Cytosolic Glutathione S-transferase Research Articles

Promotion of oxidative stress in the aquatic macrophyte Ceratophyllum demersum during biotransformation of the cyanobacterial toxin microcystin-LR

Cyanobacterial toxins have been shown to have adverse effects on mammals, birds and fish and are therefore being increasingly recognised as a potent stress and health hazard factor in aquatic ecosystems. Microcystins, which are cyclic heptapeptides and a main group of the cyanotoxins, are mainly retained within the producer-cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. The released toxins could then come into contact with a wide range of aquatic organisms including invertebrates, fish and aquatic plants. In many organisms, biotransformation of the toxins will take place via several glutathione-related conjugate. During the biotransformation process in which the toxin and the toxin conjugate are broken down, the formation of reactive oxygen species might occur. These reactive oxygen species activate several antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and also influence the glutathione-ascorbate cycle. Aim of this study was to investigate formation of the glutathione-conjugate, activation of glutathione S-transferases and the elevation of several antioxidant enzymes giving evidence for the promotion of oxidative stress by microcystins. During exposure of Ceratophyllum demersum to the cyanobacterial toxin microcystin-LR in an concentration of 5.0 μg/L, an elevation of microsomal and cytosolic glutathione S-transferase was measured, showing the beginning formation of the glutathione-toxin conjugate. The superoxide dismutase as well as in parallel the hydrogen peroxide level increased giving evidence for oxidative stress in the aquatic plant. Other reactive oxygen detoxifiying enzymes were also elevated and the glutathione pool, expressed in reduced glutathione and glutathione disulfide concentration was changed accordingly.

In Vitro Metabolism of Nitric Oxide-Donating Aspirin: The Effect of Positional Isomerism

NO-donating aspirin (NO-ASA) is a potentially important chemopreventive agent against cancer. Since positional isomerism affects strongly its potency in inhibiting colon cancer cell growth, we studied the metabolic transformations of its ortho-, meta-, and para-isomers in rat liver and colon cytosolic, microsomal, and mitochondrial fractions as well as in intact HT-29 human colon cancer cells. NO-ASA and metabolites were determined by high-performance liquid chromatography and products identified by mass spectroscopy, as required. For all three isomers, the acetyl group on the ASA moiety was hydrolyzed rapidly. This was followed by hydrolysis of the ester bond linking the salicylate anion to the spacer. The ortho- and para-isomers produced salicylic acid and a putative intermediate consisting of the remainder of the molecule, which via a rapid step generated nitrate, (hydroxymethyl)phenol, and a conjugate of spacer with glutathione. The meta-isomer, in contrast, generated salicylic acid and (nitroxymethyl)phenol, the latter leading to (hydroxymethyl)phenol and the glutathione-spacer conjugate. This metabolic pathway takes place in its entirety only in the cytosolic fraction of the tissues tested and in intact human colon cancer cells, perhaps reflecting exposure to the cytosolic glutathione S-transferase, which catalyzes the formation of the spacer-glutathione conjugate. Thus, the three positional isomers of NO-ASA differ in their metabolism and these differences correlate with their differential effects on cancer cell growth, underscoring the importance of positional isomerism in modulating drug effects.

Uptake and Effects on Detoxication Enzymes of Cypermethrin in Embryos and Tadpoles of Amphibians

A number of factors have been suggested for recently observed amphibian decreases, and one potential factor is pesticide exposure. We studied the uptake and effects of environmentally relevant concentrations of the pyrethroid insecticide cypermethrin on two different amphibian species, Bombina variegata and Rana arvalis. The uptake from water of 14C-labeled cypermethrin (0.4 microg/L) by eggs and tadpoles of B. variegata was investigated. After 24 hours of exposure, 153.9 ng cypermethrin/g fresh weight were found in embryos, thus indicating that the jelly mass of the eggs does not act as a sufficient physical barrier to protect embryos from exposure to this compound. Uptake of cypermethrin into tadpoles of both species and in all exposed individuals caused dose-dependent deformities; behavioral abnormalities such as twisting, writhing, and coordinated swimming; and mortality. In tadpoles of B. variegata and R. arvalis, the activity of microsomal and cytosolic glutathione S-transferase (mGST and sGST, respectively) were measured after treatment with cypermethrin. Activities of both GST systems increased significantly with increasing duration and concentration of cypermethrin exposure, with the reaction seeming stronger in B. variegata than in R. arvalis tadpoles. Alpha-cypermethrin--a racemic mixture of two cis isomers of cypermethrin--induced a stronger enzymatic response in the cytosolic fraction of R. arvalis tadpoles than cypermethrin at the same concentration. The observed physical and behavioral abnormities caused by environmentally relevant concentrations of cypermethrin indicate that despite detoxication of the chemical via GST-system contamination of ponds by cypermethrin could result in adverse effects on the development of amphibian embryos and tadpoles.

