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PubMed2001

The C-terminus of glutathione S-transferase A1-1 is required for entropically-driven ligand binding.

Binding of a hydrophobic glutathione product conjugate to rGST A1-1 proceeds via a two-step mechanism, including rapid ligand docking, followed by a slow isomerization to the final [GST.ligand] complex, which involves the localization of the flexible C-terminal helix. These kinetically resolved steps have been observed previously by stopped-flow fluorescence with the wild-type rGST A1-1, which contains a native Trp-21 approximately 20 A from the ligand binding site at the intrasubunit domain-domain interface. To confirm this binding mechanism, as well as elucidate the effects of truncation of the C-terminus, we have further characterized the binding and dissociation of the glutathione-ethacrynic acid product conjugate (GS-EA) to wild-type, F222W:W21F, and Delta209-222 rGST A1-1 and wild-type hGST A1-1. Although modest kinetic differences were observed between the hGST A1-1 and rGST A1-1, stopped-flow binding studies with GS-EA verified that the two-step mechanism of ligand binding is not unique to the GST A1-1 isoform from rat. An F222W:W21F rGST A1-1 double mutant provides a direct fluorescence probe of changes in the environment of the C-terminal residue. The observation of two relaxation times during ligand binding and dissociation to F222W:W21F suggests that the C-terminus has an intermediate conformation following ligand docking, which is distinct from its conformation in the apoenzyme or localized helical state. For the wild-type, Delta209-222, and F222W:W21F proteins, variable-temperature stopped-flow experiments were performed and activation parameters calculated for the individual steps of the binding reaction. Activation parameters for the binding reaction coordinate illustrate that the C-terminus provides a significant entropic contribution to ligand binding, which is completely realized within the initial docking step of the binding mechanism. In contrast, the slow isomerization step is enthalpically driven. The partitioning of entropic and enthalpic components of binding energy was confirmed by isothermal titration calorimetry with wild-type and Delta209-222 rGST A1-1.

Animals↗
PubMed1998

Photoaffinity labeling of rat liver glutathione S-transferase, 4-4, by glutathionyl S-[4-(succinimidyl)-benzophenone].

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and xenobiotic substrate, was synthesized and shown to act as a photoaffinity label of rat liver glutathione S-transferase, 4-4. A time-dependent photoinactivation occurs upon irradiation at long wavelength UV light of the complex of enzyme and GS-Succ-BP. The rate of inactivation exhibits nonlinear dependence on [GS-Succ-BP], characterized by an apparent KI of 115 microM and kmax of 0.469 min-1. Effective protection against photoinactivation by 150 microM GS-Succ-BP is provided by dinitrophenol, nitrobenzene, ethacrynic acid, and S-hexylglutathione, analogues of xenobiotic substrates and product. These results suggest that GS-Succ-BP reacts with the enzyme within the active site, probably in the xenobiotic substrate-binding site. Upon complete inactivation, reagent incorporation of about 1 mol/mol of enzyme dimer is measured by radioactivity and MALDI-TOF mass spectrometry. Isolation of modified peptides followed by gas-phase sequencing and mass spectrometry indicates that Met-112 is the only reaction target of GS-Succ-BP. Although only one subunit of the enzyme dimer is modified, catalytic activity of both subunits is lost. Molecular modeling suggests that the benzophenone moiety of the compound binds in the cleft between the two enzyme subunits and modification of Met-112 on one subunit excludes reaction of the corresponding methionine on the other subunit. It is proposed that the new compound, glutathionyl S-[4-(succinimidyl)benzophenone], may have general applicability as a photoaffinity label of other enzymes with glutathione binding sites.

Animals↗
PubMed1999

Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a.

Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases. Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties. With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes. There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH. Y6F mutations have no effect on the pKa for these enzymes. Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group. Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation. All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher. The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases. Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting.

Amino Acid Sequence↗
PubMed2006

Garlic allyl sulfides display differential modulation of rat cytochrome P450 2B1 and the placental form glutathione S-transferase in various organs.

