Cd-binding Protein Research Articles

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.

Metallothionein-Null and Wild-Type Mice Show Similar Cadmium Absorption and Tissue Distribution Following Oral Cadmium Administration

Cadmium (Cd) is an environmental pollutant and is toxic to many tissues. Food is the primary source of Cd exposure for the general population. Metallothionein (MT), a cysteine-rich, Cd-binding protein, plays an important role in Cd detoxication. However, the role of MT in Cd absorption and distribution is still controversial. For example, some reports assert that MT in the intestine decreases Cd absorption and increases its distribution to the kidney, relative to the liver. Therefore, to further clarify the role of MT in Cd absorption and tissue distribution, MT-I/II knockout (MT-null) mice and their parental background wild-type mice were given a single dose of 109Cd (1–300 μmol/kg po or 0.1–30 μmol/kg iv). Cd content in 15 organs was determined 4 h after Cd administration by gamma scintillation spectrometry. Approximately 60% of the Cd administered iv was retained in liver, and about 5% was retained in kidney in both MT-null and wild-type mice. The distribution of iv administered Cd was independent of dose. In contrast, when administered po, approximately 0.15% of the lowest dose (1 μmol/kg) and 0.75% of the highest dose (300 μmol/kg) was detected in the liver of both MT-null and wild-type mice. Similarly in kidney, approximately 0.05% of the dose was detected after the lowest dose and about 0.15% after the higher doses in both MT-null and wild- type mice. In summary, this study demonstrates that the absorption and initial distribution of orally administered Cd is dose dependent but is not influenced by MT.

Antioxidant enzymes responses to cadmium in radish tissues

To investigate the antioxidant responses of radish ( Raphanus sativus L.) to cadmium (Cd) treatment, seedlings of a tolerant variety were grown in increasing concentrations of CdCl 2, ranging from 0.25–1 mM, for up to 72 h in a hydroponic system. Analysis of Cd uptake indicated that most of the Cd accumulated in the roots, but some was also translocated and accumulated in the leaves, especially at the higher concentrations of Cd used in the experiments. Roots and leaves were analysed for catalase, glutathione reductase and superoxide dismutase activities. Catalase and glutathione reductase activities increased considerably in the roots and leaves after 24 h exposure to the metal, indicating a direct correlation with Cd accumulation. The analysis of native PAGE enzyme activity staining, revealed several superoxide dismutase isoenzymes in leaves, with the two predominant isoenzymes exhibiting increases in activity in response to Cd treatment. The results suggest that in radish, the activity of antioxidant enzymes responds to Cd treatment. The main response may be via the activation of the ascorbate–glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of Cd-binding proteins.

STUDIES ON A CADMIUM-INDUCED METALLOTHIONEIN IN HOUSEFLY LARVAE, MUSCA DOMESTICA

The induction of metallothionein (MT) was studied in the housefly larvae (Musca domestica). Upon dietary exposure to Cd, two Cd-binding proteins were isolated from the whole body homogenates, using gel filtration and ion exchange chromatography. Mass spectrometric measurement revealed that they have high purity and the molecular weight are 9045.9Da and 11560.2Da, respectively. Amino acid analysis showed that the content of cysteine is the highest, attaining to 18.2%. However, aromatic amino acid residues such as tyrosine (2.5%) and phenylalanine (3.1%) were also detected. In addition, MT from housefly larvae has strong heat stability. All these results suggest that the properties of MT isolated from housefly are very similar to that of mammalian MT, but some differences still exist.

Seasonal dependence of cadmium accumulation and Cd-binding proteins in Mytilus galloprovincialis exposed to cadmium

At different periods of the year specimens of Mytilus galloprovincialis were exposed to 0.5 μg Cd/ml seawater for 7 days. Concentrations of trace elements (Cd, Zn, Cu and Fe) and Cd-binding proteins in gills, viscera, muscle and mantle were determined after 1 weeks exposure. Cadmium accumulation was higher in May and June and was tissue dependent; it was highest in the gills and decreased in the order: gills>viscera>mantle and adductor muscle. Significant seasonal variations of zinc, copper and iron, were also found, in both exposed and control molluscs. The percentage of Cd distribution between cytosol and pellet changed during the year; a clear shift from the particulate fractions to the cytosolic fractions was measured during May and June, especially in the gills. Metallothionein (MT) was the main ligand responsible for Cd accumulation, and this protein reached a maximum between May and June. Inclusion of mercaptoethanol during the purification procedure was found to improve MT isolation by gel chromatography. In the absence of mercaptoethanol, MT showed polymerization patterns which were season dependent and temperature independent, whereas its concentration was increased in mussels kept at higher temperature.

