Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides

The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.     

Influence of selenium on toxicity of some heavy metals in the green alga Scenedesmus obliquus

The green alga Scenedesmus obliquus was incubated with various heavy metals (Cd2+, Zn2+, Mn2+, Ni2+) in presence/absence of selenium. S. obliquus exhibited higher rates of growth and some metabolic activities in cultures containing 0.1 mM Se than those containing only the heavy metals. The positive effect of Se was found in presence of Cd2+ while that in the case of presence of Ni2+ was less pronounced.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

A Functional Putative Phytochelatin Synthase from the Primitive Red Alga Cyanidioschyzon merolae

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins (PCs), which play a detoxification role in higher plants. Heterologous expression of CmPCS, a product of a PCS-like gene from the genomic DNA of the red alga Cyanidioschyzon merolae, rescued Cd²⁺-sensitive yeast from Cd²⁺ toxicity. The fact that these transformed cells synthesized PCs demonstrates that CmPCS is functional.     

Inhibition of phorbol ester binding and protein kinase C activity by heavy metals

Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ or chelator, heavy metals (Cd2+, Cu2+, Hg2+, Zn2+, in the 10 microM range) inhibit the activity of, and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of [3H]phorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalytic activity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot be surmounted by excess Ca2+ or Mg2+. The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activity of that kinase, but in a distinctly different pattern.     

Influence of selenium on toxicity of some heavy metals in the green algaScenedesmus obliquus

The green alga Scenedesmus obliquus was incubated with heavy metals (Cd2+, Zn2+, Mn2+, Ni2+) with and without selenium. S. obliquus exhibited higher rates of growth and some metabolic activities in cultures containing 0.1 mmol/L Se than those only containing the heavy metals. The positive effect of Se was found with all metals but was negligible with Mn2+.     

Function of phytochelatin synthase in catabolism of glutathione-conjugates

Detoxification of xenobiotic compounds and heavy metals is a pivotal capacity of organisms, in which glutathione (GSH) plays an important role. In plants, electrophilic herbicides are conjugated to the thiol group of GSH, and heavy metal ions form complexes as thiolates with GSH-derived phytochelatins (PCs). In both detoxification processes of plants, phytochelatin synthase (PCS) emerges as a key player. The enzyme is activated by heavy metal ions and catalyzes PC formation from GSH by transferring glutamylcysteinyl residues (gamma-EC) onto GSH. In this study with Arabidopsis, we show that PCS plays a role in the plant-specific catabolism of glutathione conjugates (GS-conjugates). In contrast to animals, breakdown of GS-conjugates in plants can be initiated by cleavage of the carboxyterminal glycine residue that leads to the generation of the corresponding gamma-EC-conjugate. We used the xenobiotic bimane in order to follow GS-conjugate turnover. Functional knockout of the two PCS of Arabidopsis, AtPCS1 and AtPCS2, revealed that AtPCS1 provides a major activity responsible for conversion of the fluorescent bimane-GS-conjugate (GS-bimane) into gamma-EC-bimane. AtPCS1 deficiency resulted in a gamma-EC-bimane deficiency. Transfection of PCS-deficient cells with AtPCS1 recovered gamma-EC-bimane levels. The level of the gamma-EC-bimane conjugate was enhanced several-fold in the presence of Cd2+ ions in the wild type, but not in the PCS-deficient double mutant, consistent with a PCS-catalyzed GS-conjugate turnover. Thus AtPCS1 has two cellular functions: mediating both heavy metal tolerance and GS-conjugate degradation.     

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase activity by cadmium, lead and mercury.

The effect of Cd2+, Pb2+ and Hg2+ on the Ca(2+)-ATPase activity of sarcoplasmic reticulum from rabbit muscle was studied. The concentration of relevant free and complex species for the assay conditions have been computed. As a result, ATP hydrolysis was found to be inhibited with an IC50 value of 950 nmol/l free Cd2+ or 95 nmol/l free Pb2+. Although calculation of the free Hg2+ was not possible, the comparison of the IC50 values for total metal ions show that Hg2+ is the strongest inhibitor of enzyme activity. The inhibition by Cd2+ seems to be independent of substrate concentration, whereas the inhibitory effect of Pb2+ is lowered in the presence of higher MgATP concentrations. Our data illustrate that the three heavy metals are potent inhibitors of the Ca2+ pump. Therefore low concentrations of these metal ions may disturb intracellular Ca2+ homeostasis and act on Ca(2+)-mediated cell functions.     

