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PubMed2003

Characterization of the electrophile binding site and substrate binding mode of the 26-kDa glutathione S-transferase from Schistosoma japonicum.

The 26-kDa glutathione S-transferase from Schistosoma japonicum (Sj26GST), a helminth worm that causes schistosomiasis, catalyzes the conjugation of glutathione with toxic secondary products of membrane lipid peroxidation. Crystal structures of Sj26GST in complex with glutathione sulfonate (Sj26GSTSLF), S-hexyl glutathione (Sj26GSTHEX), and S-2-iodobenzyl glutathione (Sj26GSTIBZ) allow characterization of the electrophile binding site (H site) of Sj26GST. The S-hexyl and S-2-iodobenzyl moieties of these product analogs bind in a pocket defined by side-chains from the beta1-alpha1 loop (Tyr7, Trp8, Ile10, Gly12, Leu13), helix alpha4 (Arg103, Tyr104, Ser107, Tyr111), and the C-terminal coil (Gln204, Gly205, Trp206, Gln207). Changes in the Ser107 and Gln204 dihedral angles make the H site more hydrophobic in the Sj26GSTHEX complex relative to the ligand-free structure. These structures, together with docking studies, indicate a possible binding mode of Sj26GST to its physiologic substrates 4-hydroxynon-2-enal (4HNE), trans-non-2-enal (NE), and ethacrynic acid (EA). In this binding mode, hydrogen bonds of Tyr111 and Gln207 to the carbonyl oxygen atoms of 4HNE, NE, and EA could orient the substrates and enhance their electrophilicity to promote conjugation with glutathione.

Aldehydes↗

PubMed1995

[Reduced nutritional status enhances ototoxicity].

BACKGROUND: This study investigates whether the nutritional state of guinea pigs is a risk factor for the ototoxic side effects of cisplatin, gentamicin, and gentamicin in combination with ethacrynic acid. METHODS: A normal nutritional state was maintained with a standard 18.5% protein diet while nutritional deficiency was produced by feeding a 7% protein diet. Hearing loss was measured by auditory evoked brainstem responses. RESULTS: Guinea pigs on the low protein diet had a significantly higher drug-induced hearing loss. Cisplatin-induced hearing loss was 32 dB in undernourished animals but 10 dB in normal animals (18 kHz). The difference for gentamicin was 74 dB versus 42 dB (18 kHz). Gentamicin in combination with 20 mg ethacrynic acid/kg body weight produced a hearing loss of 95 dB in animals on a low protein diet and 12 dB in animals on a full protein diet. The enhanced ototoxicity was not based on differences in drug pharmacokinetics since serum levels of platinum and gentamicin did not differ between the groups. CONCLUSIONS: These results demonstrate that the severity of ototoxic side effects is influenced by nutritional factors. They also imply that animals on a restricted diet may be a more appropriate model for severely compromised patients undergoing pharmacotherapy.

Animals↗

PubMed1975

Water and ion transport by the urinary bladder of the teleost Pseudopleuronectes americanus.

Water and ion transport by the isolated teleost urinary bladder were studied. The transepithelial electrical PD across sac-type bladder preparations was unstable, i.e., initially mucosa positive but becoming more negative with time. Perfused bladders maintained a low mucosa positive PD which was stable. Both Na and Cl appeared to be actively transported from mucosal side (M) to serosal side (S). Voltage clamping the bladder at 0, -50, or +50 mV had almost no effect on active or passive Na or Cl flux in either direction. Na and Cl transport seemed electrically neutral. Fluid absorption (M to S) was directly correlated with absorption of osmotically active solutes. These solutes were almost all Na and Cl. The bladder acidified and secreted K+ into the mucosal fluid. Divalent ions were concentrated in the mucosal fluid as a result of fluid absorption. Although furosemide and ethacrynic acid inhibited ion and water transport by the bladder, ouabain was effective at a much lower concentration. Ouabain (10(-4) M) inhibited active Na transport when applied only to the mucosal or only to the serosal surface. Ouabain abolished the PD only from the serosal surface.

Animals↗

PubMed1975

A Mg2+- and Ca2+-stimulated adenosine triphosphatase at the outer surface of Ehrlich ascites tumor cells.

