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.     

Partial characterization of cadmium-binding protein from roots of tomato

Cd-binding protein was extracted from tomato roots and purified on QAE-Sephadex A-25 and on Sephadex G-75 in 1 molar KCl buffer. The protein preparation was light brown and contained predominantly Cd and small amounts of Zn and Cu. Polyacrylamide gel electrophoresis at pH 6.9 removed the brown material from protein which now bound mostly Cd and some Cu. The apparent molecular weight was 3,100 daltons in high ionic strength medium (1 molar KCl buffer) and 21,500 daltons at low ionic strength. Ionic strength also affected the apparent molecular weight of the Cd-binding protein in crude root extracts. The protein contained 26% cysteine, 53% glutamic acid/glutamine, and 2.8 gram atoms (Cd+Zn+Cu)/mole. The (Cd+Zn+Cu):cysteine ratio was 1:2.3. Circular dichroism measurements indicated Cd-thiolate coordination. The tomato Cd-binding protein was more similar to phytochelatins than to animal metallothioneins.     

Isolation of a novel rat testicular metalloprotein binding cadmium and zinc

Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration of¹⁰⁹Cd- or⁶⁵Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of¹⁰⁹Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/ mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10±4 kD/pI 6.3) Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (<60 kD) protein in rat testic cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of¹⁰⁹Cd- and⁶⁵Zn-labeled cytosol demonstrated that¹⁰⁹Cd and⁶⁵Zn cochromatography with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.     

Effects of dietary zinc depletion and food restriction on intestinal transport of cadmium in the rat

The effects of Zn depletion and short-term fasting on intestinal transport of Cd were examined in perfused rat small intestines. The small intestine was isolated with its vascular network intact, then simultaneously perfused from the luminal and vascular sides. A Zn-depleted state that results in marked hypozincemia was produced in some rats by feeding a Zn-deficient diet for 4 days. Uptake of Cd from the luminal perfusate was greater in the Zn-depleted rats, whereas transport of Cd to the vascular perfusate was not affected. Fasting overnight prior to perfusion did not influence Cd transport nor alter the effect of Zn depletion on Cd uptake. The Cd concentration in the soluble fraction of intestinal mucosa from perfused intestines was not different between Zn-depleted and Zn-adequate rats. Gel filtration chromatography of the soluble fraction showed a shift in the distribution of Cd from metallothionein to high molecular weight ligands in intestines from Zn-depleted rats. The decrease in amount of metallothionein-associated Cd corresponded to a decrease of total intestinal metallothionein as measured by the Cd-binding assay. The results suggest association of Cd with intestinal metallothionein did not influence the absorption of Cd under these conditions.     

Cadmium accumulation and distribution in human lung fibroblasts

An investigation was conducted on cadmium accumulation and its molecular distribution in growing cultures of human fetal lung fibroblasts (IMR-90). For the first 24–48 h post-exposure, the amount of cadmium per cell remained low and relatively constant; more than 50% of the intracellular Cd was associated with molecular weight components less than 2000 daltons. The presence of a Cd-binding component with a molecular weight of 11 800 daltons was also detected during this initial period. Based upon its molecular size, sensitivity to trypsin, and ability to coincorporate ³⁵S along with ¹¹⁵Cd, we have concluded that this component is a protein. Its similarity to metallothionein was suggested by its molecular size, low level in cells never exposed to Cd, spectral properties, and heat stability. During the late log phase of growth, accumulation of Cd by fibroblasts occurred at a near-linear rate and the intracellular Cd level was proportional to the exogenous concentration. There was a corresponding increase in the amount of the fibroblast Cd-binding protein present, accompanied by a reduction in the amount of Cd associated with low molecular weight components. Synthesis of the Cd-binding protein appeared to occur at a more rapid rate than accumulation suggesting that its presence may be necessary for Cd transport and/or accumulation, an interpretation strengthened by the finding that cells previously passaged in Cd exhibited no lag in accumulation and accumulated 8–10-fold more Cd. By 168 h post-exposure, a plateau occurred in the intracellular Cd level as well as the amount of Cd-binding protein present. After this period, a redistribution of Cd from the metallothionein-like protein to high molecular weight proteins occurred. It is possible that this redistribution might be the cellular event that triggers the pathological changes known to occur after Cd-grown cultures have reached confluency.     

