmium carcinogenesis: Interactions of cadmium and zinc with isolated interstitial cells of the rat testes

Summary Cadmium (Cd) induces testicular tumors of interstitial cell (IC) origin in rats which can be prevented by zinc (Zn). Zn-induced synthesis of metallothionein (MT), a metal-binding protein with a high affinity for Cd, is thought to account for tolerance to Cd in most tissues by sequenstration of Cd. However, the mechanism of Zn inhibition of Cd-induced carcinogenesis in the testes is unknown. Our studies with ICs obtained by collagenase dispersion of rat testes, indicate the levels of the Cd-binding protein in ICs are unaltered by Zn. This testicular protein also was found to differe from MT in amino acid content and to have a lower affinity for Cd. Thus, MT does not seem to be involved in protection of ICs against Cd carcinogenesis. Altered Cd toxicokinetics as a possible explantation for Zn-induced tolerance was therefore explored. Cd uptake into isolated ICs had passive diffusion and nonpassive (carrier mediated or active transport or both) components. The nonpassive component of Cd accumulation was markedly reduced by addition of Zn in vitro, indicative of competition for uptake at the cellular level. These results indicate that toxicokinetic alterations leading to reduced Cd accumulation may play an important role in Zn induction of tolerance to Cd carcinogenesis in the testes. Paper presented at the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation.     

Effect of cadmium-feeding on tissue concentrations of elements in germ-free silkworm (Bombyx mori) larvae and distribution of cadmium in the alimentary canal

Silkworm (Bombyx mori) larvae were reared on an artificial diet containing cadmium (Cd) at concentrations of 5 and 80 micrograms/g wet diet from hatching to the fourth instar and then for 5 days at the fifth instar, respectively. Concentrations of Cd and other elements in the alimentary canal, Malpighian tubes, silk gland, fat body and other organs were determined simultaneously by inductively coupled argon plasma-atomic emission spectrometry. Cd was accumulated in the alimentary canal and Malpighian tubes at concentrations of 1100 and 470 micrograms/g dry wt, respectively. The distribution of Cd in the supernatants of the two highly accumulated organs were determined on an SW column by high performance liquid chromatography-atomic absorption spectrophotometry. Cd was primarily bound to inducible high molecular weight Cd-binding proteins.     

Subcellular distribution of selenium and Se-containing proteins in human liver

Selenium is an essential trace element in many living organisms. In the present paper, the subcellular distribution of selenium and Se-containing proteins in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation and column chromatography. Selenium was mainly enriched in nuclei, mitochondria and cytosol. Almost half of Se existed in the nuclei due to their large amount in liver and high Se concentration. 15-30% of Se was found in small compounds with Mr<2000 in the liver components separated by dialysis. The average abundance of Se in small molecular mass species of whole-liver was 23.6%, which suggested most of Se associated with biological macromolecules. Eight kinds of Se-containing proteins with molecular mass of 335+/-20, 249+/-15, 106+/-11, 84.6+/-5.8, 70. 5+/-5.4, 45.6+/-1.5, 14.8+/-2.6, 8.5+/-1.2 kDa were found in the subcellular fractions of human liver. Among them the 335, 84.6 and 8. 5 kDa proteins were individually present in one subcellular fraction, whereas the others coexisted in two, three or four subcellular fractions. The most abundant Se-containing proteins, 70.5 and 14.8 kDa, accounted for 33.6% and 48.5% in the whole-liver soluble Se-containing protein, respectively. The former was enriched in cytosol and the latter was mainly present in nuclei and mitochondria.     

Occurrence of various forms of metallothionein in the rat after a short-term cadmium injection regimen

1. Results are presented showing that the metal-binding metallothionein (Mt) species induced in rat liver in response to Cd administration consist of dimeric and trimeric forms of Mt. A monomeric form might be the first step in the polymerization process. 2. Two proteins (about 8 and 20 kDa mol. wt) are found in hippocampus, but not in brain cortex. 3. These proteins could not be demonstrated to cross-react with our Mt antibody, but the largest of them has strong Cd-binding capacity. 4. Our Mt antibody cross-reacts with a high metal affinity protein present in both brain cortex and hippocampus of twice the mol.wt (20 kDa) of our purified rat liver Mt standard. 5. The results indicate, however, that these Mt like proteins probably emerge from high molecular or membrane bound forms in the cells. 6. A theory is proposed that the predominant polyacrylamide gel band, matching the monomeric, rat liver Mt standard band, seen for all tissues studied in the present work originate from two sources, namely membrane bound and heavy metal induced monomeric form. 7. It is furthermore suggested that those tissues playing an active role in heavy metal metabolism and in protection against toxicity of such metals contain soluble Mts whose active metal-binding forms are oligomers.     