Stage-dependent accumulation of cadmium and induction of metallothionein-like binding activity in the testis of the Dogfish shark, Squalus acanthias.

Cadmium (Cd) is an established spermatotoxicant. Using the shark (Squalus acanthias) testis model, we investigated stage-related patterns of intratesticular Cd accumulation and effect. After a single injection of 109CdCl2, tracer was rapidly eliminated from plasma but accumulated and was retained in testis for at least 7 days. Intratesticular 109Cd was stage dependent, resulting in a 3- to 5-fold gradient: germinal zone (GZ) > premeiotic (PrM) > meiotic (M) > postmeiotic (PoM) stages. When measured as tissue:plasma ratios, the Cd-binding mechanism in GZ (71:1) was similar to that in liver (87:1) but lower than in kidney (381:1). The same intratesticular gradient was seen in untreated controls when tissue Cd levels were measured by atomic absorption spectroscopy, implying environmental exposure. A single CdCl2 injection (5 mg/kg i.v.) elevated testicular Cd > 160-fold in all stages but did not alter the direction or magnitude of the gradient. Intratesticular distribution of metallothionein-like Cd-binding protein was stage dependent (PrM = PoM > GZ = M), but the pattern differed from the Cd gradient. This binding component was Cd inducible in all but M stages, but induction did not alter the stage-dependent pattern of binding activity or Cd accumulation. Analysis of tissue subfractions after in vivo tracer injection indicated that the binding mechanism responsible for the intratesticular gradient is mainly cytosolic, but that a second less abundant component is associated with the nucleus. The functional significance of preferential Cd accumulation in GZ and PrM stages of spermatogenesis remains to be determined.

Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis.

It has been a long-lasting controversial issue as to whether or not the male genital organs, such as the testis and prostate, contain metallothioneins (MTs), a group of cysteine-rich heavy-metal-binding proteins that play a role in detoxifying heavy metals such as cadmium (Cd). Earlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on the basis of amino acid composition analysis, since MT-like proteins isolated as Cd-binding proteins did not have a characteristic MT structure. In the present study, we demonstrate that the rat testis indeed expresses Cd-binding proteins with sequences identical to those previously described for MT-1 and MT-2, the major isoforms. To confirm that MT-1 and MT-2 are present in the rat testis, we purified and isolated Cd-binding proteins by homogenization using Cd-containing buffer, followed by sequential purification using Sephadex G-75 gel filtration chromatography and anion HPLC column chromatography, which yielded Cd-binding protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-BP-1 and Cd-BP-2 were subjected to specific protein fragmentation by acids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus we believe that the present results clearly resolve the long-standing debate about the presence of MTs in the testis, at least in the rodent.

Difference between salty and sour taste transduction mechanisms in mouse taste cells

1.1. 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.2.2. 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.3.3. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein.4.4. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.

1916 Difference between salty and sour taste transduction mechanisms in mouse taste cells

1.1. 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.2.2. 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.3.3. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein.4.4. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.