Accumulation of cadmium and induction of its binding protein in the digestive tract of fleshfly (Sarcophaga peregrina) larvae

The larva of Sarcophaga peregrina ( fleshfly ) was fed with cadmium (Cd)-containing diet and the distribution of Cd among tissues was determined by separating each organ. Approximately 90% of Cd accumulated in the larva was found in the digestive tract, the fat body and the Malpighian tube being less effective tissues in its accumulation. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.     

High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco

Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.     

Macromolecular interactions with cadmium and the effects of zinc,copper, lead,and mercury ions

Interactions of cadmium (Cd) ions with bovine serum albumin (BSA), bovine hepatic metallothionein (MT), calf thymus histone and deoxyribonucleic acid (DNA), and bovine hepatic chromatins were studied in the presence and absence of divalent zinc (Zn), copper (Cu), mercury (Hg), or lead (Pb) ions, using equilibrium dialysis at pH 7 and at 37°C. The BSA had 3.5 Cd-binding sites with an apparent affinity constant of 1×10⁵. The other metal ions inhibited the binding by reducing the affinity constant and the number of Cd-binding sites in BSA. There were 6 high affinity and 13 low affinity Cd-binding sites in the MT. Zinc ions had poor efficacy in reducing the binding of Cd to the MT. However, the Cu²⁺ and Hg²⁺ ions inhibited the Cd binding to a considerable extent, the former ions being more potent in this respect. Histone did not bind Cd. There were two kinds of Cd-binding sites in DNA: One mole of Cd per four moles DNA-phosphorus at low affinity sites, and one mole of Cd per 6.7 moles DNA-phosphorus at high affinity sites. Their apparent association constants were 8.3×10⁵ and 4.4×10⁶ M, respectively. The other metal ions had inhibitory effects on the binding of Cd to DNA. Histone reduced the Cd-DNA interactions to only a minor extent. The other metal ions reduced the binding of Cd to DNA-histone complex to a small extent. Cadmium binds to the euchromatin (Euch), heterochromatin (Het), and Euch-Het mixture almost equally. The other metal ions reduced the binding maximally in Euch-Het followed next in order by Het and Euch. Cupric ions were the most potent inhibitors of the interactions of Cd with the nuclear materials.     

Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type

Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.     

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.     

Evidence for a proteolytic modification of liver pyruvate kinase in fasted rats.

Liver pyruvate kinase was purified to homogeneity from rats fed a high carbohydrate, low protein diet (LPK-C) and from rats fasted for 84 h (LPK-F). Although the enzymes have similar electrophoretic mobilities in 7% polyacrylamide disc gels, the specific activity of LPK-C was two to three times the value of the specific activity of LPK-F. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPK-C yields a single protein band of 56,000 daltons. In contrast, LPK-F yields two bands of protein. Approximately one-third of the LPK-F has an electrophoretic mobility similar to the 56,000-dalton LPK-C peptide. The remaining two-thirds of the LPK-F protein migrates as a 51,000-dalton peptide. Cyanogen bromide was used to cleave LPK-C and LPK-F. Similar peptide patterns were obtained from LPK-C and LPK-F when the cyanogen bromide fragments were resolved by 12% polyacrylamide gel electrophoresis in 7.5 m urea containing 6 mm Triton X-100 and 5% acetic acid. Separation of the two peptides from LPK-F was accomplished by selective immunologie absorption of the 56,000-dalton peptide with anti-LPK-C gammaglobulin immobilized on Sepharose 4B. Tryptic digests of LPK-C, LPK-F and the 51,000-dalton peptide yield similar peptide patterns when analyzed via sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. These results suggest that the 51,000-dalton peptide could be derived by a proteolytic cleavage or limited digestion of the 56,000-dalton subunit. Phosphorylation of LPK-C and LPK-F by [γ-³²P]ATP in vitro with cyclic AMP-activated protein kinase results in covalent incorporation of ${}^{32}$P into only the 56,000-dalton subunit. These results suggest that anin vivo proteolytic modification that yields the 51,000-dalton subunit.     

