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PubMed2005

Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Markedly decreased expression of glutathione S-transferase pi gene in human cancer cell lines resistant to buthionine sulfoximine, an inhibitor of cellular glutathione synthesis.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits an enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), which is a critical step in glutathione biosynthesis. We isolated three BSO-resistant sublines, KB/BSO1, KB/BSO2, and KB/BSO3, from human epidermoid cancer KB cells. These cell lines showed 10-to 13-fold higher resistance to BSO, respectively, and had collateral sensitivity to cisplatin, ethacrynic acid, and alkylating agents such as melphalan and nitrosourea. Cellular levels of glutathione S-transferase pi (GST-pi) and its mRNA in BSO-resistant cell lines were less than 10% of the parental cells. Nuclear run-on assay showed that the transcriptional activity of GST-pi was decreased in BSO-resistant cells, and transient transfection of GST-pi promoter-chloramphenicol acetyltransferase constructs revealed that the sequences between -130 and -80 base pairs of the 5'-flanking region wer at least partially responsible for the decreased expression of the GST-pi gene. By contrast, gamma-GCS mRNA levels were 3-to 5-fold higher in resistant cell lines than in KB cells, and the gamma-GCS gene was found to be amplified in the BSO-resistant cells lines. GST-pi mRNA levels appeared to be inversely correlated with gamma-GCS mRNA levels in BSO-resistant cells. We further established the transfectants, KB/BSO3-pi1 and KB/ BSO2-pi2, that overexpressed GST-pi, from KB/BSO3, after introducing a GST-pi expression plasmid. These two transfectants had similar levels in gamma-GCS mRNA, drug sensitivity to alkylating agents, and glutathione content at those of KB cells. These findings suggest that the cellular levels of GST-pi and gamma-GCS might be co-regulated in these novel BSO-resistant cells.

Blotting, Southern↗
PubMed1995

HCl-induced cell edema in rabbit esophageal epithelium: a bumetanide-sensitive process.

BACKGROUND & AIMS: The morphology of acid damage to esophageal epithelium is characterized by marked cell (swelling) edema. This observation suggests that, in the process of acid damage, an increase in osmotic forces develops within the cell that accounts for the increase in cell water. The aim of this study was to document that esophageal cells swell at acidic pH and to explore the nature of the osmolytes and mechanisms responsible for it. METHODS: Cell edema was assessed in sections of rabbit esophageal epithelium by correlating morphological change with change in tissue wet weight after immersion in acidic solutions for up to 4 hours. RESULTS: At pH < or = 2 for 2 hours, tissues gained weight and showed cell edema on both light and electron microscopy. In addition to being time- and pH-dependent, cell edema was dependent on bathing solution osmolytes, specifically Na+, K+, and Cl-, and could be inhibited by tissue pretreatment with bumetanide (or ethacrynic acid). CONCLUSIONS: HCl exposure can spontaneously produce cell edema in esophageal epithelium. The phenomenon is pH- and time-dependent and requires acid stimulation of osmolyte absorption through a bumetanide-sensitive process compatible with an NaK2Cl cotransporter in the epithelial cell membrane.

Animals↗
PubMed1994

Inhibition of 11 beta-hydroxysteroid dehydrogenase obtained from guinea pig kidney by furosemide, naringenin and some other compounds.

Inhibition of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) can cause excess mineralocorticoid effects and hypokalemia. Several substances causing hypokalemia (glycyrrhizic acid in licorice and gossypol) inhibit this enzyme. We tested other compounds for activity to inhibit 11 beta-OHSD in guinea pig kidney cortex microsomes with NADP as cofactor and cortisol as substrate. Furosemide was an inhibitor while bumetanide was not, indicating a mechanism for the increase K+ excretion caused by furosemide compared with bumetanide. Naringenin (found in grapefruit juice), ethacrynic acid, and chenodeoxycholic acid had inhibitor IC50 values similar to glycyrrhizic acid. We conclude that various compounds can inhibit this enzyme and may play a role in K+ metabolism and adrenocorticosteroid action.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105.