Hepatocytes from metallothionein-I and II knock-out mice are sensitive to cadmium- and tert-butylhydroperoxide-induced cytotoxicity

Metallothionein (MT) has been proposed to play an important role in heavy metal detoxication and in the scavenging of free radicals. Effects of MT on the cytotoxicity of cadmium (Cd), tert-butylhydroperoxide ( t-BHP) and N-methyl- N-tro- N'-nitrosoguanidine (MNNG) were examined using primary hepatocyte cultures from control (C57BL/6J) and MT-I and II knock-out (MT-null) mice. Compared to control hepatocytes, MT-null hepatocytes had minimal Cd-binding proteins (MT equivalents), but cellular glutathione concentration was similar to the control hepatocytes. MT-null hepatocytes were more sensitive than controls to the cytotoxic effects of Cd (50–300 μM) and f-BHP (125–500 μM), as indicated by the levels of lactate dehydrogenase released into the medium. Cd and (-BHP also produced more lipid peroxidation in MT-null hepatocytes than in control cells, as demonstrated by the abundance of thiobarbituric acid-reactive substances. However, MT-null hepatocytes were equally sensitive as controls to the cytotoxicity of MNNG (0.5–2.0 mM), suggesting that MT does not protect against MNNG-induced cytotoxicity. These results support the hypothesis that constitutive MT levels affect the sensitivity of mammalian cells to Cd and oxidative stress.

Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts.

The resistance to cadmium ions (Cd-resistance) and possible formation of cadmium-binding complexes were examined in eight different wild-type yeasts. Saccharomyces exiguus, Pichia farinosa, Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibited partial Cd-resistance, as compared to the Cd-resistant strain 301N and the Cu-resistant but Cd-sensitive strain X2180-1B of Saccharomyces cerevisiae. Saccharomyces carlsbergensis, Pichia mogii, Zygosaccharomyces rouxii and Kluyveromyces lactis were all Cd-sensitive. The partially Cd-sensitive species, with the exception of S. exiguus, accumulated Cd2+ ions in the cytoplasmic fraction to varying extents. This fraction from S. octosporus included a Cd-binding complex that contained (gamma EC)nG peptides known as cadystins or phytochelatins, while P. farinosa and T. delbrueckii synthesized Cd-binding proteins that were similar to the Cd-metallothionein produced by S. cerevisiae 301N in terms of molecular weight and amino acid composition. These results suggest that such cytoplasmic molecules play a role in the Cd-tolerance of the above three species of yeast. S. exiguus retained most cadmium in the cell wall fraction and no Cd-binding complex was found in the cytoplasm, an indication of the important role of the cell wall in its Cd-tolerance. Different modes of binding of Cd2+ ions appear to be involved in the Cd-resistance of wild-type yeasts and fungi.

Isolation of Cd-binding protein of water hyacinth (Eichhornia crassipes) grown in Nile river water

Many investigators have reported that water hyacinth plants can accumulate toxic heavy metal ions and may be useful for elutriation of polluted water. The purpose of our study was to search for the mechanism by which these plants tolerate and accumulate toxic metal ions. Cadmium was accumulated in the plants against the concentration gradient, mostly as a soluble form in the cytoplasm. Isolation and Purification of Cd-binding protein with sephadex A-25 and fractionation on sephadex G-100 showed that the accumulated Cd was associated with two major protein fractions.The first with molecular weight 25 - 20 kD contained about 35% of bound Cd. The second fraction with molecular weight 12 - 8 kD contained about 40% of bound Cd. The two forms were found also in water hyacinth cultivated in Nile water as a control, although the amount of Cd accumulated was less than those exposed to excess Cd.

Relationship between Subcellular Cadmium Distribution in Prey and Cadmium Trophic Transfer to a Predator

We tested the hypothesis that exposure-related alterations in the subcellular Cd distribution in prey relate to changes in Cd absorption by a predator. Oligochaete worms,Limnodrilus hoffmeisteri were exposed for 1 wk or 6 wk to 0.5 μg Cd 1−1, 47 μg Cd 1−1, or 140 μg Cd 1−1 (including109Cd as a tracer) and relationships between oligochaete subcellular Cd distribution and Cd absorption by a predator, the grass shrimp (Palaemonetes pugio), were determined. Concentration and duration of Cd exposure had direct effects on oligochaete subcellular Cd distribution. Changes in oligochaete subcellular Cd distribution were characterized by increases in both the amount and proportion of Cd bound to the cytosolic fraction. The induction of Cd-binding proteins (e.g., metallothioneins) were suspected to be responsible for these changes. We found 1∶1 relationships between the amount and percentage of Cd in oligochaete cytosol and the amount and percentage of Cd adsorbed by shrimp. These results demonstrate that only metal bound to the soluble fraction of prey is available to higher trophic levels, and that factors influencing subcellular metal distribution in prey will directly alter metal trophic transfer to predators.