Glyceraldehyde-3-Phosphate Dehydrogenase (NADP) from Sinapis alba L: Reversible Association of the Enzyme with a Protein Factor as Controlled by Pyridine Nucleotides in Vitro

Aggregation of glyceraldehyde-3-P dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings during gel filtration on Sepharose 6B is dependent on the presence of a fraction (“binding fraction”) which can be separated from the enzyme by precipitation with 55% ammonium sulfate. Association of the enzyme with this binding fraction is NAD-dependent whereas NADP⁺ causes release. Dithioerythritol (2 mM) has no influence on these reversible processes.

Binding fractions, partially purified by ammonium sulfate and acetone fractionation, were submitted to dodecylsulfate-polyacrylamide gel electrophoresis. They always contain one or two dominant polypeptides with apparent molecular weights 42,000 and 58,000. The 42,000 polypeptide comigrates during dodecylsulfate electrophoresis with the corresponding subunit of the enzyme. It comprises up to 70% of the total protein in partially purified binding fractions and can be regarded as a major protein in seedling extracts.

The differential transport behavior of glyceraldehyde-3-P dehydrogenase (NADP) on Sephadex G-200 in the presence of NAD⁺ and NADP⁺ can be used as a simple and effective purification procedure. The enzyme isolated in this way has an isoelectric point of about 4.5 and maintains under all tested conditions a heterogeneous subunit composition of at least three different polypeptide chains (apparent molecular weights, 39,000, 42,000, 43,000).

The present data suggest that NAD(P)-controlled aggregation of glyceraldehyde-3-P dehydrogenase (NADP) from Sinapis alba L. is due primarily to enzyme association with a separate binding fraction rather than to enzyme polymerization. It is possible that a major component of this binding fraction, the 42,000 polypeptide, consists of “surplus” nonactive enzyme subunits, which self-associate and interact with the NAD-conformer of the enzyme.

     

Cadmium speciation studies in the intestine of Lumbricus terrestris by electrophoresis of metal proteins complexes

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO₃)₂ supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (¹⁰⁹Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.     

Cadmium-binding components in soybean plants

Soybean (Glycine max L.) plants exposed to ¹⁰⁹Cd readily absorb the element. Differential centrifugation of leaf, stem, and root homogenates followed by radioassay showed that Cd was associated primarily with the 105,000g supernatant. Separation of this fraction by gel chromatography and subsequent analysis by radioassays revealed that ¹⁰⁹Cd was bound to macromolecules of >50,000, 13,800, and 2,280 molecular weights. The >50,000 and 2,280 molecular weight fractions probably are nonspecific binding of Cd to normal cell components. The 13,800 molecular weight ¹⁰⁹Cd-bound component was found to be inducible by cadmium. It had a high ultraviolet absorbance at 254 nm and a low absorbance at 280 nm at pH 8.6.     

Interaction of platinum with metallothionein-like ligands in the rat kidney after administration of cis-dichlorodiammine platinum II

After the administration of the anticancer drug cis-dichlorodiammine platinum II (cisplatin) to male rats, the Pt in the soluble fraction of the kidney is isolated, by gel filtration, in association with a high molecular weight component and a low molecular weight fraction. At 24 h, Pt is also recovered in a metallothionein-like fraction which elutes from Sephadex G-50 with a lower apparent molecular weight than endogenous (Cu, Zn)-thionein or Cd-thionein isolated from the kidneys of Cd2+-treated rats. None of these low molecular weight metal-binding fractions binds to Octyl Sepharose CL-4B. On DE-52 ion exchange chromatography, Cd-thionein is resolved into two isometallothioneins whereas the low molecular weight Pt-binding fraction is only partially purified and contains at least six components which elute at higher gradient concentrations than metallothionein. Pretreatment with Cd2+ which stimulates the synthesis of renal and hepatic metallothionein has no effect on the uptake and subcellular distribution of Pt in the liver and kidneys. Cisplatin treatment reduces the concentration of Cu and Zn in the renal metallothionein and other soluble protein fractions in the kidney. When administered to Cd2+-pretreated rats, cisplatin promotes the loss of Zn from the soluble protein fractions but causes the redistribution of Cd from the metallothionein to the high molecular weight fraction and fails to inhibit the Cd2+-induced accumulation of Cu in the kidneys and the binding of Cu to the soluble protein fractions. It is suggested that metallothionein probably does not have a significant role in the renal metabolism of Pt following the administration of cisplatin to rats.     

