Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca²⁺ in the response to high Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Hg²⁺ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca²⁺ when exposed to Cd²⁺, and to a lesser extent to Cu²⁺, but not to Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, or Hg²⁺. The response to high Cd²⁺ depended mainly on external Ca²⁺ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca²⁺ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd²⁺ was influenced by perturbations in Ca²⁺ homeostasis. Thus, the tolerance to Cd²⁺ often correlated with sharp Cd²⁺-induced cytosolic Ca²⁺ pulses, while the Cd²⁺ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca²⁺.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Cadmium uptake kinetics in intact soybean plants

The absorption characteristics of Cd²⁺ by 10- to 12-day-old soybean plants (Glycine max cv Williams) were investigated with respect to influence of Cd concentration on adsorption to root surfaces, root absorption, transport kinetics and interaction with the nutrient cations Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. The fraction of nonexchangeable Cd bound to roots remained relatively constant at 20 to 25% of the absorbed fraction at solution concentration of 0.0025 to 0.5 micromolar, and increased to 45% at solution concentration in excess of 0.5 micromolar. The exchangeable fraction represented 1.4 to 32% of the absorbed fraction, and was concentration dependent. Using dinitrophenol as a metabolic inhibitor, the `metabolically absorbed' fraction was shown to represent 75 to 80% of the absorbed fraction at concentration less than 0.5 micromolar, and decreased to 55% at 5 micromolar. At comparatively low Cd concentrations, 0.0025 to micromolar 0.3, root absorption exhibited two isotherms with K₂ values of 0.08 and 1.2 micromolar. Root absorption and transfer from root to shoot of Cd²⁺ was inhibited by Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. Analyses of kinetic interaction of these nutrient cations with Cd²⁺ indicated that Cu²⁺, Fe²⁺, Zn²⁺, and possibly Mn²⁺ inhibited Cd absorption competitively suggesting an involvement of a common transport site or process.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.     

Fluorescence detection of intracellular cadmium with Leadmium Green

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd²⁺) and lead (Pb²⁺). Leadmium Green has been used in various paradigms, such as tracking Cd²⁺ sequestration in plant cells, heavy metal export in protozoa, and Pb²⁺ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd²⁺, Pb²⁺, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd²⁺ (K_D ~600 nM) and also Pb²⁺ (K_D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca²⁺, Co²⁺, Mn²⁺ and Ni²⁺. Leadmium Green also responded to Zn²⁺ with a K_D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd²⁺ or Zn²⁺ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn²⁺ was much less than that produced by Cd²⁺. When tested in a model of oxidant-induced liberation of endogenous Zn²⁺, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd²⁺, particularly in models where Cd²⁺ accumulates rapidly, and when concomitant fluctuations of intracellular Zn²⁺ are minimal.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Resistance acquisition of Thiobacillus thiooxidans upon cadmium and zinc ion addition and formation of cadmium ion-binding and zinc ion-binding proteins exhibiting metallothionein-like properties

Through subcultivations of Thiobacillus thiooxidans WU-79A in autotrophic media in which the concentrations of Cd²⁺ and Zn²⁺ were increased successively, Cd²⁺-resistant (CDR) and Zn²⁺-resistant strains (ZNR) were obtained. The growth of WU-79A was inhibited by the addition of 25 mM Cd²⁺ as well as Zn²⁺. However, CDR and ZNR could grow without any lag phase in media containing 200 mM Cd²⁺ and 250 mM Zn²⁺, respectively. CDR and ZNR were able to grow even in media containing up to 400 mM Cd²⁺ and 600 mM Zn²⁺, respectively, although they exhibited lag phases. CDR could grow in medium containing up to 250 mM Zn²⁺, as could ZNR in medium containing up to 200 mM Cd²⁺. Cd²⁺-binding and Zn²⁺-binding proteins were isolated from CDR and ZNR, respectively, by gel filtration and ion exchange chromatography. The molecular weights of both proteins were estimated to be approximately 13,000 by gel filtration. The fact that there was no strong absorption at 280 nm of the proteins suggested that they had few aromatic amino acids. Broad absorption bands which are typical of mercaptide (metal thiolate) complexes were detected. The properties of the proteins were spectrophotometrically similar to those of metallothionein.     