Characterization of the ligandin site of maize glutathione S-transferase I.

Cytosolic GSTs (glutathione S-transferases) are a major reserve of high-capacity binding proteins and exhibit ligand-binding properties for a large variety of compounds. In the present study, the binding of two non-substrate anthraquinone dyes VBAR (Vilmafix Blue A-R) and CB3GA (Cibacron Blue 3GA) to maize (Zea mays) GST I was investigated. The results showed that the enzyme was specifically and irreversible inactivated by VBAR with a K(d) of 35.5+/-2.2 microM and a k(3) of 0.47 min(-1). Proteolytic cleavage of the VBAR-modified enzyme and subsequent separation of peptides gave only one modified peptide. Sequencing of the modified peptide revealed the target site of VBAR reaction to be Lys(41). CB3GA binds reversibly to GST I and behaves as a competitive inhibitor towards CDNB (1-chloro-2,4-dinitrobenzene) and glutathione. CB3GA binding to GST I is accompanied by a characteristic spectral change in the absorption at positive maximum (670 nm) which exhibited a hyperbolic dependence on dye concentration with a K(d) of 12.1+/-0.5 microM. Site-directed mutagenesis of selected residues (Trp(12), Phe(35), Lys(41), Asn(49), Gln(53), Ser(67) and Ile(118)) was employed, and the mutated enzymes were assessed for CB3GA binding. These results, together with molecular-modelling studies, established that the ligandin-binding site of GST I is located mainly in the hydrophobic binding site. The ability of VBAR to specifically inactivate GST I was exploited further to demonstrate the specific binding of several plant hormones and flavonoids to GST I. The inactivation of other GST isoenzymes by VBAR was also investigated, and it was concluded that VBAR may have wide applicability as an affinity label for probing structure-function relationships of GST isoenzymes.

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[ p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (γ-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1 mM CHB with 5 mM GSH at 37 °C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[ p-[ N-2-chloroethyl, N-2- S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[ p-[bis[2- S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG 2) with small amounts of the hydroxy-derivative, 4-[ p-[ N-2- S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[ p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH 2), 4-[ p-[ N-2-chloroethyl, N-2- S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG 2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.

Short-term black tea intake modulates the excretion of urinary mutagens in rats treated with 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ): role of CYP1A2 upregulation.

Rats were exposed to black tea (2.5% w/v) as their sole drinking liquid for either 1 day or 1 month, while controls were maintained on water. After this treatment period, all animals received a single oral dose IQ (2-amino-3-methylimidazo-[4,5-f]quinoline), and urine was collected for 48 h. Mutagenic activity of the urine was determined in the Ames test in the presence and absence of an activation system. The excretion of direct-acting mutagens was markedly reduced following tea intake, and was more pronounced after the 1-day treatment. Similarly, both tea treatments suppressed the excretion of indirect-acting mutagens. Furthermore, both tea treatments induced hepatic CYP1A2 activity and expression, but cytosolic glutathione S-transferase activity was only modestly induced in the group of animals receiving tea for 1 day, and only when DCNB (1,2-dichloro-4-nitrobenzene) was used as substrate; glucuronosyl activity was elevated modestly only in the animals receiving the tea for a month. It is concluded that even short-term exposure to black tea is capable of influencing the metabolic fate of IQ, and this is most likely related to the upregulation of CYP1A2.

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2).

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.

