Occurrence of acid-labile sulfide in cadmium-binding peptide 1 from fission yeast

Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l). It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field.     

Coordination and speciation of cadmium in corn seedlings and its effects on macro- and micronutrients uptake

The effect of cadmium (Cd) on both the absorption of important nutrients and the synthesis of low molecular weight thiols (LMWTs) was investigated in corn plants. The inductively coupled plasma-optical emission spectroscopy results demonstrated that the concentration of Cd in tissues (mainly in roots) increased as the concentration in the medium increased. In addition, the concentration of phosphorus increased in roots of Cd treated plants but remained at normal concentration in shoots. On the other hand, the uptake of sulfur (S) followed a similar trend as the Cd uptake. The concentration of S and the production of LMWT were found to increase significantly upon exposure to Cd. The results of the X-ray absorption spectroscopy analyses indicated that Cd within tissues was bound to S ligands with interatomic distances of 2.51–2.52 Å. These results confirm a strong linkage between S uptake and the production of LMWT upon exposure to Cd.     

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.     

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.     

Cadmium translocation and accumulation in developing barley grains

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. In order to study the transport of Cd into the developing grains, detached ears of two-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing the markers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in each part of detached spikes increased with external Cd levels, and Cd concentration in various organs over the three Cd levels of 0.5, 2, 8 μM Cd on 15-day Cd exposure was in the order: awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grains with the proportion of 51.0% relative to the total Cd amount in the five parts of detached spikes. Cd accumulation in grains increased not only with external Cd levels but the time of exposure contrast to stem, awn, rachis and glume. Those four parts of detached spike showed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 and increase again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cd concentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relative humidity (RH) also induced dramatic reduction in Cd transport to developing grains. The results indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as an important physiological process influencing the quality of harvested grains. Our results suggested that increasing RH to 90% and Zn addition in the medium at grain filling stage would be beneficial to decrease Cd accumulation in grains.     

Sulfide stabilization of the cadmium-gamma-glutamyl peptide complex of Schizosaccharomyces pombe

Addition of cadmium salts to the growth medium of Schizosaccharomyces pombe leads to synthesis of a Cd.gamma-Glu peptide complex and an enhanced generation of sulfide ions. The gamma-Glu peptide complex functions in the detoxification of heavy metal ions. Native Cd.gamma-Glu peptide complexes contain acid-labile sulfide in the metal-thiolate cluster. Two forms of the complex exist differing primarily in their sulfide content. Sulfide concentrations up to 0.2 and 1.2 mol/mol of peptide were observed in native isolates of forms I and II, respectively. Addition of sulfide to the low sulfide form I converted it to a complex similar to form II. Properties of the Cd.gamma-Glu peptide complex were altered by the incorporation of sulfide ions. Sulfide-dependent electronic transitions in the ultraviolet were evident, and the absorbance maximum of the transition was related to the sulfide content and the bound metal ion. High sulfide forms of the Cd and Zn complexes exhibited absorbance peaks at 318 nm and 255 nm, respectively. Incorporation of sulfide into the Cd.gamma-Glu peptide complex imparted greater thermodynamic stability to the complex, an increased Stokes radius, and an enhanced Cd(II) binding capacity. Sulfide generation may be a cellular response in part to enhance the effectiveness of the gamma-Glu peptide system for Cd(II) detoxification.     

Responses of the Antarctic microalga Koliella antarctica (Trebouxiophyceae,Chlorophyta) to cadmium contamination

Ultrastructural and physiological effects of exposure to 1 ppm and 5 ppm of cadmium (Cd) on cultured cells of Koliella antarctica, a green microalga from Antarctica, were investigated. The amount of Cd in the alga rose with the increase of the metal concentration in the growth medium and most Cd remained outside the cells, bound to the components of the cell walls. The increase of Cd in the microalga was concomitant with the decrease of other elements, mainly calcium (Ca). Exposure to 1 ppm Cd slowed culture growth by inhibiting cell division and also caused the development of some misshapen cells with chloroplast showing disordered thylakoids. However, this concentration did not substantially affect the chlorophyll (Chl) content or photosystem (PS) activity. At 5 ppm, Cd cell growth suddenly stopped and some cells lysed. After a week of Cd contamination, the cells were enlarged and severely damaged. The chloroplasts showed great ultrastructural alterations and a reduced Chl content. Cd exposure negatively affected PSII, whose activity was almost completely lost after four days.     