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

Binding Sites↗
PubMed1996

gamma-Aminobutyric acid increases intracellular Ca2+ concentration in cultured cortical neurons: role of Cl- transport.

The effect of gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca2+]i in a dose-dependent manner. The GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na+/K+/2Cl- cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl- pump, also inhibited the GABA-induced increased in [Ca2+]i. These results suggest a role for Cl- transport processes in the GABA response. The coapplication of GABA and high K+ led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca2+ channel blocker, and abolished by the absence of extracellular Ca2+. Results indicate that the GABA response shares a common pathway of Ca2+ movement with the high K(+)-induced response. These observations suggest that the stimulation with GABA results in Ca2+ influx through voltage-gated Ca2+ channels, and that these effects are dependent on Cl- transport systems.

Animals↗
PubMed2000

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra.

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the specific guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 microM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml(-1)) inhibited NO-mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy-PTIO (0.1 - 0.5 mM) inhibited NO-mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml(-1)). Relaxations to NO were reduced, but not abolished, by ODQ (10 microM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0. 5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L-cysteine (L-cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open. British Journal of Pharmacology (2000) 129, 53 - 62

Animals↗
PubMed1977

Diuretic effects of Bay g 2821 in patients with advanced renal failure.

Clearance studies were carried out in 18 non-oedematous patients with advanced renal failure (glomerular filtration rate less than 12 ml/min) to assess the diuretic effects of Bay g 2821 given different oral doses (120 mg to 360 mg). The results showed a significant increase in urinary volume and in clearances of sodium, calcium, magnesium, chloride, inorganic phosphorus and 51CrEDTA. There were no significant changes, however, in clearance of PAH, urea and uric acid. The diuretic response started in the first hour after drug intake, reached a maximum after 3 to 5 hours, and declined slowly in the following 3 to 4 hours. In patients with a glomerular filtration rate less than 3 ml/min the drug lost its effect. In rates between 5 to 10 ml/min the efficacy of Bay g 2821 was comparable to that of furosemide and ethacrynic acid. No side-effects were reported.

Diuretics↗
PubMed1992

[Neuronal intracellular chloride ion concentrations and their regulatory mechanisms].

Intracellular chloride ion concentration ([Cl-]i) plays an important role in cellular functions including the control of membrane potential and excitability. In neurons, Cl- equilibrium potential (ECl) is lower or higher than the membrane potential (Em), suggesting that [Cl-]i is lower or higher than that expected from passive distribution. As the mechanisms to control [Cl-]i, active outwardly or inwardly directed Cl- transport systems have been reported. The former includes Na(+)-dependent Cl-/HCO3- exchanger, K+/Cl- cotransporter and ATP-dependent Cl- pump; and the latter includes Na+/K+/2Cl- cotransporter and amino acid-dependent Na+/Cl- cotransporter. In hippocampal pyramidal cells, recent studies using a Cl(-)-sensitive fluorescent probe to monitor [Cl-]i revealed the presence of an ATP-dependent Cl- pump and a Na+/K+2Cl- cotransporter, and an uneven distribution of [Cl-]i (cell body less than dendrite) and these Cl- transport systems. Intracerebroventricular administration of an inhibitor of the ATP-dependent Cl- pump, ethacrynic acid, induces status epilepticus in mice. Thus, it appears to be necessary to elucidate cellular and molecular mechanisms of Cl- transporters and their control systems for a better understanding of Cl(-)-related functions in neurons.

Animals↗
PubMed2006

Inhibitors of cysteine proteases.