Selective suppression of cytochrome P450 gene expression by the medicinal herb, Thonningia sanguinea in rat liver

The effect of the administration of Thonningia sanguinea (T. S.) on the abundance of individual components of the cytochrome P450 monooxygenase enzyme was examined using Western blotting and competitive reverse-transcriptase-polymerase chain reaction (RT-PCR). We also investigated the time-course of inhibition of T. S. on drug metabolizing enzymes. A single intraperitoneal dose of T. S. extract (5 ml/kg) suppressed CYP, cytochrome b 5 and NADPH-CYP reductase activity by 45%, 34% and 22% respectively 24 h after T. S. adminstration. While T. S. did not have any significant effect on microsomal glutathione S-transferase activity, it inhibited p-nitrophenol hydroxylase (PNPH, CYP2E1) and 7-methoxyresorufin O-demethylase (MROD, CYP 1A2) activities by 37% and 32% respectively at 12 h post-T. S. administration. PNPH, erythromycin N-demethylase (ERDM, CYP 3A1/2) and MROD activities were inhibited by 28–36% 24 h after T. S. injection. Consistent with these observations, the levels of CYP2E1, CYP1A2 and CYP3A2 proteins were also suppressed 24 h post-T. S. administration. While CYP2E1 mRNA was unaffected by T. S. administration, CYP1A2 and CYP3A2 mRNAs were decreased by T. S. Cytosolic glutathione S-transferase activity was increased by 30%, 6 h after T. S injection. These data demonstrate that administration of T. S. differentially affect CYP isoforms in the liver of rats and that T. S. selectively suppresses CYP3A2 and CYP1A2 gene expression.

Modulation of hepatic drug metabolizing enzymes and oxidative status by rooibos (Aspalathus linearis) and Honeybush (Cyclopia intermedia), green and black (Camellia sinensis) teas in rats.

Rooibos and honeybush teas significantly (P < 0.05) enhanced the activity of cytosolic glutathione S-transferase alpha. A significant (P < 0.05) to marginal (P < 0.1) increase in the activity of the microsomal UDP-glucuronosyl transferase was obtained with unprocessed rooibos and honeybush teas, respectively. Oxidized glutathione (GSSG) levels were significantly (P < 0.05) reduced in the liver of all tea treated rats while reduced glutathione (GSH) was markedly increased in the liver of the herbal tea treated rats. These changes resulted in a significant (P < 0.05) increase in the GSH/GSSG ratio by the unprocessed, processed rooibos and unprocessed honeybush teas. Green and black teas markedly to significantly decreased the oxygen radical absorbance capacity in liver homogenates, respectively. Modulation of phase II drug metabolizing enzymes and oxidative status in the liver may be important events in the protection against adverse effects related to mutagenesis and oxidative damage.

The role of glutathione-S-transferase in anti-cancer drug resistance.

Glutathione-S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs are divided into two distinct super-family members: the membrane-bound microsomal and cytosolic family members. Microsomal GSTs are structurally distinct from the cytosolic in that they homo- and heterotrimerize rather than dimerize to form a single active site. Microsomal GSTs play a key role in the endogenous metabolism of leukotrienes and prostaglandins. Human cytosolic GSTs are highly polymorphic and can be divided into six classes: alpha, mu, omega, pi, theta, and zeta. The pi and mu classes of GSTs play a regulatory role in the mitogen-activated protein (MAP) kinase pathway that participates in cellular survival and death signals via protein : protein interactions with c-Jun N-terminal kinase 1 (JNK1) and ASK1 (apoptosis signal-regulating kinase). JNK and ASK1 are activated in response to cellular stress. GSTs have been implicated in the development of resistance toward chemotherapy agents. It is plausible that GSTs serve two distinct roles in the development of drug resistance via direct detoxification as well as acting as an inhibitor of the MAP kinase pathway. The link between GSTs and the MAP kinase pathway provides a rationale as to why in many cases the drugs used to select for resistance are neither subject to conjugation with GSH, nor substrates for GSTs. GSTs have emerged as a promising therapeutic target because specific isozymes are overexpressed in a wide variety of tumors and may play a role in the etiology of other diseases, including neurodegenerative diseases, multiple sclerosis, and asthma. Some of the therapeutic strategies so far employed are described in this review.