Cadmium-binding proteins from a tunicate, Pyura stolonifera

The determination of cadmium levels in tissues of the tunicate Pyura stolonifera collected from uncontaminated sites revealed highest concentrations in the liver. After keeping P. stolonifera under laboratory conditions in Cd-containing water for 15 days, cadmium accumulated most markedly in liver tissue. Liver tissue of Cd-exposed specimens was used for the isolation of Cd-binding proteins. Five Cd-binding proteins, which differ in their Chromatographic properties, could be purified. At least four of these Cd-binding proteins are heat stable and cysteine-rich. The N-terminal sequence (Met-Asp-Pro-Cys-Asn-Cys-Ala-Glu…) of at least two of these peptides resembles fish (plaice) metallothionein. Unlike vertebrate metallothioneins, P. stolonifera Cd-binding proteins are not N-terminally blocked by acetylation of methionine.

Cadmium accumulation and Cd-binding proteins in the bivalve Scapharca inaequivalvis

In the arcid clam, Scapharca inaequivalvis, cadmium was accumulated in the kidneys, gills, viscera, mantle, foot, muscle and red blood cells. Maximum levels were reached in the kidney (725.70 μ/g dry weight) after 4 weeks exposure to 0.5 μg Cd/ml sea-water. The accumulation was higher during the warm rather than in the cold season and in animals exposed to 0.5 as compared with 0.05 μg Cd/ml sea-water. Concentrations of zinc and copper were unaffected by cadmium treatment, whereas iron levels changed during cadmium exposure. When molluscs were returned to natural sea-water, cadmium levels fell slowly. The metal was found in the cytosolic fraction, bound to a low molecular weight ligand, shown by gel filtration to be a Cd-protein with an apparent molecular weight of 10,000 Da, possibly a metallothionein. Ion exchange chromatography of this protein isolated from the viscera revealed the presence of at least two Cd-binding isoforms; the -SH: Cd ratio of the main isoform was 2.8, the UV spectra presented a strong absorption band at 250 nm.

Synthesis of a bifunctional metallothionein/β‐glucuronidase fusion protein in transgenic tobacco plants as a means of reducing leaf cadmium levels

Chimeric genes under the control of a CaMV 35S promoter with a doubled enhancer (35S2) that encode a mammalian metallothionein (hMTII), or an Escherichia coli beta-glucuronidase (GUS), or a hMTII/GUS fusion protein were introduced into the genome of tobacco (Nicotiana tabacum cv. PBD6). Transcripts and Cd-binding proteins of expected size were observed in plants expressing either the 35S2-hMTII or the 35S2-hMTII/GUS gene, and in the latter plants a protein with GUS activity that was larger than the native GUS enzyme was observed. Thus, plants expressing the hMTII-GUS gene synthesize a bifunctional protein, with both GUS and Cd-binding activity. In an in vitro assay, seedlings expressing either one of these genes had 60-70% lower Cd concentration in their shoots than controls, and Cd translocation to the shoot system was reduced (approximately 20% of Cd absorbed was translocated), compared with that in controls expressing a 35S2-GUS gene, where approximately 50% of the Cd absorbed was translocated.

Mechanism for the decrease in the accumulation of cadmium (Cd) in Cd-resistant Chinese hamster V79 cells.