Genes for all metals—a bacterial view of the Periodic Table

Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH₄ ⁺, K⁺, Mg²⁺, Co²⁺, Fe³⁺, Mn²⁺, Zn²⁺ and other trace cations, PO₄ ^3-, SO₄ ^2- and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag⁺ AsO₂ ^-, AsO₄ ^3-, Cd²⁺, Co²⁺, CrO₄ ²⁻, Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺, TeO₃ ²⁻, TI⁺ and Zn²⁺. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd²⁺-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with ³²P from [γ-³²P]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson’s disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides, ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex functions as a chemiosmotic cation/protein exchanger to efflux Cd²⁺, Zn²⁺ and Co²⁺. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together. Received 08 August 1997/ Accepted in revised form 01 November 1997     

Effect of Fe2+, Mn2+, Zn2+ and Pb2+ on H+/K+ fluxes in excisedPistia stratiotes roots

Pistia stratiotes is used for the epuration of domestic sewage in the Biyem Assi phytopurification station. During the process, Fe²⁺, Mn²⁺, Zn²⁺ and Pb²⁺ are absorbed in substantial amounts by the plant. These metals modify the H⁺/K⁺ exchange system at the root level. H⁺ efflux is inhibited by Fe²⁺ and by Zn²⁺ and enhanced by Mn²⁺ and Pb²⁺. K⁺ influx is inhibited by Fe²⁺, by Zn²⁺ and by Pb²⁺ and enhanced by Mn²⁺. It is shown that the purification capacity ofPistia stratiotes can vary with the composition of the heavy metals in the surrounding medium.     

Cadmium ion stimulation of growth and versicolorin synthesis in a mutant strain ofAspergillus parasiticus

Cultures ofAspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn²⁺ content of the growth medium. With suboptimal Zn²⁺ (0.8 μM) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd²⁺ (1–100 μM) in the Zn²⁺-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd²⁺, like Zn²⁺, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd²⁺ uptake may be the same as that for Zn²⁺, as judged byin vivo competition studies. Cd²⁺ is a competitive inhibitor of Zn²⁺ uptake, with K_i = 20 μM.     

Development of a broad-spectrum fluorescent heavy metal bacterial biosensor

Bacterial biosensors can measure pollution in terms of their actual toxicity to living organisms. A recombinant bacterial biosensor has been constructed that is known to respond to toxic levels of Zn²⁺, Cd²⁺ and Hg²⁺. The zinc regulatory gene zntR and zntA promoter from znt operon of E. coli have been used to trigger the expression of GFP reporter protein at toxic levels of these ions. The sensor was induced with 3–800?ppm of Zn²⁺, 0.005–4?ppm of Cd²⁺ and 0.001–0.12?ppm of Hg²⁺ ions. Induction studies were also performed in liquid media to quantify GFP fluorescence using fluorimeter. To determine the optimum culture conditions three different incubation periods (16, 20 and 24?h) were followed. Results showed an increased and consistent fluorescence in cells incubated for 16?h. Maximum induction for Zn²⁺, Cd²⁺ and Hg²⁺ was observed at 20, 0.005 and 0.002?ppm, respectively. The pPROBE-zntR-zntA biosensor reported here can be employed as a primary screening technique for aquatic heavy metal pollution.     

