Native Cadmium-Metallothionein from the Yeast Saccharomyces cerevisiae: Its Primary Structure and Function in Heavy-Metal Resistance

A Cd-resistant strain of yeast (Saccharomyces cerevisiae, strain30IN) accumulated Cd with the concomitant synthesis of a Cd-bindingprotein of low molecular weight when grown in Cd²⁺-containingmedium. Analysis of the amino acid composition, N-terminal sequenceand immunological properties of the protein revealed its structuralhomology to Cu-metallothionein (Cu-MT) in S. cerevisiae 2186,a Cu-resistant strain of yeast (Winge et al. 1985). The synthesisof MT in Cu-resistant strains of yeast is known to be underthe strict control of Cu²⁺ ions, while that in 301N was inducedboth by Cd²⁺ and Cu²⁺ ions. When 301N was precultured for 48h in the presence of 1 mM CuSO₄, its resistance to Cu²⁺ andthe synthesis of MT in response to Cu²⁺ were enhanced whileanalogous responses to Cd²⁺ were conversely reduced. These resultssuggest that the synthesis of MT is controlled by Cd²⁺ and Cu²⁺in a counteractive manner in strain 301N and, therefore, theregulation of the synthesis of MT plays a role in the adaptationof this strain to conditions when either metal is present. (Received November 1, 1990; Accepted February 22, 1991)     

Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus.

Resistance of Staphylococcus aureus strain 17810R to Cd2+ appears to be due to a plasmid-coded Cd2+ efflux system. Complete efflux of Cd2+ after transfer of preloaded cells into Cd2+-free medium occurred in the resistant strain 17810R, but not in the plasmidless derivative strain 17810S. Net efflux was blocked by 2,4-dinitrophenol, N,N,-dicyclohexylcarbodiimide (DCCD), and incubation at 4 degrees C. The inhibition of Cd2+ efflux by DCCD paralleled a stimulation of net uptake in the resistant cells by this agent. Cd2+ efflux by the resistant strain was accompanied by a reversal of inhibition of respiration, whereas in the sensitive strain, inhibition of respiration was not reversed after transfer to Cd2+-free medium. Net Cd2+ uptake by strain 17810R was inhibited by p-chloromercuribenzoate. In Cd2+ contrast, Cd2+ uptake by the plasmidless strain 17810S was affected neither by p-chloromercuribenzoate nor by DCCD when added alone, but was blocked by a combination of these two agents. Valinomycin had no effect on the reduced Cd2+ uptake by the resistant strain, whereas nigericin stimulated uptake to values comparable to those of the untreated sensitive cells. With sensitive cells, valinomycin reduced Cd2+ uptake by about 50%, whereas nigericin was without effect. A possible mechanism of Cd2+ movements in both strains is discussed.     

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.     

Cadmium accumulation in the chloroplast of Euglena gracilis

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl₂, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd²⁺, since uptake of added CdCl₂ in isolated chloroplasts exhibited a hyperbolic type of kinetics with a K_m of 57 µ M and V_max of 3.7 nmol (mg protein)⁻¹ min⁻¹. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd²⁺, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.     

Heavy metals resistant plasmid-mediated utilization of solar by Pseudomonas aeruginosa AA301

Solar-degrading bacteria, Pseudomonas aeruginosa strains, were isolated from Egyptian soil by Mineral Salt Medium (MSM) supplemented with Solar (motor fuel) from different oil-contaminated sites in Sohag province. The strain AA301 of Pseudomonas aeruginosa showed appreciable growth in MSM medium containing high concentrations of Solar ranging from 0.5 to 3% (v/v), with optimum concentration at 1.5%. Solar was used as a sole carbon source and a source of energy by the bacterium. The ability to degrade Solar was found to be associated with a single 60-kb plasmid designated pSOL15. The plasmid-cured variant, which was obtained by culturing in LB broth with kanamycin, lost the plasmid indicative the ability to degrade Solar must depend on this plasmid. The wild type isolate, Pseudomonas aeruginosa AA301 and transformant strain, have maximum growth (OD600 = approximately 2) on Solar, however the plasmid-cured variant did not have any significant growth on Solar. Moreover, resistance to a wide range of heavy metals such as Mn2+, Hg2+, Mg2+, Cd2+, Zn2+, and Ni2+ was also 60-kb plasmid-mediated. Therefore, the strain AA301 could be good candidate for remediation of some heavy metals and oil hydrocarbons in heavily polluted sites.     

Cd2 removal by Azomonas agilis PY101, a cadmium accumulating strain in continous aerobic culture

Azomonas agilis PY101 removed approximately >90% of Cd2+ from solution in continuous aerobic culture. After the unsteady state, approximately 5 days after inoculation, Cd2+ concentration in the artificial wastewater containing 1 mM CdCl2 remarkably decreased to below 92 to 96% of initial value during the growth of A. agilis PY101. A. agilis PY101 cells grown into the artificial wastewater actively uptaked Cd<2+ (from 2.37 to 6.28 mM/g dry weight cells) in the cell. © Rapid Science Ltd. 1998     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Involvement of thioredoxin peroxidase type II (Ahp1p) of Saccharomyces cerevisiae in Mn2+ homeostasis

To identify new proteins involved in Mn2+ homeostasis, we isolated Mn(2+)-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (delta cmp1 delta cmp2). The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca(2+)-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a delta cmp1 delta cmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane.     