The roles of cysteine proteases (CP) as protein degrading and protein processing enzymes both in physiological and pathological processes of mammals are well known. Furthermore, the key roles of CP;s in the life cycles of infectious agents like protozoa and viruses turn them into new important targets for anti-infective drugs. Thus, the effective inhibition of pathologically relevant cysteine proteases has raised increasing interest in drug development. One strategy to create CP inhibitors is the use of electrophilic moieties, which covalently bind to the cysteine residue of the active site of the target protease. In a previous approach we have selected the aziridine-2,3-dicarboxylic acid as weak electrophilic inhibitor fragment. In order to achieve effective enzyme inhibition this electrophile was incorporated into peptidic or peptidomimetic sequences addressing the substrate binding sites of the protease. High selectivity could be obtained with compounds, which bind into both the primed and non-primed substrate binding pockets. In a second approach the alpha,beta-unsaturated ketone of the well-known diuretic drug ethacrynic acid was found to be another appropriate electrophilic moiety. Derivatives thereof turned out to be new non-peptidic CP inhibitors. Results of inhibition assays obtained with these two inhibitor series on various proteases of human, protozoan, and viral origin, theoretical studies to investigate binding modes and inhibition mechanisms, and structure-activity relationships are presented. Furthermore, the results of in vitro assays on respective pathogens as well as the results of first toxicity studies are summarized.

Animals↗
PubMedDate not supplied

Sites and mechanisms of action of diuretics in the kidney.

Utilization of classical clearance methodology for the determination of diuretic mechanism and site of action is based on four kinds of observations: (1) the effects of diuretic agents on the concentrating and diluting mechanism; (2) an analysis of the pattern of anionic excretion produced by the drug; (3) determination of the action of the agent on acid excretion and on acid-base status; (4) an evaluation of the effects of the diuretic on potassium excretion. Agents (such as acetazolamide) that act in the proximal convoluted tubule cause an enhancement of solute-free water generation (CH2O), induce a phosphaturia and an increase in bicarbonate excretion, alkalinize the urine, and cause a kaliuresis. Those agents which inhibit sodium chloride transport in the loop of Henle (for example, furosemide and ethacrynic acid) reduce both CH2O and the abstraction of tubular water from the collecting duct (TCH2O). They are the most potent natriuretic agents currently available, causing increments in the excretion of sodium in the urine of 15 to 25 per cent of filtered load. Those drugs which act in the early portion of the distal convolution (the thiazides, metolazone) reduce CH2O modestly or not at all and have no effect on TCH2O. They are capable of increasing urinary sodium by 5 to 8 per cent of the filtered load. The special-purpose agents such as triamterene and spironolactone are only mildly effective as natriuretic agents. While they augment fractional sodium excretion by only 2 to 3 per cent, they are useful because of their capacity to reduce urinary potassium excretion, either by a direct renal tubular effect (triamterene) or by competitive inhibition of aldosterone (spironolactone).

Bicarbonates↗
PubMed2000

The three mechanisms of intracellular chloride accumulation in vascular smooth muscle of human umbilical and placental arteries.

Recordings of membrane potential (Em) and intracellular [Cl-] ([Cl-]i) were made from the smooth muscle of human umbilical and placental arteries, using double-barrelled, ion-sensitive microelectrodes. In both arteries, [Cl-]i was above equilibrium with Em. In the umbilical artery, [Cl-]i was 33.8+/-0.9 mM (+/-SD, n=19) and Em -54.9+/-1.3 mV and in the placental artery respectively 35.1+/-0.7 mM (n=17) and -50.6+/-0.9 mV. In both arteries, [Cl-]i was reduced and Em hyperpolarised significantly by successive additions of 100 microM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), 10 microM bumetanide and 1 mM acetazolamide, thus revealing the presence of Cl-/HCO3- exchange, (Na+K+Cl) cotransport and "pump III". In the presence of all three inhibitors, [Cl-]i was in equilibrium with Em. As in earlier studies on rat arterial smooth and cardiac muscle, pump III was unaffected by DIDS, bumetanide, metolazone and the removal of Na+, partly inhibited by chlorothiazide and fully inhibited by ethacrynic acid. The results are discussed in terms of the possibility that of chloride accumulating systems may regulate vasomotor tone in the foetoplacental unit.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1975

[Phalloidin antagonists 4th communication: Thioctic acid, SH-compounds, rifampicin, choleretics, dexamethasone, estradiol, unspecific inhibitors, and ineffective compounds (author's transl)].

