Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S bimane S-glutathione - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP-SG S-(2,4-dinitrophenyl)glutathione - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydrazone - LTC₄ cysteinyl leukotriene - MDR multidrug transporter - MRP multidrug resistance protein - NEM-SG N-ethylmaleimide glutathione We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom     

Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol

The microtiter plate technique reported by Baker and colleagues for the glutathione reductase-DTNB recycling assay of total glutathione (GSx) and glutathione disulfide (GSSG) has been modified according to Anderson's recommendations, in order to improve the reliability and accuracy of this miniaturized method for the measurement of glutathione status in cultured/isolated cells. Dilute HCl (10 mmol/L) has been used to lyse cells, before protein removal by centrifugation in the presence of 1.3% sulfosalicylic acid. The final DTNB, GSSG-reductase and NADPH concentrations in the reaction mixture have been increased to 0.7 mmol/L, 1.2 IU/ml and 0.24 mmol/L, respectively. The procedure specificity has been tested by spiking and dilution assays, showing that about 90% of the expected GSx amounts could actually be recovered, while no changes of GSSG concentrations were caused in the cells. Accuracy has been assessed by analysis of within-series precision as well as of intra- and interassay reproducibility, showing coefficient variation of <10%. Glutathione changes measured either in control rat hepatocytes or in primary cultures treated with paracetamol or menadione were in good agreement with well-known literature data. These data suggest that the experimental conditions reported in this paper are suitable for the analysis of total glutathione and glutathione disulfide concentrations in cultured/isolated cells.Abbreviations BSO dl-butionine-(S,R)-sulfoximine - DTNB 5,5-dithiobis-2-nitrobenzoic acid - GSSG glutathione disulfide - GSx total glutathione - SSA 5-sulfosalicylic acid     

Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant chinese hamster ovary cells

Summary Arsenic-resistant Chinese hamster ovary (CHO) cells were established by progressively increasing the concentration of sodium arsenite in culture medium. One of the resistant clones, SA7, was also cross-resistant to As(V), Zn, Fe(II), Co, and Hg. The susceptibilities to sodium arsenite in parental CHO cells, revertant SA7N cells, and resistant SA7 cells were correlated with their intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activity. The resistance in SA7 cells was diminished by depletion of GSH in cells after treatment with buthionine sulfoximine. Furthermore, after reexposure of revertant SA7N cells to sodium arsenite, the intracellular GSH levels, GST activity, and resistance to sodium arsenite were raised to the same levels as SA7 cells. These data indicate that the elevation of intracellular GSH levels and GST activity in SA7 cells may be responsible for the resistance to arsenite. A p25 protein, which could be a monomer subunit of GST, accumulated in SA7 cells. In addition, an outward transport inhibitor, verapamil, indiscriminately increased the arsenite toxicity in resistant and parental cells. This work was supported in part by grant NSC77-0201-B001-31 from the National Science Council, Republic of China.     

The pyrogallol assay for superoxide dismutase: Absence of a glutathione artifact

Reduced glutathione was found to affect the assay for superoxide dismutase when autooxidation of epinephrine, but not pyrogallol, was used as the indicator. Glutathione concentrations in the micromolar range, which correspond to levels in erythrocyte extracts, were capable of perturbing the epinephrine assay method and causing overestimation of enzyme content. The pyrogallol method was not significantly affected by large excesses of glutathione and appears to be a superior method for tissue extracts likely to be rich in glutathione.     

Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: Chemical nature of cellular metal

Copper-sensitive (Cu^s) and copper-resistant (Cu^r) strains of Pseudomonas aeruginosa were characterized in terms of Cu²⁺ sensitivity, uptake and its compartmentalization in the possible cell sectors. Minimum inhibitory concentrations (MICs) of Cu²⁺ for the Cu^r strain (3.2 mM and 0.12 mM in enriched- and in minimal-medium, respectively) were almost 5-fold higher over that of its sensitive counterpart. While Cu^s strain accumulated Cu²⁺ to a maximum of 1.8 mol mg^–1 protein, Cu^r strain increased it to 2.37 mol mg^–1 protein. Both the strains also demonstrated energy- and pH-dependent Cu²⁺ uptake through the broad-substrate range divalent cation (Zn²⁺, Mg²⁺, Co²⁺) uptake system as well as through the system specific for Cu²⁺. Cell-fractionation study revealed that in Cu^r strain, periplasm and membrane are the main Cu²⁺ binding sites, whereas, in case of Cu^s strain, it is the cytoplasm. The overall observations indicate that the Cu^r strain restricted Cu²⁺ sequestration exterior to the cytoplasm as the possible strategy for Cu-resistance. The chemical nature of Cu²⁺ deposition in the respective strains was also ascertained by X-ray powder diffraction analysis.     