Production of Two Chitosanases from a Chitosan-Assimilating Bacterium, Acinetobacter sp. Strain CHB101

A bacterial strain capable of utilizing chitosan as a sole carbon source was isolated from soil and was identified as a member of the genus Acinetobacter. This strain, designated CHB101, produced extracellular chitosan-degrading enzymes in the absence of chitosan. The chitosan-degrading activity in the culture fluid increased when cultures reached the early stationary phase, although the level of activity was low in the exponential growth phase. Two chitosanases, chitosanases I and II, which had molecular weights of 37,000 and 30,000, respectively, were purified from the culture fluid. Chitosanase I exhibited substrate specificity for chitosan that had a low degree of acetylation (10 to 30%), while chitosanase II degraded colloidal chitin and glycol chitin, as well as chitosan that had a degree of acetylation of 30%. Rapid decreases in the viscosities of chitosan solutions suggested that both chitosanases catalyzed an endo type of cleavage reaction; however, chitosan oligomers (molecules smaller than pentamers) were not produced after a prolonged reaction.     

Extracellular release of recombinant alpha-amylase from Escherichia coli using pulsed electric field

It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space. In this study, we demonstrated that recombinant alpha-amylase can be released from recombinant E. coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF). When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660=0.7, the amount of released alpha-amylase was about 30% of the total amount of alpha-amylase produced in the cells. As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the alpha-amylase, which was identical with intracellular alpha-amylase, was released by applied PEF cultivation. PEF treatment could be useful for easy release of periplasmic protein with selectivity.     

Interactions between cadmium,selenium and glutathione peroxidase in rat testis

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

Long term immune responses to pandemic influenza A/H1N1 infection in solid organ transplant recipients

In solid organ transplant (SOT) recipients it is unknown if natural infection with influenza confers protection from re-infection with the same strain during the next influenza season. The purpose of this study was to determine if infection with pandemic influenza A/H1N1 (pH1N1) resulted in a long-term immunologic response. Transplant recipients with microbiologically proven pH1N1 infection in 2009/2010 underwent humoral and cell-mediated immunity (CMI) testing for pH1N1 just prior to the next influenza season. Concurrent testing for A/Brisbane/59/2007 was done to rule-out cross-reacting antibody. We enrolled 22 adult transplant patients after pH1N1 infection. Follow up testing was done at a median of 7.4 months (range 5.8-15.4) after infection. After excluding those with cross-reactive antibody, 7/19 (36.8%) patients were seroprotected. Detectable pH1N1-specific CD4+ and CD8+ interferon-γ producing T-cells were found in 11/22 (50%) and 8/22 (36.4%) patients respectively. Humoral immunity had a significant correlation with a CD4 response. This is the first study in transplant patients to evaluate long-term humoral and cellular response after natural influenza infection. We show that a substantial proportion of SOT recipients with previous pH1N1 infection lack long-term humoral and cellular immune responses to pH1N1. These patients most likely are at risk for re-infection.     

Cadmium induces nuclear translocation of β-catenin and increases expression of <Emphasis Type="Italic">c-myc</Emphasis> and <Emphasis Type="Italic">Abcb1a</Emphasis> in kidney proximal tubule cells

Cadmium (Cd²⁺) induces renal proximal tubular (PT) damage, including disruption of the E-cadherin/β-catenin complex of adherens junctions (AJs) and apoptosis. Yet, chronic Cd²⁺ exposure causes malignant transformation of renal cells. Previously, we have demonstrated that Cd²⁺-mediated up-regulation of the multidrug transporter Abcb1 causes apoptosis resistance in PT cells. We hypothesized that Cd²⁺ activates adaptive signaling mechanisms mediated by β-catenin to evade apoptosis and increase proliferation. Here we show that 50 μM Cd²⁺, which induces cell death via apoptosis and necrosis, also causes a decrease of the trans-epithelial resistance of confluent WKPT-0293 Cl.2 cells, a rat renal PT cell model, within 45 min of Cd²⁺ exposure, as measured by electric cell-substrate impedance sensing. Immunofluorescence microscopy demonstrates Cd²⁺-induced decrease of E-cadherin at AJs and redistribution of β-catenin from the E-cadherin/β-catenin complex of AJs to cytosol and nuclei after 3 h. Immunoblotting confirms Cd²⁺-induced decrease of E-cadherin expression and translocation of β-catenin to cytosol and nuclei of PT cells. RT-PCR shows Cd²⁺-induced increase of expression of c-myc and of the isoform Abcb1a at 3 h. The data prove for the first time that Cd²⁺ induces nuclear translocation of β-catenin in PT cells. We speculate that Cd²⁺ activates β-catenin/T-cell factor signaling to trans-activate proliferation and apoptosis resistance genes and promote carcinogenesis of PT cells.     