Feminization of wild carp, Cyprinus carpio, in a polluted environment: plasma steroid hormones, gonadal morphology and xenobiotic metabolizing system

Wild carp, Cyprinus carpio, were sampled in January and March 2000 in a section of the Anoia River (NE Spain) known to be polluted by estrogenic compounds. At each sampling time, three groups were distinguished: (1) apparently normal males; (2) apparently normal females; and (3) affected fish. The latter were characterized by the simultaneous development of male and female tissue in their gonads at a macroscopical level (six out of 31 fish sampled at this particular point), or testicular atrophy (three out of 31). Plasmatic and hepatic vitellogenin (VTG) levels and plasma testosterone (T) and estradiol (E2) were measured to observe the particular estrogenic response of the affected fish. Moreover, the response in the xenobiotic metabolizing capacity in liver was tested. This involved the analysis of mixed function oxygenase (MFO) system such as: total cytochrome P450 content, NAD(P)H cytochrome c reductases and the associated CYP1A1, EROD activity. Also, glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) as detoxifying enzymes were measured. Our results showed: (1) a highly variable VTG content in all fish groups; (2) an increase in sex hormones content in March for the female group; and (3) an enhanced xenobiotics metabolism in the affected fish group, measured as total cytochrome P450, EROD activity in the January survey and cytosolic GST in March. The observed increase in VTG, sex hormones and in most of the enzymatic activities from January to March that could also be attributed to higher water temperature.

Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis.

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.

Glutathione Transferase Superfamily Behaves Like Storage Proteins for Dinitrosyl-Diglutathionyl-Iron Complex in Heterogeneous Systems

Electron paramagnetic resonance and kinetics experiments have been made to determine the formation, stability, and fate of the natural nitric oxide carrier, dinitrosyl-diglutathionyl-iron complex (DNDGIC), in heterogeneous systems approaching in vivo conditions. Both in human placenta and rat liver homogenates DNDGIC is formed spontaneously from GSH, S-nitroso-glutathione, and trace amounts of ferrous ions. DNDGIC is unstable in homogenates depleted of glutathione S-transferase (GST); an initial phase of rapid decomposition is followed by a slower decay, which is inversely proportional to the concentration. In the crude human placenta homogenate, GSTP1-1, which represents 90% of the cytosolic GST isoenzymes, is the preferential target for DNDGIC. It binds the complex almost stoichiometrically and stabilizes it for several hours (t1/2 = 8 h). In the presence of an excess of DNDGIC, negative cooperativity in GSTP1-1 opposes the complete loss of the usual detoxicating activity of this enzyme. In the rat liver homogenate, multiple endogenous GSTs (mainly Alpha and Mu class isoenzymes) bind the complex quantitatively and stabilize it (t1/2 = 4.5 h); negative cooperativity is also seen for these GSTs. Thus, the entire pool of cytosolic GSTs, with the exception of the Theta GST, represents a target for stoichiometric amounts of DNDGIC and may act as storage proteins for nitric oxide. These results confirm the existence of a cross-link between NO metabolism and the GST superfamily.

Effect of Silica Exposure on Urinary Excretion of Copper and Zinc

Occupational exposure to silica could lead to renal histological alterations in the glomeruli and proximal tubules. We investigated urinary excretion of copper and zinc and its possible relationship to renal alterations and work duration among nonsmoking, silica-exposed workers. Thirty-six control subjects (age, 39.08 +/- 6.65 years) and 48 silica-exposed workers (age, 36.79 +/- 7.33 years) were included in the present study. The exposed workers were grouped into workers with <10 years' work duration (n = 9; age, 29.0 +/- 1.50 years) and workers with > or =10 years' work duration (n = 39; age, 38.59 +/- 6.94 years). Glomerular function was assessed by measuring urinary level of microalbumin, and proximal tubular reabsorption function was assessed by measuring urinary level of alpha1-microglobulin. Structural integrity of proximal tubules was studied by measuring urinary activity of cytosolic enzyme glutathione S-transferase (GST). Also, urinary levels of copper, zinc, and creatinine were measured. All measured urinary parameters were significantly increased among silica-exposed workers compared with control subjects. A significant correlation was observed between urinary levels of each of copper and zinc with the glomerular and proximal tubular functional and structural urinary parameters as well as work duration. Occupational silica exposure may lead to increased urinary loss of copper and zinc as protein-metal complex, and the urinary level of these essential trace elements may be a marker for renal dysfunction. The urinary excretion of GST may be a useful marker for proximal tubular injury.

Novel class of bivalent glutathione S-transferase inhibitors.