Cadmium and mineral nutrient accumulation in potato plantlets grown under cadmium stress in two different experimental culture conditions

In order to evaluate the effect of cadmium (Cd²⁺) toxicity on mineral nutrient accumulation in potato (Solanum tuberosum L.), two cultivars named Asterix and Macaca were cultivated both in vitro and in hydroponic experiments under increasing levels of Cd²⁺ (0, 100, 200, 300, 400 and 500 μM in vitro and 0, 50, 100, 150 and 200 μM in hydroponic culture). At 22 and 7 days of exposure to Cd²⁺, for the in vitro and hydroponic experiment, respectively, the plantlets were separated into roots and shoot, which were analyzed for biomass as well as Cd²⁺, and macro (Ca²⁺, K⁺ and Mg²⁺) and micronutrient (Cu²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) contents. In the hydroponic experiment, there was no reduction in shoot and root dry weight for any Cd²⁺ level, regardless of the potato cultivar. In contrast, in the in vitro experiment, there was an increase in biomass at low Cd²⁺ levels, while higher Cd²⁺ levels caused a decrease. In general, Cd²⁺ decreased the macronutrient and micronutrient contents in the in vitro cultured plantlets in both roots and shoot of cultivars. In contrast, the macronutrient and micronutrient contents in the hydroponically grown plantlets were generally not affected by Cd²⁺. Our data suggest that the influence of Cd²⁺ on nutrient content in potato was related to the level of Cd²⁺ in the substrate, potato cultivar, plant organ, essential element, growth medium and exposure time.     

Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd2+, Zn2+, Cu2+, and NaCl in transgenic yeast

A metallothionein-like gene, ThMT3, encoding a type 3 metallothionein, was isolated from a Tamarix hispida leaf cDNA library. Expression analysis revealed that mRNA of ThMT3 was upregulated by high salinity as well as by heavy metal ions, and that ThMT3 was predominantly expressed in the leaf. Transgenic yeast (Saccharomyces cerevisiae) expressing ThMT3 showed increased tolerance to Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress. Transgenic yeast also accumulated more Cd²⁺, Zn²⁺, and NaCl, but not Cu²⁺. Analysis of the expression of four genes (GLR1, GTT2, GSH1, and YCF1) that aid in transporting heavy metal (Cd²⁺) from the cytoplasm to the vacuole demonstrated that none of these genes were induced under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in ThMT3-transgenic yeast. H₂O₂ levels in transgenic yeast under such stress conditions were less than half those in control yeast under the same conditions. Three antioxidant genes (SOD1, CAT1, and GPX1) were specifically expressed under Cd²⁺, Zn²⁺, Cu²⁺, and NaCl stress in the transgenic yeast. Cd²⁺, Zn²⁺, and Cu²⁺ increased the expression levels of SOD1, CAT1, and GPX1, respectively, whereas NaCl induced the expression of SOD1 and GPX1.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Sorption of Heavy Metal Cations by Corn Mitochondria and the Effects on Electron and Energy Transfer Reactions

The passive sorption of Pb⁺², Cd⁺², Zn⁺², Co⁺², Ni⁺², and Mn⁺² by isolated corn mitochondria was determined, and, except for Pb⁺², the maximum sorption for each cation was about 58 nmol per milligram of protein. Sorption of Pb⁺² was apparently ten times greater, but precipitation may have been the cause of this larger value. The effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² on acceptorless rates of electron transport for three substrates were determined. Greater than 50% inhibitions of oxidation were observed for succinate after additions of >0.1 mM Cd⁺², Zn⁺², or Pb⁺²: for NADH after additions of >0.5 mM Cd⁺² or Zn⁺²; and for malate + pyruvate after additions of >0.1 mM Cd⁺². Some inhibition of the rate of substrate oxidation was observed for most cations at higher concentrations. Coupling, as measured by ADP/O ratios, was inhibited at lowest concentrations by Cd⁺² or Zn⁺² and at higher concentrations by Co⁺² or Ni⁺². Substantial swelling of mitochondria oxidizing succinate was observed following additions of O.1 mM Cd⁺² or Pb⁺², Correlations are drawn between the effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² and their sorption to mitochondrial membranes.