Cadmium-copper interaction in intestinal mucosal cell cytosol of mice

In vivo as well as in vitro protein-metal interaction was studied in cytosolic fractions from intestinal mucosal cells. Female Swiss-Webster mice wre pretreated with cadmium (25 ppm) or copper (100 ppm) in drinking water for 3 weeks. Treatment groups were divided into subgroups receiving Cd or Cd+Cu for an additional 6 weeks. In the in vitro study, mucosal cytosol obtained from pretreated animals was incubated with Cd-109 or Cd-109+Cu. Proteins were separated by gel filtration chromatography and metals determined by furnace AAS or gamma-spectrometry. Cadmium-induced synthesis of metallothionein-like proteins (MTP) in cytosol was indicated by increased Cd in those eluted fractions corresponding to the molecular weight of purified equine renal metallothionein. This cadmium level reached a plateau after 3 weeks of cadmium treatment. In addition, an increased amount of cadmium bound to MTP was noted when copper was added to cadmium in drinking water of mice pretreated with copper. This was not the case for Cd-pretreated animals. The in vitro experiments produced similar results, in that MTP fractions retained a greater percentage of Cd when animals were pretreated with copper compared to controls. Cadmium pretreatment resulted in even higher amounts of cadmium bound to MTP. The existence of a Cd as well as a separate Cu MTP, each with specific metal-binding properties, is suggested.     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen,antiestrogen and progesterone administration

A synthetic progestin, 16α-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with K_D values of 1.2 nM (at 0–4°C) and 2.3 nM (at 15°C), respectively. Administration of estradiol-17β or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34 000 to 120 000 (estradiol-17β) and 80 000 (tamoxifen) receptors/ cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18 000 to 48 000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18 000 to 35 000 receptor/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70 000 vs. 30 000, and 40 000 vs. 17 000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Toxicity of Cadmium to Amoeba Proteus: A Biochemical Approach

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

Cd2+ transport and storage in the chloroplast of Euglena gracilis

Euglena gracilis lacks a plant-like vacuole and, when grown in Cd2+-containing medium, 60% of the accumulated Cd2+ is located inside the chloroplast. Hence, the biochemical mechanisms involved in Cd2+ accumulation in chloroplast were examined. Percoll-purified chloroplasts showed a temperature-sensitive uptake of the free 109Cd2+ ion. Kinetics of the uptake initial rate was resolved in two components, one hyperbolic and saturable (Vmax 11 nmol 109Cd2+ min(-1) mg protein (-1), Km 13 microM) and the other, linear and non-saturable. 109Cd2+ uptake was not affected by metabolic inhibitors or illumination. Zn2+ competitively inhibited 109Cd2+ uptake (Ki 8.2 microM); internal Cd2+ slightly inhibited 109Cd2+ uptake. Cadmium was partially and rapidly released from chloroplasts. These data suggested the involvement of a cation diffusion facilitator-like protein. Chloroplasts isolated from cells grown with 50 microM CdCl2 (ZCd50 chloroplasts) showed a 1.6 times increase in the uptake Vmax, whereas the Km and the non-saturable component did not change. In addition, Cd2+ retention in chloroplasts correlated with the amount of internal sulfur compounds. ZCd50 chloroplasts, which contained 4.4 times more thiol-compounds and sulfide than control chloroplasts, retained six times more Cd2+. The Cd2+ storage-inactivation mechanism was specific for Cd2+, since Zn2+ and Fe3+ were not preferentially accumulated into chloroplasts.     

Baicalein-Induced Apoptosis via Endoplasmic Reticulum Stress Through Elevations of Reactive Oxygen Species and Mitochondria Dependent Pathway in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Studies were designed to investigate the effects of baicalein on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca²⁺, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca²⁺ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca²⁺ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca²⁺ modulates the baicalein-induced cell death via a Ca²⁺-dependent mitochondrial death pathway in N18 cells.     

Accumulated forms of thallium and cadmium in Lemna minor. I. Distribution in aqueous soluble and insoluble fractions

Abstract

The distribution and chemical forms of thallium (Tl) and cadmium (Cd) in Lemna minor have been investigated using extractants of different polarity, enzyme digestion and ultrafiltration and chromatographic methods. Over 80% of Tl and 60% of Cd taken up by the plant was found in aqueous soluble forms. Water was more efficient than ethanol in extracting both elements; about 30% of bound Cd was released by dilute HCI treatment and Cd was mainly bound to pectins and proteins in the cell wall fractions but only a small proportion of Tl was associated with these components. In the aqueous soluble extracts a sizeable proportion of Cd was complexed with soluble moieties, including proteins; whereas Tl seems to be mainly present in the free ionic form.