To investigate whether the regulation of garlic allyl sulfides on biotransformation enzyme expression is tissue-specific, the expression of cytochrome P450 2B1 (CYP 2B1) and the placental form of glutathione S-transferase (PGST) in liver, lung, and intestine, which are the three major organs responsible for drug metabolism, was examined. Rats were orally administrated 0.5 or 2 mmol/kg BW diallyl sulfide (DAS) or 0.5 mmol/kg BW diallyl disulfide (DADS) or diallyl trisulfide (DATS) three times per week for 6 weeks. The final body weights and the body weight ratio of liver and lung were not changed by any of these three allyl sulfide treatments as compared to the control rats. An 11- and 12-fold increase of 7-pentoxyresorufin O-dealkylase (PROD) activities was noted in rats treated with 0.5 or 2 mmol/mg BW DAS, respectively, as compared with the controls (P < 0.05). In contrast, DADS and DATS significantly increased hepatic PGST activity toward ethacrynic acid by 30 and 40%, respectively, as compared with the control rats (P < 0.05). An increase in PGST activity was only noted at 2 mmol/kg BW DAS group (P < 0.05). In addition, similar increases in PGST activity due to DADS and DATS were also noted in lung and jejunum tissue (P < 0.05). Immunoblot assay shows that the changes in CYP 2B1 and PGST proteins due to the three garlic allyl sulfide treatments on liver, lung, and jejunum were consistent with those observed for PROD and PGST activities. Northern blot further revealed that the DADS and DATS increased PGST mRNA levels in both liver (2.9- and 3.0-fold, respectively) and lung (4.1- and 2.6-fold, respectively) and DAS dose-dependently increased CYP 2B1 mRNA levels in the liver. Garlic allyl sulfides differentially induced CYP 2B1 and PGST expression, and this up-regulation of these two biotransformation enzymes is tissue-specific.

Allyl Compounds↗
PubMed2002

The organochlorine pesticide residues and antioxidant enzyme activities in human breast tumors: is there any association?

The levels of some organochlorine pesticides (OCP)s (hexachlorobenzene, HCB, alpha-hexachlorocyclohexane, alpha-HCH, beta-HCH, gamma-HCH, heptachlorepoxide, HE, bis (4-chlorophenyl)-1,1-dichloroethene, p.p'DDE, bis (4-chlorophenyl)-1,1,1-trichloroethane, p.p' DDT and total DDT (E-DDT) and antioxidant enzyme activities namely Cu, Zn superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px), total glutathione peroxidase (T-GSH-Px), selenium independent glutathione peroxidase (GSH-Px II), glutathione reductase (GRd), level of reduced glutathione (GSH) and lipid peroxidation (LP), glutathione S-transferase (GST) activity toward several substrates including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP) were measured in tumor and surrounding tumor free tissues of 24 female breast cancer patients and was evaluated whether there exist any association between the levels of OCPs and antioxidants. The mean levels of GSH, alpha-BHC, gamma-BHC and HE, and activities of SOD, Se-GSH-Px, T-GSH-Px, GSH-Px II,GRd, GST CDNB, and GST DCNB were significantly higher in tumors than in controls. In tumors, significant correlations were noted between: SOD and y-BHC; Se-GSH-Px and gamma-BHC; T-GSH-Px and gamma-BHC; GSH-Px II and alpha-BHC, gamma-BHC; GSH and alpha-BHC, gamma-BHC, HE; GRd and alpha-BHC; CDNB GST and alpha-BHC, gamma-BHC. These results show that free-radical mediated oxidative stress is, at least partly, associated with some of these OCP residues in human breast tumors.

Antioxidants↗
PubMed1976

Multiple pumps for sodium reabsorption by the perfused kidney.

Several distinct transport mechanisms responsible for sodium reabsorption by the rat kidney can be identified by studying the function of isolated perfused kidneys. Approximately one-half of the fractional sodium reabsorption by the isolated perfused rat kidney appears to depend on Na-K-adenosine triphosphatase (AT-Pase) and is inhibited by ouabain. About 10 to 20% is associated with the reabsorption of bicarbonate and is blocked by acetazolamide. This fraction of transported sodium is unaffected by ouabain and therefore does not involve Na-K-ATPase. Neither furosemide nor ethacrynic acid produce further inhibition of sodium reabsorption in a kidney already exposed to ouabain and acetazolamide. Most of the residual transport of sodium is inhibited by cooling the perfused kidney, suggesting that it is powered by metabolic rather than physical sources of energy.

Acetazolamide↗
PubMed1980

Renal bicarbonate reabsorption during bicarbonate loading.

To examine bicarbonate reabsorption at different GFR's, we varied the renal perfusion pressure in anesthetized dogs after inhibiting autoregulation by expanding extracellular volume and infusing ethacrynic acid. At a plasma bicarbonate concentration (PHCO3) of 28 +/- 1 mM, bicarbonate reabsorption varied in proportion to GFR (glomerulotubular balance). When PHCO3 was raised to 52 +/- 2 mM at constant PCO2 and hematocrit, bicarbonate reabsorption was reduced at all levels of filtered load. When plotted against GFR, different results were obtained dependent on the GFR level examined. At the control GFR,. bicarbonate loading reduced bicarbonate reabsorption by 30 +/- 5%. At a GFR level about 50% below the control GFR, bicarbonate loading increased reabsorption by about one third because the inhibitory effect of raising PHCO3 and extracellular pH was not sufficient to counteract the stimulatory effect of a higher filtered load. At intermediate levels of GFR, a rise in PHCO3 did not alter bicarbonate reabsorption. The finding that acetazolamide (30 mg/kg of body wt) at high PHCO3 failed to reduce bicarbonate reabsorption supports the hypothesis that the depressive effect of high extracellular pH on bicarbonate reabsorption may be attributed to reduced net tubular hydrogen ion secretion.