A Mg2+- and Ca2+-stimulated adenosine triphosphatase (ATPase) at the outer surface of intact Ehrlich ascites tumor cells is described. A surface-bound adenosine triphosphate (ATP)-splitting activity at a lower rate was also demonstrated in the absence of Ca2+ but with Mg2+, Na+, and K+ present in the isotonic medium. Hence, when part of the Mg2+ was exchanged for Ca2+, a marked increase of the ATP-splitting activity was observed. The stimulatory effect of Ca2+ was seen only if both Na+ and K+ were present in the isotonic incubation medium. Thus, the enzyme activity was Mg2+- and Ca2+-dependent. Ca2+, together with the monovalent cations was inhibitory compared with Mg2+ under similar conditions. The apparent Km for ATP for the Mg2+-stimulated ATPase is 0.05 mM, while that of the Mg2+- and Ca2+-stimulated enzyme is 0.10 mM. The Vmax of the former is 0.8 mu-mole per 100 mg Schneider protein per 30 sec compared with 1.92 mu-moles per 100 mg Schneider protein per 30 sec for the latter. The calculated Km for the Mg2+- and Ca2+-stimulated ATPase after subtraction of the Mg2+-stimulated part is 0.22 mM. Ethacrynic acid and N-ethylmaleimide both inhibited the Mg2+- and Ca2+-stimulated ATPase by about 10 percent, while the ouabain inhibition was 15 percent. Cytochalasin B did not influence the enzyme activity, whereas La3+ had a slight stimulatory effect.

Adenosine Triphosphatases↗

PubMed1995

TNF-induced apoptosis in multidrug resistant friend erythroleukemia is not influenced by the P-glycoprotein and glutathione status of the cell line.

The effects that TNF-alpha exerts on Friend erythroleukemia (FLC) and on one multidrug resistant variant (FLC-DXR) of the cell line were studied. Resistance to doxorubicin of FLC-DXR entails two mechanisms: overexpression of P-glycoprotein; and increased glutathione-related activities. Both these might also decrease the effects of the cytokine. Nonetheless, TNF caused even greater cytotoxicity and apoptosis, with no induction of differentiation markers, in FLC-DXR. In addition, TNF produced minor changes of the levels of reduced and oxidized glutathione in the cell lines, and its cytotoxic effects were not inluenced by agents that modify the cell glutathione content such as buthionine sulfoximine, ethacrynic acid, or N-acetyl cysteine. We can exclude that the mechanisms of drug resistance of FLC-DXR prevent the response to the cytokine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts.

A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy.

1-Propanol↗

PubMed1991

Inhibition of adenylate-cyclase-coupled G protein complex by ototoxic diuretics and cis-platinum in the inner ear of the guinea pig.

The enzyme adenylate cyclase produces the second messenger cAMP and is located in the mammalian inner ear, predominantly in the stria vascularis and to a lesser extent in the organ of Corti. It is coupled to hormone receptors and regulating G proteins in the outer cell membrane. By means of immunofluorescence in cryostat sections of the guinea pig cochlea, we could demonstrate the G proteins Gs and Gi, which belong to the adenylate cyclase complex. These proteins had their highest density in the stria vascularis. In membrane preparations of this tissue, the adenylate cyclase complex was inhibited by ototoxic drugs (furosemide, ethacrynic acid and cis-platinum). Stimulation at different sites of the enzyme system showed that the target of these drugs was probably the regulating G protein complex and not the enzyme molecule itself. Inhibition depended on the concentration of the drug and the incubation time.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice.

The effect of glutathione depletion on the viability of freshly isolated synaptosomes from whole brain was investigated in young and aged mice. Aging did not influence the GSH and ATP levels and the viability of these synaptosomes. However depletion of glutathione caused by the cytosolic glutathione inhibitor diethyl maleate (1 mM) resulted in a significant decline, after 60 min of incubation, in ATP levels and viability in the synaptosomes from aged mice but not in those from young mice. When synaptosomes were incubated in the presence of the mitochondrial glutathione inhibitor ethacrynic acid (0.2 mM) there was a similar decline in glutathione, ATP levels and synaptosomal viability, both in young and aged mice. These results emphasize the relative importance of the cytosolic glutathione pool for the maintenance of the plasma membrane integrity in synaptosomes from aged mice.

Adenosine Triphosphate↗

PubMed1976

[Formation of the inner ear lymphs. Permeability of inner ear membranes (author's transl)].

1. The endolymphatic system is morphologically a close system. The inner surface of the wall is tightly lined with an epithelium of ectodermal origin. The perilymphatic spaces are enlarged intercellular spaces which are built from the embryonic mesenchyme. 2. The perilymph ist an ultrafiltrate of plasma. There is probably a flow from the cerebrospinal fluid which is constantly renewed. The diffusion in the perilymph is dependent on the concentration and the size of the molecules. The endolymph is mainly a perilymph-filtrate. The "secretory" epithelia (e.g. stria vascularis cells and other tissues) of the endolymphatic system perform an important role to sustain the potassium and sodium concentrations. The ionic concentrations regulate the water movement also the volume of the endolymphatic spaces. They are maintained by anoxy-sensitive pumps. 3. The DC potential within the endolymphatic spaces represents the movement of certain electrical charge through membranes. By applying various inhibitors it is possible to distinguish the pumping mechanisms, and to observe the continuous changes of potassium and sodium concentrations with Na+ specific electrodes and K+ specific electrodes. There are probably three interdependent sources of driving-forces: a. A positively electrogenic K+-pump which is anoxia-sensitive and can be inhibited by Ethacrynic acid. This mechanism is more active in stria cells and less so in utricle and saccule. b. A negatively electrogenic Na+-K+ exchange-pump in all parts of the endolymphatic spaces is inhibited by Ouabain or anoxia. c. The passive diffusion of potassium-ions from endolymph to perilymph results an electro-negative effect.

