Induction of glutathione S-transferases in Arabidopsis by herbicide safeners

Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledenous crops. The reason(s) for this difference in safening is unknown. However, safener-induced protection in cereals is associated with increased expression of herbicide detoxifying enzymes, including glutathione S-transferases (GSTs). Treatment of Arabidopsis seedlings growing in liquid medium with various safeners similarly resulted in enhanced GST activities toward a range of xenobiotics with benoxacor, fenclorim, and fluxofenim being the most effective. Safeners also increased the tripeptide glutathione content of Arabidopsis seedlings. However, treatment of Arabidopsis plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Each safener produced a distinct profile of enhanced GST activity toward different substrates suggesting a differential induction of distinct isoenzymes. This was confirmed by analysis of affinity-purified GST subunits by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AtGSTU19, a tau class GST, was identified as a dominant polypeptide in all samples. When AtGSTU19 was expressed in Escherichia coli, the recombinant enzyme was highly active toward 1-chloro-2,4-dinitrobenzene, as well as chloroacetanilide herbicides. Immunoblot analysis confirmed that AtGSTU19 was induced in response to several safeners. Differential induction of tau GSTs, as well as members of the phi and theta classes by safeners, was demonstrated by RNA-blot analysis. These results indicate that, although Arabidopsis may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

Multiple roles of glutathione binding-site residues of glutathione s-transferase

This study was designed to characterize residues in the glutathione binding site of AdGSTD4-4 from the mosquito malaria vector Anopheles dirus. The data revealed that Leu33, His38 and His50 each play a role in enzyme catalysis and glutathione binding. The mutants of these three residues also displayed differences in hydrophobic substrate specificity, suggesting that changes in the active site conformation occurred. Differences in conformations was also suggested by protein stability changes. These results indicate that residues in the glutathione binding site are not only important in the catalytic function but also play a role in the structural integrity of the enzyme.     

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.     

Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an approximately 2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue.     

Microplate assay of glutathione s-transferase activity for resistance detection in single-mosquito triturates

1. Optimum conditions are described for a simple, rapid microplate assay that measures glutathione s-transferase (GST) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 10 assay replicates were possible for individual adults and larvae. Concentration of GST activity in the head/thorax region allows blood-fed mosquitoes with abdomens removed to be used in assays. 3. The method allows the use of GST activity as a biochemical character in comparative studies of populations. 4. The microplate assay detects elevated GST activities associated with DDT resistance in Anopheles arabiensis.     

Developmental studies on Drosophila melanogaster glutathione s-transferase and its induction by oxadiazolone

Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene was detected in various developmental stages of Drosophila melanogaster. The specific activity of the enzyme was 110, 35, 25 and 15 nmol/min/mg protein in crude extracts prepared from eggs, larvae, pupae and adult stages respectively. The enzymes from larval, pupal and adult stages were purified and compared. Incorporation of the widely used herbicide oxadiazolone at concentrations of 375 and 563 part/million into the culture media caused 4- and 2.5-fold increase in the enzyme activity in pupal and adult stages respectively.     

Acrolein-detoxifying isozymes of glutathione transferase in plants

Main conclusion

Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K _M isozyme from spinach.

Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K _M value for acrolein 93 μM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.

     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O₂ and greatest at 0.06%, the lowest O₂ concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O₂ level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-¹⁴C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [¹⁴C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.     

Forced evolution of a herbicide detoxifying glutathione transferase

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

单宁酸对杨小舟蛾谷胱甘肽S-转移酶活性的诱导

利用分光光度酶动力学的方法,确定杨小舟蛾Micromelalopha troglodyte(Graeser)谷胱甘肽S-转移酶(GSTs)的最适反应条件,并进一步研究单宁酸对杨小舟蛾GSTs活性的诱导。结果表明:杨小舟蛾GSTs测定的最适反应pH为6.5,最适反应温度为25℃。杨小舟蛾GSTs的米氏常数(KmCDNB和KmGSH)为2.63±2.32和0.61±0.10mmol/L,最大反应速度(VmaxCDNB和Vmax GSH)分别为556.26±380.02和234.12±12.84nmol/(min.mg)。单宁酸对杨小舟蛾GSTs诱导具有明显的剂量效应和时间效应关系。有效成分为0.01,0.05,0.10,0.50和1.00mg/mL的单宁酸作用48h后,杨小舟蛾体内GSTs活性分别增加了1.13,0.89,0.94,0.86和0.85倍;同样有效成分的单宁酸作用72h后,杨小舟蛾GSTs活性分别增加了1.49,1.06,1.55,1.63和0.93倍;而作用96h后,GSTs活性则分别增加了2.04,1.61,1.12,1.56和2.03倍。     

Induction of anionic glutathione transferases in rat liver by propylthiouracil

The treatment of rats with propylthiouracil (PTU) resulted in an induction of not only cationic but also anionic glutathione (GSH) transferases. DE-52 column chromatography of the anionic GSH transferases divided into five main peaks (I-V). Peaks I-IV had a high activity toward 1-chloro-2,4-dinitrobenzene (CDNB). Peak V, which is a new form of the enzyme, showed high activity to ethacrynic acid. PTU induced peaks I and V, whereas phenobarbital increased the activity of only peak I.     

Induction of carboxylesterase isozymes in Bombyx mori by E. coli infection.

Many proteins, including antibacterial peptides in the hemolymph, are induced by bacterial infections. We found two bacterially inducible carboxylesterases (CEs) in the hemolymph of the silkworm, Bombyx mori. CEs Est-1 and 2 were induced by lipopolysaccharide injection after 6 hours as well as E. coli infection. We found that bacterially inducible CEs clearly differed from noninducible CEs, including juvenile hormone esterases, in pI values, migration on analytical native PAGE, and inhibitor sensitivity. We are now studying the features and functions of these CEs.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with ⁷⁵Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Induction and suppression of glutathione transferases by interferon in the mouse

The administration of interferon-alpha/beta to female nude (nu/nu) mice caused significant changes in the levels of the cytosolic hepatic glutathione transferases. Antibodies raised against rat subunits, Ya, Yc, Yb1, Yb2, and Yk, and the subunits of the human transferases, mu (YbYb), lambda (YfYf), and epsilon (B1B1) all reacted with enzymes in the mouse and were used to demonstrate suppression and induction of transferase levels. Western blot analysis followed by semiquantitation by laser scanning showed the Ya, Yb1, Yb2, Yc, Yk, mu, and B1 subunits to be suppressed by 11, 11, 44, 30, 12, 14, and 47%, respectively, by interferon treatment. In contrast to these findings, the Yf subunit was induced 5-7-fold. A concomitant 220% increase was observed in the specific activity of the hepatic cytosol for ethacrynic acid, a substrate for the Yf subunit. Changes in the levels of transferase enzymes in normal and tumor cells may have significant implications when cytotoxic drugs are used in combination with interferons in cancer therapy. The Yf subunit, an enzyme found in human tumors and in placenta (Polidoro, G., Di Mio, C., Del Boccio, G., Zulli, P., and Fererici, G. (1980) Biochem. Pharmacol. 29, 1677-1680) has also been shown to be elevated in hepatic preneoplastic lesions (Kitahara, A., Satoh, K., Nishimura, K., Ishikawa, T., Ruike, K., Sato, K., Tsuda, H., and Ito, N. (1984) Cancer Res. 44, 2698-2703). These data indicate that the Yf subunit represents a potentially important interferon-inducible gene product.     

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.     

Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.