The Zn- and Cd-clusters of recombinant mammalian MT1 and MT4 metallothionein domains include sulfide ligands

Recombinant (E. coli ) synthesis of mammalian MT1 and MT4 domains as separate peptides in Zn(II) and Cd(II) enriched growth media has rendered metal complexes containing sulfide anions as additional ligands. The Cd preparations show higher sulfide content than the Zn preparations. Also, the betaMT1 and betaMT4 fragments exhibit higher sulfide/peptide ratios than the respective alpha fragments. Titration of Zn3-betaMT1 with Cd(II) followed by addition of several sodium sulfide equivalents shows that the Cd(II)-betaMT1 species can incorporate sulfide ligands in vitro, with a concomitant evolution of their UV-vis and CD fingerprints to those characteristic of the Cd-S2- chromophores. Current results have also provided full understanding of previous data collected by this group in the characterization of the Cd-betaMT1 preparations obtained from large-scale fermentor synthesis by allowing identification of at least 2S2- ligands per Cd-betaMT1 species. Furthermore, the results here presented have revealed that synthesis of betaMT4 in Cd-supplemented cultures yielded Cd,S(2-)-containing clusters instead of the proposed heterometallic Zn,Cd-betaMT4 complexes. Finally, a global evaluation of our results suggests that the higher the Cu-thionein character of a MT peptide, the higher is its tendency to harbor nonproteic ligands (i.e., sulfide anions) when building divalent metal clusters, especially Cd-MT complexes.     

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.     

Inducible cadmium binding complexes of cabbage and tobacco

Cadmium complexes with apparent molecular weights of 10,000 were observed in aqueous extracts of Cd-treated cabbage (Brassica capitata L., cv. red danish) and tobacco (hybrid of Nicotiana glauca and N. langsdorffii) plants. The amount of complex (as Cd) recovered was found to be dependent on the concentration of the metal in the growth medium and the total time of exposure of plants to the metal. Induction of the complex at moderate levels of ¹¹²Cd exposure was monitored after labeling the complex with ¹⁰⁹Cd in vitro. The constitutive nature of the ligand of the complex in cabbage and tobacco leaves was suggested when control plant extracts were exposed to ¹⁰⁹Cd. Such extracts contained ¹⁰⁹Cd, which eluted from Sephadex G-50 in the region of Cd complex. Simultaneous labeling with ¹¹²Cd and ³⁵S or ³²P indicated that the complex contained sulfur but probably not phosphorus. The amount of ³⁵S which eluted coincident with ¹¹²Cd complex increased during complex induction. No evidence was found for the presence of 10,000 molecular weight Cd complex in stem exudates (vascular sap) of Cd-treated plants.     

Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.     

Glutathione and phytochelatin contents in tomato plants exposed to cadmium

The effect of cadmium on growth and contents of glutathione (GSH) and phytochelatins (PCs) were investigated in roots and leaves of tomato plants (Lycopersicon esculentum Mill. cv. 63/5 F1). The accumulation of Cd increased with external Cd concentrations and was considerably higher in roots than in leaves. Dry mass production decreased under Cd treatment especially in leaves. In both roots and leaves, exposure to Cd caused an appreciable decline in GSH contents and increase in PCs synthesis proportional to Cd concentrations in the growth medium. At the same Cd concentration, PCs production was higher in roots than in leaves. The implication of glutathione in PC synthesis was strongly suggested by the use of buthionine sulfoximine (BSO). The major fraction of Cd accumulated by tomato roots was in the form of a Cd-PCs complex.     

Induction of metallothionein in a human trophoblast cell line by cadmium and zinc

The induction of metallothionein (MT) in a cell line derived from a malignant trophoblastic tumor (JAr cells) was demonstrated using the Cd/heme radioassay following exposure to Cd or Zn. Cd at an optimal concentration of 1 microM produced a 30-fold increase in MT following a 24 hr incubation. Induction by Cd was both time and dose dependent, with a significant increase in MT noted as early as 3 hr, with levels continuing to increase up to 24 hr. Zn was also quite effective in inducing MT synthesis in this cell line. Exposure to 80 microM Zn for 24 hr produced a 70-fold increase in MT. Although Cd was a more potent inducer of MT, exposure to Zn resulted in a greater magnitude of induction. The magnitude of MT induction in JAr cells was much greater than that seen in cultured trophoblasts from term chorion laeve. The degree of induction seen in this cell line makes it an interesting model for the study of MT's role in trophoblast function. MT induction in trophoblasts may reflect a protective mechanism against heavy metal toxicity and/or an integral aspect of normal zinc homeostasis.     

Microbial production of water-soluble non curdlan type exopolymer-B with controlled composition by agrobacteriumsp

The cell growth, production of exopolymers, and the molar ratio of glucose to mannose in the water-soluble non curdlan type exopolymer-B (WSNCE-B), which is one of three exopolymers purified from the culture of Agrobacterium sp., varied with carbon source, culture medium, and initial medium pH. The molar percentage of rhamnose, a minor component in WSNCE-B, varied up to 13%, dependent on physiological conditions. No rhamnose was found in the WSNCE-B purified from the culture with initial medium pH > 6.8. The relative amount of mannose in WSNCE-B increased regularly with the amount of yeast extract added to the mineral salts medium. The relative amounts of glucose, mannose, and rhamnose in the WSNCE-B can be controlled by varying culture conditions.     