Removal of heavy metals from water effluents using supermacroporous metal chelating cryogels

Applications of IDA in, for example, immobilized metal ion affinity chromatography for purification of His-tagged proteins are well recognized. The use of IDA as an efficient chelating adsorbent for environmental separations, that is, for the capture of heavy metals, is not studied. Adsorbents based on supermacroporous gels (cryogels) bearing metal chelating functionalities (IDA residues and ligand derived from derivatization of epoxy-cryogel with tris(2-aminoethyl)amine followed by the treatment with bromoacetic acid (defined as TBA ligand)) have been prepared and evaluated on capture of heavy metal ions. The cryogels were prepared in plastic carriers, resulting in desired mechanical stability and named as macroporous gel particles (MGPs). Sorption and desorption experiments for different metals (Cu2+, Zn2+, Cd2+, and Ni2+ with IDA adsorbent and Cu2+ and Zn2+ with TBA adsorbent) were carried out in batch and monolithic modes, respectively. Obtained capacities with Cu2+ were 74 μmol/mL (TBA) and 19 μmol/mL gel (IDA). The metal removal was higher for pH values between pH 3 and 5. Both adsorbents showed improved sorption at lower temperatures (10°C) than at higher (40°C) and the adsorption significantly dropped for the TBA adsorbent and Zn2+ at 40°C. Desorption of Cu2+ by using 1 M HCl and 0.1 M EDTA was successful for the IDA adsorbent whereas the desorption with the TBA adsorbent needs further attention. The result of this work has demonstrated that MGPs are potential treatment alternatives within the field of environmental separations and the removal of heavy metals from water effluents.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Effects of heavy metal on rat liver microsomal Ca2(+)-ATPase and Ca2+ sequestering. Relation to SH groups.

In isolated hepatic microsomal vesicles the heavy metals Cd2+, Cu2+, and Zn2+ inhibit Ca2+ uptake and evoke a prompt efflux of Ca2+ from preloaded vesicles in a dose-dependent manner. N-Ethylmaleimide also inhibits Ca2+ uptake and causes Ca2+ release, but it is less effective in these respects than the heavy metals. Measurement of mannose-6-phosphatase activity indicate that the heavy metal-induced Ca2+ efflux is not caused by a general increase in membrane permeability. Heavy metals also inhibit the Ca2(+)-ATPase activity and the formation of the phosphorylated intermediate of the enzyme. In contrast, the sulfhydryl modifying reagent, N-ethylmaleimide inhibits the Ca2(+)-ATPase activity while it has a relatively small effect on Ca2+ release. Thus, the effects of these agents on Ca2+ sequestering and Ca2(+)-ATPase activity are not strictly proportional. The sulfhydryl group reducing agent dithiothreitol protects the microsomes from the effects of heavy metals, while glutathione is less protective. Addition of vanadate to vesicles, at a concentration which completely blocked the activity of the Ca2(+)-ATPase, resulted in a small and slow release of the accumulated Ca2+. Subsequent additions of heavy metals evoked a massive Ca2+ release. Thus, the effects of heavy metals on Ca2+ efflux cannot be due entirely to their inhibition of the Ca2+ pump. The heavy metal-induced Ca2+ efflux is not inhibited either by ruthenium red or tetracaine.     

Petiolar felt-sheath of palm: a new biosorbent for the removal of heavy metals from contaminated water.

Biosorption of heavy metals such as Pb2+, Ni2+, Cd2+, Cu2+, Cr3+ and Zn2+ by petiolar felt-sheath of palm (PFP) from contaminated water was examined. PFP was found to efficiently remove all the toxic metal ions with selectivity order of Pb2+ > Cd2+ > Cu2+ > Zn2+ > Ni2+ > Cr3+. The uptake was rapid, with more than 70% completed within 15 min. The bound metal ions were successfully desorbed and the PFP fibrous-biomass remained effective after several adsorption-desorption cycles.     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

Biosorption of Cu2+, Cd2+, Pb2+, and Zn2+ using dried marine green macroalga Caulerpa lentillifera

The sorption of Cu2+, Cd2+, Pb2+, and Zn2+ by a dried green macroalga Caulerpa lentillifera was investigated. The removal efficiency increased with pH. The analysis with FT-IR indicated that possible functional groups involved in metal sorption by this alga were O-H bending, N-H bending, N-H stretching, C-N stretching, C-O, SO stretching, and S-O stretching. The sorption of all metal ions rapidly reached equilibrium within 20min. The sorption kinetics of these metals were governed by external mass transfer and intraparticle diffusion processes. The sorption isotherm followed the Langmuir isotherm where the maximum sorption capacities was Pb2+>Cu2+>Cd2+>Zn2+.     

Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco

Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757?bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum. KEY MESSAGE: Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.     

Oxidation of cysteines activates cGMP-dependent protein kinase.

The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.