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.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn⁺² (50, 100, or 150 μM) or Cd⁺² (3, 6, or 12 μM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn⁺² toxicity and a lesser but significant effect on Cd⁺² toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn⁺² or Cd⁺² exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.     

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.     

重金属铜、锌、镉复合胁迫对麻疯树幼苗生理生化的影响

该研究以Cu~(2+)、Zn~(2+)、Cd~(2+)单一胁迫为对照,探讨不同浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫对麻疯树幼苗生理生化指标的影响。结果表明:随着Cu~(2+)、Zn~(2+)、Cd~(2+)浓度的增加,麻疯树幼苗叶片中的蛋白质(Pro)、丙二醛(MDA)含量均逐渐增加,其叶片叶绿素含量随着Zn~(2+)胁迫浓度的增加呈现出先降后升的趋势,在中等浓度(100 mg·L-1)的Zn~(2+)胁迫时含量最低、随着Cu~(2+)胁迫浓度的增加叶绿素含量先升高后降低,在Cu~(2+)浓度为200 mg·L-1时含量最高,达到1 200 mg·g-1FW; Cd~(2+)胁迫对叶绿素含量和根系活力无明显影响。根系活力在Zn~(2+)浓度为100 mg·L~(-1)时最强,随着Cu~(2+)浓度的增加而减弱。低浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)对过氧化物酶活性和可溶性糖含量都具有促进作用。Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫时对可溶性蛋白、叶绿素和丙二醛含量均无明显影响,随着复合胁迫时浓度的增加,可溶性糖含量和根系活力先增后减。这表明麻疯树对三种重金属的胁迫具有一定的抗性,过高浓度的胁迫会影响麻疯树幼苗生理生化的一些指标,但是麻疯树可以通过自身的防御系统使伤害降到最小。此外,重金属复合胁迫可以在一定程度上减轻单一胁迫对麻疯树幼苗造成的毒害作用。     

Effects of heavy metals on the Ca2+-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.)

1. Heavy metals (Hg²⁺, Cu²⁺, Cd²⁺, Zn²⁺, Pb²⁺) at micromolar concentrations strongly inhibit the Ca²⁺-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate.2. All the heavy metals tested inhibit the Ca²⁺-ATPase activity, the effect following the order: Hg²⁺ > Pb²⁺ > Cu²⁺ > Cd²⁺ > Zn²⁺; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals.3. The inhibitory effects of Cu²⁺ on Ca²⁺-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 μM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.     

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd²⁺), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd²⁺ can be transported as phytochelatin‐Cd²⁺ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd²⁺ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 μM), and high (200 μM) Cd²⁺ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ?). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low‐Cd²⁺ conditions, an inorganic pyrophosphatase and a γ‐tonoplast intrinsic protein (γ‐TIP) were up‐regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance‐associated macrophage protein (NRAMP), responsible for vacuolar Fe²⁺ export was increased. CAX1a might play a role in vacuolar Cd²⁺ transport. An increase in NRAMP activity leads to a higher cytosolic Fe²⁺ concentration, which may prevent the exchange of Fe²⁺ by toxic Cd²⁺. Additionally, an ABC transporter homolog to AtMRP3 showed up‐regulation. Under high Cd²⁺ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd²⁺ conditions, was up‐regulated. Interestingly, AtMRP3 is able to partially rescue a Cd²⁺‐sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd²⁺ detoxification.     

Analysis of Metal-Regulated Metallothionein and Heat Shock Gene Expression in HeLa-Derived Cadmium-Resistant Cells

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd²⁺and Zn²⁺exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd²⁺-exposed H454 cells at levels comparable to the ones present in Cd²⁺-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

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.     

Accumulation of cadmium in pancreatic β cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd²⁺ and the effects of this ion on secretory activity and metabolism were investigated in β cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 μmol/kg dry wt. After 60 min of incubation in a Ca²⁺-deficient medium containing 2.5 μM Cd²⁺ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca²⁺. The incorporation of Cd²⁺ was stimulated either by raising the concentration of glucose to 20 mM or K⁺ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K⁺. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd²⁺-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd²⁺ efflux into a Ca²⁺-deficient medium. Concentrations of Cd²⁺ up to 2.5 μM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd²⁺ concentration was above 10 μM. Basal insulin release was stimulated by 5 μM Cd²⁺. At a concentration of 160 μM, Cd²⁺ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the β cell uptake of Cd²⁺ is facilitated by the activation of voltage-dependent Ca²⁺ channels. Apparently, the accumulation of Cd²⁺ mimics that of Ca²⁺ also involving a component of intracellular sequestration promoted by glucose.     

The effects of copper,cadmium and zinc on particle filtration and uptake of glycine in the pacific oyster Crassostrea gigas