Exploiting the principle of bivalent binding, we have designed symmetrical, bifunctional inhibitors to simultaneously occupy both active sites of cytosolic glutathione S-transferase, with enhanced specificity for the P1-1 isoform. We have prepared two series of compounds that differ in their binding domains-the first is a series of bis-glutathione conjugates, and the second is a series of compounds each possessing two equivalents of Uniblue A, an analogue of Cibacron Blue. For each series, a monofunctional reference compound was also prepared to determine the relative advantage of the bivalent inhibitors. Within each series, the most potent inhibitors exhibited IC(50) values 2 orders of magnitude lower than the relevant reference compounds. Moreover, within the bis-glutathionyl series, a 10-fold increase in selectivity was achieved for GST P1-1 over the A1-1 isoform. Isothermal titration calorimetry with a representative bis-glutathione conjugate and a monofunctional reference compound indicates that the bivalent inhibitor exhibits the expected increase in intrinsic affinity and decrease in stoichiometry relative to the monofunctional compound, supporting the overall design strategy.

The Anopheles gambiae glutathione transferase supergene family: annotation, phylogeny and expression profiles.

BackgroundTwenty-eight genes putatively encoding cytosolic glutathione transferases have been identified in the Anopheles gambiae genome. We manually annotated these genes and then confirmed the annotation by sequencing of A. gambiae cDNAs. Phylogenetic analysis with the 37 putative GST genes from Drosophila and representative GSTs from other taxa was undertaken to develop a nomenclature for insect GSTs. The epsilon class of insect GSTs has previously been implicated in conferring insecticide resistance in several insect species. We compared the expression level of all members of this GST class in two strains of A. gambiae to determine whether epsilon GST expression is correlated with insecticide resistance status.ResultsTwo A. gambiae GSTs are alternatively spliced resulting in a maximum number of 32 transcripts encoding cytosolic GSTs. We detected cDNAs for 31 of these in adult mosquitoes. There are at least six different classes of GSTs in insects but 20 of the A. gambiae GSTs belong to the two insect specific classes, delta and epsilon. Members of these two GST classes are clustered on chromosome arms 2L and 3R respectively. Two members of the GST supergene family are intronless. Amongst the remainder, there are 13 unique introns positions but within the epsilon and delta class, there is considerable conservation of intron positions. Five of the eight epsilon GSTs are overexpressed in a DDT resistant strain of A. gambiae.ConclusionsThe GST supergene family in A. gambiae is extensive and regulation of transcription of these genes is complex. Expression profiling of the epsilon class supports earlier predictions that this class is important in conferring insecticide resistance.

Hematopoietic prostaglandin D synthase

The biological actions of prostaglandin (PG) D 2 include vasodilatation, bronchoconstriction, inhibition of platelet aggregation, and recruitment of inflammatory cells. Characterization of DP receptor null mice in which antigen-induced airway and inflammatory responses are attenuated and identification of CRTH2 as a novel PGD 2 receptor have shed light on the role of PGD 2 in the immune and inflammatory responses. Hematopoietic PGD synthase (H-PGDS) is a cytosolic enzyme that isomerizes PGH 2, a common precursor for all PGs and thromboxanes, to PGD 2 in a glutathione-dependent manner. H-PGDS is expressed in mast cells, antigen-presenting cells, and Th2 cells, and is the only mammalian member of the Sigma class of cytosolic glutathione S-transferases. In this review, we focus on the molecular biology of H-PGDS, the determination of its three-dimensional structure, characterization of the regulation of its gene expression, and information gleaned from transgenic animals.

Hepatic glutathione S-transferases from lamprey ( Petromyzom marinus): purification and characterization

Glutathione S-transferases constitute a very important family of biotransformation enzymes, which catalyse the conjugation of glutathione to a broad spectrum of xenobiotics. In this study, cytosolic glutathione S-transferases enzymes were purified from lamprey ( Petromyzon marinus) liver, using an affinity chromatography method. Enzymatic activity was determined towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1,2-dichloro-4-nitrobenzene. The application of a HPLC system associated to electrospray ionization mass spectrometry allowed the identification of three different subunits, with Mrs between 22,300 and 25,300 Da. The one with the low Mrs was the main form, with a retention time of 29.5 min, a pi-related class isoenzyme.

Phosphorylation Enhances Mitochondrial Targeting of GSTA4-4 through Increased Affinity for Binding to Cytoplasmic Hsp70

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.