Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B₁ (AFB₁), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB₁-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB₁, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB₁ detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8 kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.     

Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster

Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B₁ (AFB₁) and GST dysfunction is a known risk factor for susceptibility towards AFB₁. Turkeys are one of the most susceptible animals known to AFB₁, which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB₁-8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB₁ susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway.     

Biochemical factors underlying the age-related sensitivity of turkeys to aflatoxin B(1)

Poultry are some of the most sensitive species to the toxic effects of aflatoxin B(1) (AFB(1)), and younger poultry are more sensitive to this mycotoxin. To elucidate the mechanisms for this age-related susceptibility, various enzyme activities relevant to AFB(1) were measured in liver microsomes prepared from male turkeys 9, 41 and 65 days of age. Hepatic microsomal o-dealkylation of methoxy- and pentoxyresorufin significantly increased, while that of ethoxyresorufin decreased with age. Microsomal AFB(1) activation to the reactive AFB(1)-8,9-epoxide (AFBO) was most efficient in the youngest birds, with apparent K(m) and V(max) values of 168 and 19, 110 and 6, and 116 microM and 10 nmol/mg/min for 9, 41 and 65-day-old birds, respectively. The activity of hepatic cytosolic glutathione S-transferases (GSTs) was deficient in the youngest age group, but were higher in the older groups. There was also an age-related increase in the expression of GST isoforms Yc, Yc(2), as well as AFB(1)-aldehyde reductase (AFAR). However, livers from all ages lacked specific GST-mediated conjugation of AFBO, indicating that turkeys are deficient in this key AFB(1)-detoxification pathway. Our data indicate that efficient activation may underlie the extreme sensitivity of young turkeys to the toxic effects of AFB(1).     

Proteomic and Immunochemical Characterization of Glutathione Transferase as a New Allergen of the Nematode Ascaris lumbricoides

Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules.Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.     

Cytotoxicity of aflatoxin B1 and its chemically synthesised epoxide derivative on the A549 human epithelioid lung cell line

Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin found in feeds and in airborne grain dusts. Aflatoxin B₁ requires biotransformation to the AFB₁-8,9 epoxide (AFBO) by a bioactivation system and subsequent covalent binding to DNA or proteins, to exert its carcinogenic potential. The lung contains cytochrome P₄₅₀, prostaglandin-H-synthase, lipoxygenase, epoxide hydrolase and other bioactivation enzymes, and is thus a potential target for the effects of AFB₁ via the routes of inhalation and ingestion. The A549 human epithelioid lung cell line and the methylthiazol tetrazolium (MTT) bioassay were used to investigate the cytotoxicity of AFB₁ and its chemically synthesised epoxide (AFBO) in vitro. Statistical analysis of the MTT results indicated that there were overall significant differences between the control and both the AFB₁-treated (p < 0.0001) and AFBO-treated cells (p = 0.00 2). However, there was no significant difference between AFB₁ and AFBO-treated cells, when the entire range of concentrations were assessed against each other (p = 0.2877). Whenanalysed at each concentration, only at 0.01 mM was there a significant difference between the effects of AFB₁ and AFBO (p = 0.0358). The results of this investigation show that AFB₁ and AFBO are both cytotoxic in the A549 cell line.This revised version was published online in October 2005 with corrections to the Cover Date.     

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate

Summary. Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.     

Characterization of a novel alpha-class anionic glutathione S-transferase isozyme from human liver

A novel, alpha-class glutathione S-transferase (GST) isozyme has been isolated from human liver using glutathione (GSH) affinity chromatography, DEAE-cellulose ion-exchange chromatography, and immunoaffinity chromatography. The isozyme is a dimer of approximately 25,000 Mr with blocked N termini. Structural, kinetic, and immunological properties of this enzyme indicate that it belongs to the alpha class of GSTs. Noticeable differences between the properties of this enzyme and the other alpha-class GSTs of human liver are its anionic nature (pI 5.0), GSH peroxidase activity toward hydrogen peroxide, and relatively higher GSH conjugating activities toward CDNB and epoxide substrates as compared to other alpha-class GSTs. Results of these studies indicate that anionic GST omega characterized previously (Y. C. Awasthi, D. D. Dao, and R. P. Saneto, 1980, Biochem. J. 191, 1-10) from human liver is a mixture of GST pi and a novel alpha-class GST. We have, therefore, reassigned the name GST omega to this new alpha-class anionic GST of human liver.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B₁ (AFB₁)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB₁-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB₁ totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB₁. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB₁-induced DNA damage in the Hepa-1 cell by direct trapping of AFB₁. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB₁ metabolites.     

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.     

Dual localization of glutathione S-transferase in the cytosol and mitochondria: implications in oxidative stress, toxicity and disease

Glutathione (GSH) conjugating enzymes, glutathione S-transferases (GSTs), are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus and plasma membrane. The regulation and function of GSTs have implications in cell growth, oxidative stress as well as disease progression and prevention. Of the several mitochondria localized forms, GSTK (GST kappa) is mitochondria-specific since it contains N-terminal canonical and cleavable mitochondria targeting signals. Other forms like GST alpha, mu and pi purified from mitochondria are similar to the cytosolic molecular forms or 'echoproteins'. Altered GST expression has been implicated in hepatic, cardiac and neurological diseases. Mitochondria-specific GSTK has also been implicated in obesity, diabetes and related metabolic disorders. Studies have shown that silencing the GSTA4 (GST alpha) gene resulted in mitochondrial dysfunction, as was also seen in GSTA4 null mice, which could contribute to insulin resistance in type 2 diabetes. This review highlights the significance of the mitochondrial GST pool, particularly the mechanism and significance of dual targeting of GSTA4-4 under in vitro and in vivo conditions. GSTA4-4 is targeted in the mitochondria by activation of the internal cryptic signal present at the C-terminus of the protein by protein-kinase-dependent phosphorylation and cytosolic heat shock protein (Hsp70) chaperone. Mitochondrial GST pi, on the other hand, has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N-terminal region. Both physiological and pathophysiological implications of GST translocation to mitochondria are discussed in the review.     