石油烃对沙蚕镉生物富集特性及金属硫蛋白诱导的影响

多毛类沙蚕作为海陆交错带的关键性物种,常被作为环境监测的指示生物.我国海陆交错带沙蚕常见种——双齿围沙蚕经常遭受各种污染物\[包括重金属镉(Cd)和石油烃\]的胁迫.本研究采用慢性微宇宙试验,以暴露于单一和复合Cd和石油烃条件下的沙蚕为研究对象,重点考察石油烃对沙蚕Cd生物富集特性及诱导金属硫蛋白的影响.结果表明: 单一Cd胁迫条件下,沙蚕对重金属Cd的生物累积随暴露浓度的升高而显著上升,生物富集系数随暴露时间的延长呈上升趋势;复合胁迫条件下,与对照组相比,石油烃的添加可以显著增加沙蚕对重金属Cd的生物累积.Cd可以诱导沙蚕金属硫蛋白(MT)的表达,但当沙蚕体内Cd含量达到180 mg·kg^-1DM时,MT基本饱和;石油烃不能显著诱导沙蚕MT合成,与单一Cd处理相比,石油烃的添加能够显著影响Cd诱导的MT含量.这说明尽管石油烃本身不能直接影响MT合成,但在与Cd共存时可以调节MT的表达,因此在利用野生沙蚕MT作为监测指标时要具体区分蛋白表达的潜在诱导因素.     

ADP-ribosyl transferase activity of cholera toxin polypeptide A1 and the effect of limited trypsinolysis

Cd-binding peptide 1 (Cd-BP1) from $\text{S.}\text{pombe}$ (1) has been purified and characterized. Cd-BP1 has a characteristic shoulder at 265 nm in UV absorption spectrum, and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These are not found in the other Cd-thioneins (2) and are unique properties of Cd-BP1 from $\text{S.}\text{pombe}$. With acidification (pH 2) and successive neutralization, a shoulder at 265 nm in UV spectrum and a Cotton band at 275 nm disappeared, and the molecular weight changed from 4 000 to 1 800. In connection with these changes, two molecular forms of Cd-binding peptide are considered.     

Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata.

Cultures of Candida glabrata treated with CdCl2 form intracellular Cd(II) complexes that evolve with the time of culturing. Initially, glutathione (gamma ECG) appears to be the major buffering component. One type of Cd(II)-glutathione complex exists as a cadmium:sulfide (CdS) crystallite coated with glutathione. A time dependent change in the coating of the CdS particles occurs with a decrease in the (gamma ECG) content and a corresponding increase in the abundance of (gamma EC)nG peptides with (gamma EC)2G becoming the predominant peptide. The des-Gly variant (gamma EC)2 appears in significant concentration only in late cultures. The evolution in isopeptide coating appears to be dependent on the sulfide content of the CdS particles. Cellular conditions that enhance the generation of sulfide ions facilitate the conversion from gamma ECG to (gamma EC)2G.     

Toxicity of cadmium to Helisoma anceps and its effect on kidney ultrastructure

When specimens of Helisoma anceps were exposed to water containing 0.1, 1, and 5 ppm cadmium, mortality was proportional to concentration but tissue levels of Cd were not. The shell was the site of highest concentration and total amount of Cd due to adsorption and active accumulation. Concentrations of Cd in the kidney and mantle were equal to or greater than the remaining soft tissue. Ultrastructural changes in the kidney associated with Cd exposure were almost exclusively confined to the nephrocytes. Graded transformation of the residual body to a secondary lysosome was proportional to the Cd concentration of the medium and length of exposure. Atypical nucleoli and reticular bodies appeared in the 1-ppm Cd dose. Only after autolysis of the nephrocytes in 5 ppm did ultrastructural changes occur in the ureter cells. Ultrastructural changes in the kidney epithelia were proportional to Cd dosage but were not necessarily indicative of the tissue burden of Cd.     

Ca-releasing action of beta, gamma-methylene adenosine triphosphate on fragmented sarcoplasmic reticulum.

beta,gamma-Methylene adenosine triphosphate (AMPOPCP) has two effects on fragmented sarcoplasmic reticulum (FSR), i.e., inhibition of the rate of Ca uptake and the induction of Ca release from FSR filled with Ca. The Ca release brought about by AMPOPCP has many features in common with the mechanism of Ca-induced Ca release: i) it is inhibited by 10 mM procaine; ii) the amount of Ca release increases with increase in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the medium facilitates the release of Ca. However, no facilitation of Ca release upon decrease of Mg concentration in the medium is observable. AMPOPCP and caffeine potentiate each other remarkably in their Ca-releasing action, irrespective of the kind of substrate. From the mode of action of AMPOPCP on the rate of Ca uptake, the amount of phosphorylated intermediate (EP), and the effect on Sr release, it is suggested that the state of the FSR-ATP complex is crucial for Ca-induced Ca release.