Effect of N,N-diallyl-2,2-dichloroacetamide (R-25788) on efflux and synthesis of glutathione in tobacco suspension cultures

The low level of glutathione synthesis observed in tobacco suspension cultures grown under heterotrophic conditions was stimulated by the addition of t     

Regulation of cytoplasmic and vacuolar volumes by plant cells in suspension culture

Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.     

Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH-stress strategy

AIMS: To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. METHODS AND RESULTS: When C. utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1.5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5.5 to 1.2 at 24 h and maintained at 1.2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02-08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1.2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. CONCLUSIONS: Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197.3 mg l-1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2.11 to 2.67% (w/w), and the total GSH concentration could reach 737.1 mg l-1 and increased by 24.9%. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.     

Glutathione metabolism in primate lenses: A phylogenetic study of glutathione synthesis and glutathione redox cycle enzyme activities

Lens wet weights, soluble protein, and activities of γ-glutiamylcysteine synthetase, glutathione synthetase, glutathione peroxidase, and glutathione reductase were determined in primate lenses. The primary sources of lenses were middle-aged adult animals. The Primates, from 23 genera, were categorized into six superfamilies: hominoids (five species), Old World monkeys (seven species), New World monkeys (five species), tarsiers (two species), lemurs (six species), and lorisids (three species). Significant differences between various groups or combinations of groups were noted for γ-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase activities. Lenticular γ-glutamylcysteine synthetase activity was very low in the Old World simian lenses and highest in the prosimians. Glutathione peroxidase activity was extraordinarily high in lenses of Old World monkeys. Glutathione reductase activity was low in all the prosimians but tenfold higher in hominoid lenses with intermediate values in monkeys of both the Old World and New World. Glutathione synthetase activity was variable, and no clear pattern which might be useful for primate classification was noted. Lenticular activity ratios of glutathione synthetase:γ-glutamylcysteine synthetase were highest in the Old World simians and lowest in the prosimians. These data with emphasis upon Aotus and the tarsiers were examined with regard to phylogenetic relationships. © 1994 Wiley-Liss, Inc.     

Recent advances in protein engineering and biotechnological applications of glutathione transferases

Glutathione transferases (GSTs, EC 2.5.1.18) are a widespread family of enzymes that play a central role in the detoxification, metabolism, and transport or sequestration of endogenous or xenobiotic compounds. During the last two decades, delineation of the important structural and catalytic features of GSTs has laid the groundwork for engineering GSTs, involving both rational and random approaches, aiming to create new variants with new or altered properties. These approaches have expanded the usefulness of native GSTs, not only for understanding the fundamentals of molecular detoxification mechanisms, but also for the development medical, analytical, environmental, and agricultural applications. This review article attempts to summarize successful examples and current developments on GST engineering, highlighting in parallel the recent knowledge gained on their phylogenetic relationships, structural/catalytic features, and biotechnological applications.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

Phenotypic changes in Arabidopsis caused by expression of a yeast vacuolar Ca2+/H+ antiporter

In plants, cytosolic Ca²⁺ levels are tightly regulated, and changes in cytosolic Ca²⁺ have been implicated in converting numerous signals into adapted responses. Vacuolar ion transporters are thought to be key mediators of cytosolic Ca²⁺ concentrations. In an attempt to interpret the role of vacuolar Ca²⁺ transport in plant processes, we have expressed the yeast vacuolar Ca²⁺/H⁺ antiporter, VCX1, in Arabidopsis and tobacco. This transporter localizes to the plant vacuolar membrane. VCX1-expressing Arabidopsis plants displayed increased sensitivity to sodium and other ions. These ion sensitivities could be suppressed by addition of calcium to the media. VCX1-expressing plants demonstrated increased tonoplast-enriched Ca²⁺/H⁺ antiport activity as well as increased Ca²⁺ accumulation. These results suggest that VCX1 expression in Arabidopsis could be a valuable tool with which to experimentally dissect the role of Ca²⁺ transport around the plant vacuole.     

Co-induction of a glutathione-S-transferase, a glutathione transporter and an ABC transporter in maize by xenobiotics

Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by four herbicides (2,4-D, atrazine, metolachlor and primisulfuron) and a herbicide safener (dichlormid) on the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect on gene expression varies with both chemicals and genes. The expression of ZmGST27 and ZmMRP1 was up-regulated by all five compounds, whereas that of ZmGT1 was increased by atrazine, metolachlor, primisulfuron and dichlormid, but not by 2,4-D. For all chemicals, the inducing effect was first detected on ZmGST27. The finding that ZmGT1 is activated alongside ZmGST27 and ZmMRP1 suggests that glutathione transporters are an important component in the xenobiotic detoxification system of plants.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.