Absorption↗
PubMed1981

Mechanism of osmotic diuresis.

Mannitol might inhibit paracellular reabsorption of water and sodium chloride in the proximal tubules by reducing the osmotic driving force. We examined this hypothesis in anesthetized dogs. Bicarbonate reabsorption was kept constant by sodium bicarbonate infusion, and transcellular sodium chloride reabsorption was inhibited by ethacrynic acid. The glomerular filtration rate (GFR) was varied by altering renal perfusion pressure. Mannitol infusion reduced sodium chloride reabsorption from 62 +/- 5% to 33 +/- 5% of the filtered load. The calculated increase in reabsorbate osmolality, averaging 82 +/- 6 mOsm/kg H2O, was due to sodium bicarbonate and equalled the increase in plasma osmolality. Mannitol concentration averaged 81 +/- 7 mM in plasma and 101 +/- 12 mM in urine. A linear relationship between reabsorption and GFR (glomerulo-tubular balance) was maintained over the same range of GFR before and after mannitol infusion. Mannitol infusion reduced sodium chloride reabsorption from 2.6 to 1.4 moles for each mole of sodium bicarbonate reabsorbed. During mannitol infusion, acetazolamide inhibited sodium bicarbonate reabsorption as in control experiments, but reduced sodium chloride reabsorption less. We conclude that reduced water reabsorption increases sodium bicarbonate concentration in the paracellular fluid as much as mannitol concentration is raised in the plasma and glomerular filtrate. Along the proximal tubules, net osmotic force is progressively reduced as mannitol concentration rises, accounting for reduced water and sodium chloride reabsorption.

Acetazolamide↗
PubMed1984

Glomerular filtration rate and plasma pH as determinants of phosphate reabsorption.

Glomerular filtration rate (GFR) was altered by varying renal perfusion pressure in volume-expanded, anesthetized dogs infused with ethacrynic acid. Phosphate reabsorption varied linearly with GFR (r greater than 0.9), 0.83 of the increase in filtered load being reabsorbed. Phosphate reabsorption at comparable filtered loads was not significantly changed by raising plasma bicarbonate concentration from 30 to 55 mM and adjusting PCO2 to keep plasma pH constant. Plasma pH was altered by inducing hyper- and hypocapnia or infusing bicarbonate. Plasma phosphate concentration varied with plasma pH before phosphate infusion and was kept constant at 3.4 +/- 0.1 mM in intact and thyroparathyroidectomized dogs; some of which were also examined during hyperchloremic acidosis. At comparable GFR, phosphate and bicarbonate reabsorption correlated (r greater than 0.9), except during acidosis when the filtered load of bicarbonate became inadequate. In all experiments phosphate reabsorption and plasma pH correlated (r greater than 0.85). Compared with control values at plasma pH 7.4, phosphate reabsorption increased by about 40% during acidosis (pH 7.1) and decreased by about 50% during alkalosis (pH 7.8) both in intact and thyroparathyroidectomized dogs. We propose that net hydrogen ion secretion is the common determinant of phosphate and bicarbonate reabsorption.

Absorption↗
PubMed2005

Combined effects of GSTP1 and MRP1 in melanoma drug resistance.

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Selective modifiers of glutathione biosynthesis and 'repriming' of vascular smooth muscle photorelaxation.

Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a finite molecular store that can be depleted by irradiating pre-contracted arteries with visible light. The ability of an 'exhausted' vessel to respond to a further period of illumination is lost temporarily but then recovers slowly as the photosensitive store is reconstituted in the dark. The recovery process, termed repriming, displays an absolute requirement for endothelium-derived NO and is inhibited by pre-treating arteries with ethacrynic acid, a thiol-alkylating agent. Here we demonstrate that agents that up- or down-regulate glutathione (GSH) biosynthesis influence the extent to which the store is regenerated in the dark. Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE] +/- s.e.mean: 5. 78+/-0.46 microM) and periodically exposed to laser light (lambda=514.5 nm, 6.3 mW cm(-2) for 6 min). Photorelaxations of control RTAs were compared with those from either (a) vessels taken from animals previously injected i.p. with buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (three injections, 100 mg kg(-1) at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine (10(-4) M OXO for 60 min). RTAs from BSO-treated animals exhibited attenuated photorelaxations: the mean (+/-s.e.mean) amplitude of the response recorded after 72 min recovery in the dark was 12.4+/-1.6% versus 21.4+/-2.9% for control arteries (n=5; P<0. 01). Conversely RTAs treated with OXO and allowed to recover for a similar period showed enhanced photorelaxations, 32.6+/-6.3% as compared to 21.4+/-2.9% for control arteries (n=5; P<0.01). A hyperbolic curve fit to repriming curves for BSO-treated and control arteries returned asymptote values (maximum photorelaxations) of (mean +/- s.e.mean) 24.2+/-3.2% and 55.2+/-8.5%, respectively. The level of GSH in RTA extracts was measured by high-pressure liquid chromatography (HPLC). Injecting animals with BSO decreased GSH to 85% of control levels (P<0.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P<0.05). Thus, drug-induced changes in RTA GSH levels were positively correlated with altered photorelaxations. The results lead us to postulate that the photosensitive store in VSM is generated, at least in part, from intracellular GSH which becomes converted to S-nitrosoglutathione (GSNO) by nitrosating species that are formed ultimately from endothelium-derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.

Animals↗
PubMed2001

DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration.

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.

Action Potentials↗
PubMed2003

A photosensitive vascular smooth muscle store of nitric oxide in mouse aorta: no dependence on expression of endothelial nitric oxide synthase.

(1) Photorelaxation is the reversible relaxation of vascular smooth muscle (VSM) when irradiated with ultraviolet (UV) light resulting from the release of nitric oxide (NO). In this study we characterize the involvement of endothelial nitric oxide synthase (eNOS) in the photorelaxation response of thoracic aorta from endothelial NOS deficient (-/-) and control (C57BL/6j) mice. (2) Cirazoline contracted aortae were repeatedly exposed to 30 s of UV light every 3-4 min. Equal levels of photorelaxation (45+/-2%; n=34) was observed in both strains. (3) 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), K(+), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), 4-aminopyridine (4-AP) and ethacrynic acid significantly reduced the photorelaxation response. In C57BL/6j mice diethyldithiocarbamate (DETCA) also reduced photorelaxation. (4) Control endothelium-intact and -denuded aorta and L-NAME (100 micro M) treated and untreated eNOS (-/-) aortae were repeatedly exposed to UV light for 5 min every 10 min until no photorelaxation response was observed. After 1 h of rest in the dark the vessels showed between 30-70% recovery of the photorelaxation response indicating regeneration of the store in the absence of the endothelium and eNOS. (5) The results of this study suggest that photorelaxation in mouse aorta VSM results from the release of NO from a stable store of RSNOs, which activates soluble guanylate cyclase (sGC), leading to cGMP-dependent relaxation that is partially mediated by an increase in K(V) channel activation and hyperpolarization. In addition, the eNOS isoform is not essential for the formation of the photorelaxation store and a non-NOS source of NO may be involved in the maintenance of this store.

Animals↗
PubMed1981

Kinetic studies of the mechanism of pig kidney aldehyde reductase.

Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

Alcohol Oxidoreductases↗
PubMed1984

Purification and properties of glutathione S-transferases from larvae of Wiseana cervinata.

The glutathione S-transferases from the porina moth, Wiseanna cervinata, were purified by affinity chromatography, cation-exchange chromatography and preparative isoelectrofocusing. The major transferase (IV) was purified to homogeneity by a factor of 530-fold with a yield of 83%. Other transferases present were purified to a smaller degree (approx. 50-fold) to a stage of near-homogeneity. The transferases examined all had Mr values about 45 000-50 000. They appeared to be homodimers of either of two types of subunit, of Mr 22 800 and 24 600. Enzymes consisting of the different types of subunit were not immunologically cross-reactive. The major enzyme fractions separated by cation-exchange chromatography were both active with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and iodomethane, but were inactive with 4-nitropyridine N-oxide, 1,2-epoxy-3-(p-nitrophenoxy)propane, bromosulphophthalein and p-nitrobenzyl chloride. The kinetics of the enzyme-catalysed reaction with enzyme IV were non-Michaelean with respect to both substrates. Both products were inhibitory. The results appear to be compatible with a random steady-state mechanism. It is concluded that these enzymes are very similar, in their physical and chemical constitution, in their catalytic properties and in their relationships with each other, to those enzymes that have been isolated from vertebrate organisms.

Amino Acids↗
PubMed1985

Isoenzymes of glutathione transferase in rat kidney cytosol.

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a 'new' form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

Animals↗
PubMed1985

Kinetic independence of the subunits of cytosolic glutathione transferase from the rat.

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

Animals↗