Testicular toxicity induced by dietary cadmium in cocks and ameliorative effect by selenium

Cadmium (Cd) is an ubiquitous environmental pollutant that has been associated with male reproductive toxicity in animal models. However, little is known about the reproductive toxicity of Cd in birds. To investigate the toxicity of Cd on male reproduction in birds and the protective effects of selenium (Se) against subchronic exposure to dietary Cd, 100-day-old cocks received either Se (as 10 mg Na₂SeO₃ per kg of diet), Cd (as 150 mg CdCl₂ per kg of diet) or Cd + Se in their diets for 60 days. Histological and ultrastructural changes in the testis, the concentrations of Cd and Se, amount of lipid peroxidation (LPO), the activities of the antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx), and apoptosis and serum testosterone levels were determined. Exposure to Cd significantly lowered SOD and GPx activity, Se content in the testicular tissue, and serum testosterone levels. It increased the amount of LPO, the numbers of apoptotic cells and Cd concentration and caused obvious histopathological changes in the testes. Concurrent treatment with Se reduced the Cd-induced histopathological changes in the testis, oxidative stress, endocrine disorder and apoptosis, suggesting that the toxic effects of cadmium on the testes is ameliorated by Se. Se supplementation also modified the distribution of Cd in the testis.     

Maleate induced change in the kidney binding of mercury in rats pretreated with cadmium

The kidney uptake of Hg2+ was increased by Cd2+-pretreatment when Hg2+ was given intraperitoneally but not subcutaneously. Subsequent s.c. administration of maleate increased Hg2+ release from the kidneys only if Hg2+ was given subcutaneously. Neither the effect of Cd2+, nor that of maleate, on the distribution of Hg2+ among the renal soluble protein fractions was affected by the route of Hg2+ administration. The protective effect of Cd2+-pretreatment against the nephrotoxic effect of Hg2+ was also independent of the route of Hg2+ administration. Maleate given in nephrotoxic doses removed Hg2+ and copper, but not Cd2+ from the renal metallothionein fraction. Mercury in the urine, however, was not complexed by proteins with the molecular weight of thionein, but was bound to high molecular weight proteins and diffusible molecules. These findings are discussed in relation to the role of metallothionein in the interaction between Cd2+ and Hg2+.     

Effects of copper, cadmium and chromium cations on the freshwater fish Clarias batrachus L

The data on the effects of cations such as Cu2+, Cd2+ and Cr6+ on the changes in the biochemical parameters in a freshwater fish, Clarias batrachus L., showed an increase of the protein content in the liver, kidney, stomach, intestine, testis and ovary, and a decrease in the muscle after Cu2+ and Cd2+ treatment as compared with control data; but the Cr6+ did not cause any changes of protein concentration in the kidney and testis. The administration of Cu2+ and Cd2+ increased the concentration of free amino acids in all the fish organs, whereas the Cr6+ did not changes this concentration in the muscle. A decrease in dry weight, and an increase in tissue permeability after these treatments were recorded in all the organs studied. In general, the above biochemical parameters of the organs were affected by treatments of the above cations in the following order: Cd greater than Cu greater than Cr over control values of C. batrachus, and their effects were markedly pronounced in the liver and kidney, followed by the intestine, stomach, muscle, testis and ovary in this species.     

Metal-protein binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) during short term cadmium stress

Cd-binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) have been analysed in response to the administration of a single dose of Cd ions. Four main components, with apparent molecular weights of 3-4, 12, 20 and greater than 70 K daltons, have been isolated from soluble cell fractions. The variation in molar metal/protein ratios of these components is discussed in terms of the uptake, transfer and detoxification of Cd in the hepatopancreas. A model is proposed which describes Cd flux at the subcellular level and indicates that metallothionein-like proteins may mediate in this process.     

Accumulation of cadmium and induction of its binding protein in the digestive tract of fleshfly (Sarcophaga peregrina) larvae

The larva of Sarcophaga peregrina ( fleshfly ) was fed with cadmium (Cd)-containing diet and the distribution of Cd among tissues was determined by separating each organ. Approximately 90% of Cd accumulated in the larva was found in the digestive tract, the fat body and the Malpighian tube being less effective tissues in its accumulation. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.