1. Thioctic acid used clinically in poisoning by A. phalloides, protected perfused livers and also isolated hepatocytes against phalloidin, when given in high concentrations. 2. Some SH-compounds like coenzyme A, dimercaprol, cysteine and cysteamine were found to be protective in different concentrations. 3. Rifampicin protects mice against lethal doses of phalloidin, and inhibits poisoning of isolated hepatocytes at low concentrations. 4. Some choleretic drugs like dehydrocholate, temoebilin (extr. cucumae xanth.), ethacrynic acid influenced phalloidin poisoning by inhibition of binding. 5. Doses of 0.2 to 4.0 mg dexamethasone added to 100 ml of perfusion medium did not protect perfused rat livers against 0.5 mg phalloidin. 6. Pretreatment of female rats with estrogens effected protection against phalloidin in vivo. The same procedure resulted in moderate decrease of phalloidin effects when the livers of pretreated animals were poisoned in vitro. In male rats estrogen pretreatment was less effective. Castration did not augment the protective effect. 7. Secophalloidin, a biologically inactive derivative, did not influence phalloidin poisoning in perfused livers, even when applied in excessive concentrations. 8. Concanavalin A, probably bound in the neighborhood of binding sites for phalloidin, did not protect perfused livers against phalloidin. 9 Diethyldithiocarbamate, a compound protecting livers against carbon tetrachloride and halothane, was ineffective in phalloidin poisoning. 10. Further protective actions of Evans blue, of some phenanthrolines and of EDTA are discussed. 11. Pretreatment of animals with hepatotoxic compounds (CCl4, CHCl3, cinchophen) decreased the toxicity of phalloidin in vivo. Possible mechanisms are discussed.

Animals↗
PubMed1997

Mechanism-based phage display selection of active-site mutants of human glutathione transferase A1-1 catalyzing SNAr reactions.

A library of active-site mutants has been constructed by targeting selected amino acid residues in human glutathione transferase (GST) A1-1 for random mutagenesis. The mutated residues are suitably positioned for interaction with the second, electrophilic substrate, in particular chloronitrobenzene derivatives undergoing SNAr reactions. DNA representing the GST A1-1 mutant library was fused with DNA encoding gene III protein, a component of the coat of filamentous phage. Phage display was used for affinity selection of GST A1-1 mutants with altered catalytic properties. The affinity ligand used was the sigma-complex of 1,3,5-trinitrobenzene and glutathione immobilized to Sepharose. The complex was designed to mimic the transition state of SNAr reactions catalyzed by GSTs. The selection system is based on the combination of affinity for the sigma-complex as well as the ability to promote its formation, thus mimicking two salient features of the assumed catalytic mechanism for the SNAr reactions. Many of the GST A1-1 mutants selected and analyzed contained an aromatic amino acid residue in one of the mutated positions, suggesting favorable interactions with the trinitrocyclohexadienate moiety of the affinity ligand. A mutant C36 was selected for more detailed studies. Its catalytic efficiency with several chloronitrobenzene substrates was 20-90-fold lower than that of wild-type GST A1-1, but fully comparable to naturally evolved GSTs of different classes, providing a 10(5)-fold rate enhancement over the uncatalyzed reaction. In the conjugation of ethacrynic acid, a Michael addition reaction, mutant C36 was 13-fold more efficient than the wild-type enzyme. Within experimental error, the quotient between the KF values for wild-type GST A1-1 and mutant C36 is the same as that between the kcat/KM values determined with 1-chloro-2,4-dinitrobenzene for the two enzyme forms. This result indicates that sigma-complex formation is rate-limiting for the catalyzed reaction. Thus, the principle of transition-state stabilization as a component of catalysis has been successfully exploited in affinity selection of catalytically competent GST A1-1 mutants. This mechanism-based procedure also selects for the ability to promote sigma-complex formation, and serves as a probe of the catalytic mechanism.

Bacteriophages↗
PubMed1992

Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system.

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

Amino Acid Sequence↗
PubMed2000

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage.

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.

Amino Acid Sequence↗
PubMed1996

Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues.

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247-250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with l-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of l-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

Benzene Derivatives↗