The mechanism for the decrease in the accumulation of cadmium (Cd) in Cd-resistant Chinese hamster V79 (Cdr) cells in culture was investigated in a comparison with Cd-sensitive (Cds) cells. Both Cdr and Cds cells took up Cd in a time-dependent manner but the rate of uptake of Cd by Cdr cells was about 15% of that by Cds cells. Kinetic studies of the uptake of Cd showed that the Vmax values for Cdr and Cds cells were 0.31 and 0.46 pmol Cd/h per mg protein, respectively. The Km values were 31.95 microM for Cdr cells and 3.15 microM for Cds cells. Mersalyl acid, a sulfhydryl (SH) blocker to which cells are impermeable, inhibited the uptake of Cd by Cds cells at subtoxic concentrations while Cdr cells were insensitive to inhibition by mersalyl acid, suggesting that SH groups in the plasma membrane play a role in the uptake of Cd. Uptake of Cd by Cds cells was dependent on the pH of the incubation medium and the rate of uptake was very high at pH 7.4 and pH 8.0 relative to the rates at pH 6.0 and pH 6.8. By contrast, the uptake of Cd by Cdr cells was lower at all pH values than that by Cds cells. The decrease in the rate of uptake of Cd by Cdr cells could not be ascribed to an increase in the efflux of Cd. A Cd-blotting technique was used to detect plasma membrane proteins with high affinity for Cd. Two major differences in terms of Cd-binding proteins (Cd-BPs) were observed between Cdr and Cds cells. A 110-kDa Cd-BP, detected in Cds cells, was found at a reduced level in Cdr cells, while an 82-kDa Cd-BP, which was not observed in Cds cells, was detected in Cdr cells.

Responses to cadmium toxicity during In Vitro growth in Arachis hypogaea

Toxic effects of increasing concentration of cadmium (Cd) in the environment due to rapid industrialization has become a major environmental concern. Cadmium is readily taken up by the plant system which retards the growth and yield of crops. It has been implicated inhibition of the photosynthetic mechanism as well as nodulation, nitrogen fixation, and consequently the growth of leguminous plants. Cell cultures are convenient systems for the study of mechanism of metal toxicity, as they eliminate the interfering processes of translocation and organ-specific trapping of metal ions. Genetic manipulations for metal tolerance can improve the responsiveness to the environmental stress and promote productivity of plants. To determine the extent of tolerance and raise resistant callus lines, cultures of Arachic hypogaea (groundnut), an important oil-yielding crop, were subjected to different concentrations of Cd in the media. The callus growth during the different passages as well as the percentage of dividing cells and mitotic abnormalities was recorded. Also, the protein profiles, quantitative and qualitative, were studied to detect whether synthesis of Cd-binding protein occurs.

Subcellular Cd accumulation and Cd-binding proteins in the netted dog whelk, Nassarius reticulatus L.

The subcellular accumulation of Cd in different tissues of the marine gastropod Nassarius reticulatus was determined following exposure to 0.1, 0.3 and 0.9 mg Cd/litre seawater (nominal concentrations) and the importance of cytosolic Cd-binding components for the sequestration and immobilisation of the metal evaluated. Different tissues from exposed whelks were removed, pooled, homogenised, the homogenates subjected to subcellular fractionation and fractions measured for Cu, Zn and Cd by atomic absorption spectrophotometry. Cytosols were further separated and heat-stable Cd-binding capacity determined. All subcellular fractions in different tissues accumulated Cd at all exposures. The total cytosolic protein concentration in some tissues was strongly affected, but there was no concentration-dependent increase in Cd-binding capacity of the heat-stable protein pool. The major cytosolic Cu- and Cd-binding components in all four tissues were low molecular weight ( M r 5–10 kDa), whereas most cytosolic Zn coeluted with components of very low molecular weight.

Evidence for a charge variant of Cd-BP 14, a cadmium-binding protein from Allolobophora caliginosa (Oligochaeta, Annelida)

1. 1. In Allolobophora caliginosa , a Cd-binding protein distinct in charge from Cd-BP 14, a Cd-binding protein previously isolated from the same oligochaete species [Nejmeddine et al. (1992) Comp. Biochem. Physiol. 101C, 601–605], has been purified by a three-step chromatographic procedure including gel permeation and cation-exchange chromatography. 2. 2. This Cd-binding protein exists in a monomeric form with a molecular weight of 14 kDa and does not contain carbohydrate. 3. 3. The purified protein significantly absorbed at 280 nm and its amino acid composition revealed the presence of a high level of aromatic amino acids and a lack of cysteine, indicating that the molecule is distinct from metallothioneins. 4. 4. By contrast, except for its chromatographic behavior on an ion-exchange chromatography column, the metalloprotein was found to be similar to Cd-BP 14. We thus conclude that it represents a charge-variant of Cd-BP 14.