Glutathione S-transferases of human lung: characterization and evaluation of the protective role of the alpha-class isozymes against lipid peroxidation.

Glutathione S-transferase (GST) isozymes of human lung have been purified, characterized, quantitated, and, based on their structural and immunological profiles, identified with their respective classes. The tau-, mu-, and alpha-class GSTs represented 94, 3, and 3% activities of total human lung GSTs toward CDNB, respectively, and 60, 10, and 30% of total GST protein, respectively. Both the mu- and the alpha-class GSTs of human lung exhibited heterogeneity. The two mu-class GSTs of human lung had pI values of 6.5 and 6.25 and were differentially expressed in humans. Significant differences were seen between the kinetic properties of these two isozymes and also between the lung and liver mu-class GSTs. The alpha-class GST isozymes of lung resolved into three peaks during isoelectric focusing corresponding to pI values of 9.2, 8.95, and 8.8. All three alpha-class GSTs isozymes had blocked N-termini and were immunologically similar to human liver alpha-class GSTs. Peptide fingerprints generated by SV-8 protease digestion and CNBr cleavage indicated minor structural differences between the liver and the lung alpha-class GSTs. The three alpha-class GSTs of lung expressed glutathione peroxidase activities toward the hydroperoxides of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with Km values in the range of 22 to 87 microM and Vmax values in the range of 67-120 mol/mol/min, indicating the involvement of the alpha-class GSTs in the protection mechanisms against peroxidation. All three classes of lung GSTs expressed activities toward leukotriene A4 methyl ester and epoxy stearic acid but the mu-class GSTs had relatively higher activities toward these substrates.     

Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.     

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.     

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.     

Effects of aflatoxin B1 on the liver and kidney of broiler chickens during development

Aflatoxin B₁ (AFB₁) negatively affects chicken (Gallus domesticus) growth. This effect is more severe during development. We studied the influence of age on the toxic effects of AFB₁ on plasma, renal and hepatic enzymes, under two protocols, in adult and in developing Arbor-Acres chickens. Protocol A: 100 male 4-week-old chickens (640 g), received AFB₁, 0.5, 1.0, or 2.0 μg/g of feed (daily p.o.), a fourth group received an aflatoxin-free diet. Five birds/group were slaughtered at 7, 14, 21 and 28 days of treatment. Body, hepatic and renal weights, succinate-dehydrogenase (SDH) and glutamate-dehydrogenase (GluDH) in plasma and liver were measured. Hepatic SDH and GluDH decreased (P<0.05). Protocol B: two groups of 24 male 1-week-old chickens (106 g) received either aflatoxin-free feed (n=24) or AFB₁ feed (2.0 μg/g). At days 7, 14, 21 and 28, the same parameters of Protocol A were measured. AFB₁ markedly reduced body weight gain (20–30%), plasma proteins, albumin, renal and hepatic protein content (P<0.05) and increased absolute and relative weights of the kidney (P<0.05). SDH and GluDH were reduced (P<0.05), while total renal γ-glutamyltranspeptidase (GGT) increased (P<0.05). Results suggest that serum proteins, SDH and GluDH are sensitive early indicators of this toxicity that was more severe in developing chickens. Decrease in serum albumin might be used as an early and suitable indicator of the deleterious effect of this mycotoxin in developing chickens.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

Binding and protection of porphyrins by glutathione S-transferases of Zea mays L.

Glutathione S-transferases (GSTs) are multi-functional enzymes, known to conjugate xenobiotics and degrade peroxides. Herein, we report on the potential of four Zea mays GST isoforms (Zm GST I–I, Zm GST I–II, Zm GST II–II and Zm GST III–III) to act as binding and protection proteins. These isoforms bind protoporphyrin IX (PPIX), mesoporphyrin, coproporphyrin, uroporphyrin and Mg-protoporpyhrin, but do not form a glutathione conjugate. The binding is non-covalent and inhibits GSTs enzymatic activity, dependent on the type of the porphyrin and GST isoform tested. I₅₀ values are in the range of 1 to 10 μM for PPIX, the inhibition by mesoporphyrin and Mg-protoporphyrin (Mg-PPIX) is two to five times less. The mode of binding is non-competitive for the hydrophobic substrate and competitive for glutathione. Binding affinities (K_D values) of the GST isoforms are between 0.3 and 0.8 μM for coproporphyrin and about 2 μM for mesoporphyrin.Zm GST III–III prevents the nonenzymatic autoxidation of protoporphyrinogen to the phytotoxic PPIX. Zm GST II–II can reduce the oxidative degradation of hemin. This points to a specific ligand role of distinct GST isoforms to protect tetrapyrroles in the plant cell.