芦苇抗镉污染机理研究

研究了芦苇幼苗体内 Cd的积累、亚细胞微区分布、存在形态和其诱导蛋白以及植物络合素合成抑制剂 (BSO)对芦苇光合作用和生长的影响。在 Cd污染条件下 ,芦苇幼苗植株和根皮层细胞中可积累大量的Cd,但 Cd在芦苇各器官和根皮层细胞亚细胞结构中的分布显著不均 ;Cd在芦苇幼苗体内的分配为 :根 >叶片 >茎 >地下茎 ,在根皮层细胞中的分布为 :细胞间隙 >细胞壁 >液泡 >细胞质。受 Cd污染的芦苇幼苗体内的 Cd以不同化学形态存在 ,其中 Na Cl提取态的 Cd在根和叶片中占的比例均为最大 ,其次为根内的醋酸提取态 ;在叶片中以水提取态为主 ,其它形态的含量相对较低。层析结果表明 ,根和叶片中各存在一种Cd结合蛋白 ,其中根内的 Cd结合蛋白可能是一种植物络合素聚合体。受 Cd诱导 ,芦苇幼苗根中还新合成了一种小分子蛋白或多肽 ,但另有一种蛋白因 Cd影响而消失。此外 ,BSO实验证明了植物络合素对 Cd的解毒作用。可见 ,芦苇的抗 Cd机理与以下几个方面有关 :根部截留 ,细胞间隙积累 ,细胞壁沉淀 ,液泡区域化 ,形成活性较低的难溶化合物 ,形成 Cd结合蛋白     

Combined application of a laser ablation-ICP-MS assay for screening and ESI-FTICR-MS for identification of a Cd-binding protein in Spinacia oleracea L. after exposure to Cd

We have studied the binding of the toxic element Cd to plant proteins and have used for this purpose spinach (Spinacia oleracea L.) plants treated with 50 μM Cd(II) as a model system. Laser ablation ICP-MS has been applied for the screening of Cd-binding proteins after separation by native anodal polyacrylamide gel electrophoresis (AN-PAGE) and electroblotting onto membranes. The main Cd-carrying protein band was isolated and investigated by nano-electrospray ionization-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry after tryptic digestion. By this procedure, the main Cd-binding protein was identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The latter enzyme has been discussed in the literature to be affected in its activity by oxidative stress induced by Cd. However, in this paper it is demonstrated for the first time that RuBisCO directly binds Cd and thus may be directly altered by this toxic element. A commercially available protein standard was used to verify direct binding of Cd(II) to the protein, even without metabolisation. The resulting metal-protein complex was shown to be stable enough to survive AN-PAGE separation and electroblotting. By the use of size exclusion chromatography coupled with ICP-MS it was demonstrated that the RuBisCO protein standard shows similar metal binding properties to Cd. Furthermore, essential elements such as Mn(II), Fe(II) and Cu(II), which are known to possibly replace the RuBisCO activator Mg(II), were investigated in addition to Zn(II). Again, similar binding properties in comparison to the plant protein were observed.     

Studies of Sulfate Utilization by Algae: 9. Fractionation of a Cell-free System from Chlorella into Two Activities Necessary for the Reduction of Adenosine 3'-Phosphate 5'-Phosphosulfate to Acid-Volatile Radioactivity

Further properties of the enzymatic system obtained from Chlorella pyrenoidosa (Emerson strain 3) which reduces adenosine 3′-phosphate 5′-phosphosulfate-³⁵S to acid-volatile radioactivity, when fortified with Mg²⁺ and 2, 3-dimercaptopropan-1-ol as reductant, are described. Optimal concentrations of adenosine 3′-phosphate 5′-phosphosulfate-³⁵S and Mg²⁺ and the pH optimum have been determined. 2,3-Dimercaptopropan-1-ol can be replaced by dithiothreitol, mercaptoethanol, reduced glutathione, cysteine, and cysteamine. Treatment of the crude extracts with ammonium sulfate and alumina C-gamma gel yields two fractions, designated “S” and “A,” which must be recombined to obtain acid-volatile radioactivity. Further fractionation of fraction S by ammonium sulfate gradient elution and diethylaminoethyl cellulose chromatography yields approximately a 50-fold increase in specific activity compared to that found in the crude extract. This material appears to contain an active component with a molecular weight estimated by agarose gel chromatography of about 330,000.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.