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.     

Iron promotes cadmium binding to citrate.

Iron-cadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd2+] in physiological saline solution at pH 7.4, we show that Fe2+ promotes Cd2+ binding to citrate thereby decreasing the availability of free Cd2+. This suggests the formation of high molecular weight Cd2+-Fe2+-citrate complexes. We confirm this suggestion by showing that 109Cd2+ is retained by 1 kDa cut off filters when present with total 50 microM Fe2+ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd2+ absorption. As citrate is part of the diet, we suggest that these iron-cadmium interactions may contribute to the protective effect of iron against cadmium toxicity.     

Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa. Subunits of 97 to greater than 200 kDa have been observed in these preparations. Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl. Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related. Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl. Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein. Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis. The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors. Neither polyclonal nor monoclonal antibodies against E. gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.     

Isolation of a novel type-I fatty-acid synthetase from Euglena gracilis. Specific derepression in streptomycin-bleached cells.

A novel, high-molecular-mass fatty-acid synthetase (FAS) complex has been isolated from streptomycin-bleached Euglena gracilis cells. The enzyme was purified 250-fold from the crude cell homogenate and subsequently migrated upon SDS/PAGE as a single band of molecular mass 270 kDa. This apparent subunit size of the purified protein contrasted with a smaller size of only 200 kDa which was exhibited by the same protein upon immunoblotting of the crude cell extract. The purified Euglena FAS complex cosediments in a sucrose density gradient with yeast FAS and, from this, both enzymes were concluded to have the same overall molecular mass of 2.3 MDa. The enzyme described in this paper appears to be a typical type-I FAS multienzyme which clearly differs from the E. gracilis FAS so far described. Instead, it appears to be organized structurally similar to the type-I FAS multienzymes of lower fungi. In vitro, the purified Euglena FAS complex synthesizes mainly palmitic acid, or its CoA ester, from acetyl CoA and malonyl CoA as substrates. The Km values for acetyl CoA and malonyl CoA are 20 microM and 31 microM, respectively. Similar to the FAS enzymes of other lower eucaryotes, the Euglena type-I FAS is a flavoprotein. In contrast to yeast FAS, however, the flavin cofactor appears to be covalently attached to the enzyme protein. By immunological techniques, the enzyme was shown to be absent in green as well as in etiolated E. gracilis cells, while being rapidly induced upon streptomycin bleaching of heterotrophically growing green cells. The data suggest an inverse correlation between organellar development and derepression of this FAS complex.     

Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position.

We sequenced the chloroplast 16S rRNA gene of two Euglena gracilis mutants which contain streptomycin-resistant chloroplasts (Smr 139.12/4 and Smr 139.20/2). These mutants are known to contain a single intact rrn operon per circular chloroplast genome. Nucleotide sequence comparison between a 16S rRNA gene of wild type Euglena gracilis, strain Z, with streptomycin-sensitive chloroplasts, and the 16S rRNA gene of both Smr-strains reveals a single base change (C to T) at position 876. This position is equivalent to the invariant position 912 of the E. coli 16S rRNA gene. The analogous position is also conserved in all chloroplast small subunit RNA genes from lower and higher plants sequenced so far. Light dependent protein synthesis with purified chloroplasts from streptomycin-resistant cells is not inhibited by streptomycin. Based on the results reported here we postulate linkage between the observed point mutation on the 16S rRNA gene and streptomycin-resistance of chloroplast 70S ribosomes.     

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.     

Cadmium-induced synthesis of HSP70 and a role of glutathione in Euglena gracilis

The effect of cadmium-induced oxidative stress, with or without glutathione supplementation, was investigated in the single cell eukaryotic phytoflagellate, Euglena gracilis strains Z and its achlorophyllous mutant SMZ as experimental models. Both these strains actively synthesize thiols to prevent or resist cadmium toxicity. The content of glutathione, as a representative antioxidant, was also examined in both strains. Exposure to cadmium induced heat-shock protein 70 (HSP70) synthesis in both strains of E. gracilis. Glutathione supplementation also induced HSP70. Overall, these results indicate that glutathione was closely linked to the induction of stress-related proteins. The sensitivity to cadmium-stress was higher in strain Z than SMZ. The results suggest that chloroplasts may have a role in the regulation of HSP70 expression. The relationship between HSP70 and GSH levels is still far from understood, and further research may shed light upon their up-regulation in the presence of Cd.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

Nucleotide sequence of a Euglena gracilis chloroplast gene coding for the 16S rRNA: homologies to E. coli and Zea mays chloroplast 16S rRNA

The nucleotide sequence of 16S rDNA from Euglena gracilis chloroplasts has been determined representing the first complete sequence of an algal chloroplast rRNA gene. The structural part of the 16S rRNA gene has 1491 nucleotides according to a comparative analysis of our sequencing results with the published 5'- and 3'-terminal "T1-oligonucleotides" from 16S rRNA from E. gracilis. Alignment with 16S rDNA from Zea mays chloroplasts and E. coli reveals 80 to 72% sequence homology, respectively. Two deletions of 9 and 23 nucleotides are found which are identical in size and position with deletions observed in 16S rDNA of maize and tobacco chloroplasts and which seem to be characteristic for all chloroplast rRNA species. We also find insertions and deletions in E. gracilis not seen in 16S rDNA of higher plant chloroplasts. The 16S rRNA sequence of E. gracilis chloroplasts can be folded by base pairing according to the general 16S rRNA secondary structure model.     

Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii

Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem>root>leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the contrary, Cd exposure strongly induced the production of PCs (mainly PC(2) and PC(3)) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii.     

Comparison of the structure of the extrinsic 33 kDa protein from different organisms

The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane. The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da. The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants. The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments. The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa). The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants). Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types.     

Involvement of reactive oxygen stress in cadmium-induced cellular damage in Euglena gracilis

Inorganic cadmium (Cd) causes cellular damage to eukaryotes and to tissues of higher organisms, including DNA strand breaks and intracellular membrane damage, as a result of reactive oxygen stress. We previously reported cadmium chloride (CdCl2)-induced abnormal cell morphologies in the unicellular eukaryote Euglena gracilis Z (a plant cell model) and its achlorophyllous mutant SMZ strain (an animal cell model). The present study was undertaken to examine whether exposure of both strains to CdCl2 would lead to similar cellular responses, especially with regard to reactive oxygen stress loading and cellular damage. The results indicate that CdCl2 exposure can induce morphological alteration, linked to reactive oxygen stress. Both E. gracilis Z and SMZ cells subjected to short-term, high-dose CdCl2 exposure showed long 'comet lengths' in the so-called 'Comet' assay, indicating DNA strand breaks. Similarly, short-term, high-dose CdCl(2)-exposed cells and CdCl(2)-induced morphologically altered cells showed intense fluorescence of dihydrofluorescein (HFLUOR) after incubation with dihydrofluorescein diacetate (HFLUOR-DA). Positive data on the generation and involvement of intracellular reactive oxygen species (ROS) were obtained from long-term, low-dose CdCl(2)-exposed E. gracilis Z and SMZ, by thiobarbituric acid (TBA)-malondialdehyde (MDA) complex analyses.     

Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.     

Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.

The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.     

S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase.

An amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas azotoformans IAM 1603 and characterized. The enzyme acted S-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined. The gene encoding the S-stereoselective piperazine-2-tert-butylcarboxamide amidase was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 2.1 kb of genomic DNA revealed the presence of two ORFs, one of which (laaA) encodes the amidase. This enzyme, LaaA is composed of 310 amino acid residues (molecular mass 34 514 Da), and the deduced amino acid sequence exhibits significant similarity to hypothetical and functionally characterized proline iminopeptidases from several bacteria. The laaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant LaaA enzyme in cell-free extracts of E. coli was 13.1 units.mg(-1) with l-prolinamide as substrate. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and two column chromatography steps. On gel-filtration chromatography, the enzyme appeared to be a monomer with a molecular mass of 32 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylhydrazine, Zn2+, Ag+, Cd2+ or Hg2+. LaaA had hydrolyzing activity toward L-amino acid amides such as L-prolinamide, L-proline-p-nitroanilide, L-alaninamide and L-methioninamide, but did not act on the peptide substrates for the proline iminopeptidases despite their sequence similarity to LaaA. The enzyme also acted S-stereoselectively on (R,S)-piperidine-2-carboxamide, (R,S)-piperazine-2-carboxamide and (R,S)-piperazine-2-tert-butylcarboxamide. Based on its specificity towards L-amino acid amides, the enzyme was named L-amino acid amidase. E. coli transformants overexpressing the laaA gene could be used for the S-stereoselective hydrolysis of (R,S)-piperazine-2-tert-butylcarboxamide.     

Isolation and properties of intracellular peptidase from Brevibacterium]

The intracellular peptidase of Brevibacterium E531, a lysine-producing bacterial species, was purified 6500-fold by chromatography on DEAE-cellulose and the affinity adsorbent H-Thr(But)-Phe-Pro-hexamethylene-diamine-Sepharose 4B and by gel filtration on Sephadex G-200. The enzyme displayed the maximum activity towards proline p-nitroanilide at pH 7.7-7.9 and readily split glycine, alanine and proline from di-, tri- and tetrapeptides but did not practically hydrolyze oligopeptides of a greater chain length. The enzyme was not inhibited by complexons (EDTA, 8-oxiquinoline and 1.10-phenanthroline). The peptidase was not activated by divalent metal ions and was inhibited by Zn2+; Cd2+, Hg2+ and Cu2+. Data brom gel filtration on Sephadex G-200 suggest that the molecular mass of the enzyme is no less than 250 kDa. In the presence of sodium dodecyl sulfate the molecular mass of the enzyme is 43 kDa, which is suggestive of the presence of a quaternary structure. One peculiarity of the enzyme is its activation by alkaline metal halogenides and sodium nitrate which reaches a maximum at the 0.05-0.1 M concentration of the salts.     

伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。