1. The filtration rate (volume of water completely cleared of collodial carbon per unit time) by control oysters is 36.60 ml/g hr ± 7.68 (sd).2. Filtration rates decrease with increasing concentrations of Cd²⁺ and Zn²⁺.3. In 8–16 mg/l Cu²⁺, filtration rates are significantly higher than the control, but in Cu²⁺ concentrations above 32 mg/l, filtration rates are lower than controls.4. Influx of ¹⁴C-glycine is characterized by Michaelis-Menten kinetics with J_max and K_t values of 1.85 ± 0.097 μmol/g hr and 33.7 ± 4.6 μM respectively.5. The uptake rate of glycine from 1 μM solution is 37.79 μmol/g hr.6. In order of degree of inhibition of glycine uptake, Cu²⁺ > Cd²⁺ > Zn²⁺.7. In 128 mg/l Cu²⁺, glycine uptake rate is reduced to 3.96 nmol/g hr or 10.5% of control.8. The rate of glycine uptake by filter feeding bivalves is dependent on rate of water pumping rate.9. The volume specific glycine transport (amount of glycine transported/unit volume of seawater completely cleared of colloidal carbon) by control oysters in 1 μM glycine concentrations is 1.03 μmol/l.10. The volume specific glycine transport remains constant in increasing Zn²⁺ concentrations, and declines in increasing Cu²⁺ concentrations, suggesting differential effects of the metals on particle filtration and the epithelial amino acid carriers.11. The apparent volume specific glycine transport increases to 2.14 μmol/l in 128 mg/l Cd²⁺. This volume specific transport greater than the glycine concentration in the medium suggests that there may be uptake of cadmium complexed glycine by the oysters.     

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd²⁺ shares the same uphill uptake system with Zn²⁺. Both heavy metals inhibited growth, respiration, H⁺/glucose uptake, and glucose-induced proton extrusion, Cd²⁺ being a 10–15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H⁺-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd²⁺ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn²⁺, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd²⁺. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 μM, Cd²⁺ brought about loss K⁺ (18 K⁺/1 Cd²⁺) from energized, but not from deenergized cells since Cd²⁺ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd²⁺ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn²⁺ had virtually no effect below 1 mM concentration, and it again weakened the K⁺-releasing effect of Cd²⁺. Cd²⁺ caused a strong loss of K⁺ also from K⁺-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K⁺ loss sixfold. Received: 13 November 1995 / Accepted: 31 January 1996     

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.     

Effect of Zn2+ on water and K+ fluxes in detopped maize plants

Water and K⁺ fluxes were examined in detopped plants ofZea mays L. (cv. White Horse Tooth), which were grown and exuded on half-strength Long Ashton nutrient solution containing the appropriate concentration of Zn²⁺ at 20 °C. In light-grown plants, 100 and 500 μM Zn²⁺ increased both water and K⁺ fluxes in detopped maize plants whereas 1 000 μM Zn²⁺ inhibited both fluxes. In the dark-pretreated plants, 1 000 μM Zn²⁺ in the medium stimulated K⁺ flux. The fluxes of K⁺, Zn²⁺, Ca²⁺ and Mg²⁺ were usually higher in detopped plants than in intact ones. At 1 000 μM Zn²⁺ in the exudation medium, Zn²⁺ concentration was higher in the xylem exudate of dark-pretreated plants than in roots of plants maintained in light. The results are discussed in relation to the influence of Zn²⁺ on the membrane permeability and transport in plants.     

Antagonistic effect of calcium, zinc and selenium against cadmium induced chromosomal aberrations and micronuclei in root cells of Hordeum vulgare

The antagonistic effect of calcium (Ca²⁺), zinc (Zn²⁺) and selenium (Se⁴⁺) at different concentrations (10⁻²–10⁻⁶ M) against cadmium (Cd²⁺) induced genotoxic effects in root cells of Hordeum vulgare were studied. The results showed that 10⁻³–10⁻⁵ M could induce chromosomal aberrations and micronuclei formation. But in the treatment with 10⁻²–10⁻⁶ M of Ca²⁺, Zn²⁺ and Se⁴⁺ together with Cd²⁺ (10⁻³–10⁻⁵ M), respectively, the frequencies of chromosomal aberrations and micronuclei effectively decreased after 48 h of treatment. The treatment with 10⁻⁴–10⁻⁶ M of Ca²⁺ together with 10⁻⁴–10⁻⁵ M Cd²⁺, 10⁻⁶ M of Zn²⁺ together with 10⁻⁵ M Cd²⁺ and 10⁻⁶ M of Se⁴⁺ together with 10⁻⁵ M Cd²⁺ suggested rather obvious antagonistic effects. The order of the antagonisms of Ca²⁺, Se⁴⁺ and Zn²⁺ against Cd²⁺ toxicity was Ca²⁺>Se⁴⁺>Zn²⁺. The degree of antagonisms of Ca²⁺, Se⁴⁺ and Zn²⁺ against Cd²⁺ related to their concentration ratio.     

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn²⁺ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn²⁺ and Cd²⁺ chelator with capacity to bind 10 Zn²⁺ ions per C terminus. When AtHMA4 is expressed in a Zn²⁺-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn²⁺ and Cd²⁺ tolerance and lowered Zn²⁺ and Cd²⁺ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn²⁺ and Cd²⁺ chelator (sensor) and as a regulator of the efficiency of Zn²⁺ and Cd²⁺ export. The identification of a post-translational handle on Zn²⁺ and Cd²⁺ transport efficiency opens new perspectives for regulation of Zn²⁺ nutrition and tolerance in eukaryotes.     

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells

Mouse P₂A₂-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (K_a). ¹²⁵I-IGF-I, ¹²⁵I-IGF-II, and ¹²⁵I-R³-IGF-I bind only to the type 1 IGF receptor on these cells. Zn²⁺ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd²⁺ significantly increased binding of ¹²⁵I-IGF-I, although by only 8%. La³⁺ and Cr³⁺ did not effect binding. Au³⁺ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn²⁺ increased the strength of only the high affinity sites. Au³⁺ decreased the affinity of both high and low affinity sites. Zn²⁺ increased binding with a half-maximal effect between 40 μM and 60 μM. Half-maximal dose of Au³⁺ was > 130 μM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn²⁺. J. Cell. Physiol. 176:392–401, 1998. © 1998 Wiley-Liss, Inc.