Animals↗

PubMed1991

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase.

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.

Base Sequence↗

PubMed1995

Ouabain-insensitive, Na(+)-stimulated ATPase activity in rabbit cardiac sarcolemma.

The rabbit cardiac sarcolemma shows an ouabain, Na,K-stimulated ATPase activity and an ouabain-insensitive, Na-stimulated ATPase activity. The Na-ATPase has the following characteristics: (i) It is also stimulated by other monovalent cations. (ii) It is inhibited by 2 mM Furosemide and by 2 mM ethacrynic acid. (iii) It reaches maximal values (Vmax) at around 20 mM Na+. (iv) The apparent Km is around 5 mM. Except for the monovalent cation stimulation, the main characteristics of this ATPase are very similar to those of the ouabain-insensitive, Na-stimulated ATPase of mammalian kidneys.

Adenosine Triphosphatases↗

PubMed1978

Generation of transepithelial potentials by isolated perfused reptilian distal tubules.

Transepithelial potentials were measured in the most distal segments of garter snake (Thamnophis spp.) distal tubles perfused in vitro. The segments generated high lumen-negative potentials when sodium was in the lumen. The size of the potentials was a saturable function of luminal sodium concentrations between 0 and 32 mM. The potentials were stable with time only when sodium concentrations in the lumen were less than 30 mM. Perfusion with high sodium concentrations resulted in transient potentials. Stable potentials changed markedly when the lumen sodium concentration or the bath potassium concentration was altered suddenly, but they were independent of lumen potassium concentrations and bath sodium concentrations. Amiloride stimulated or inhibited the potentials, ouabain partially depressed them, and ethacrynic acid and cyanide inhibited them slowly and often irreversibly. We conclude that distal transepithelial potentials reflect sodium transport from lumen to bath across a tight asymmetric epithelium which differs from other sodium-transporting epithelia in that stable transepithelial potentials are maintained only with luminal sodium concentrations less than 30 mM.

Amiloride↗

PubMed1990

[Synergism between noise and ototoxic drugs: new experimental data].

Results of experiments investigating, in the guinea pig, the effects of combinations of gentamicin (GM), ethacrynic acid (EA) and acoustic stimulations are reported. They indicate that functional and morphological alterations develop only after GM has penetrated specifically into the sensory cells, that this penetration is indirectly facilitated by EA, since it is known to increase GM concentration in endolymph, and directly by functional depolarization of the cell, as it occurs during normal acoustic stimulation.

Animals↗

PubMed1975

[Fluorescence study of lymphocyte plasma membranes].

ANS binding parameters--dissociation constant, number of binding sites, rotation freedom--are measured by fluorescence studies of a complex between ANS and lymph node cell plasma membranes. Divalent ions, Mg++ and Ca++, enhance the complex fluorescence intensity without shifting its maximum wavelength : this enhancement is induced by affinity and quantum yield increases, while the number of binding sites remains constant. The complex fluorescence quenching by ethacrynic acid shows the presence of free SH groups in the ANS binding site. An energy transfer takes place between membrane protein tryptophan residues and bound ANS ; the energy transfer yield is unaffected by Ca++ ions. A correlation of these results is postulated with the biological activity of the membrane.

Anilino Naphthalenesulfonates↗

PubMed1990

[Subcellular distribution of glutathione S-transferase in Chinese fetal liver].

Subcellular fractions were isolated from Chinese fetal liver at 4-8 months of age for the determination of glutathione S-transferase (GST). Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, GST activity was found to be 66 +/- 34 nmol/(min.mg protein), mainly in the cytosol. The GST activities were detected principally in microsomes and their values were 66 +/- 31 and 144 +/- 83 nmol/(min.mg protein), respectively, when assayed with p-nitrobenzyl chloride (PNB) and ethacrynic acid (EA) as substrates. There were no age and sex-related differences in GST activities for any of the substrates studied during fetal development. The Km values of GST for CDNB, PNB and EA were 1112, 1039 and 205 mumol/L, respectively. The conjugation of GST may play an important role in fetal hepatic metabolism of toxic electrophiles.

Cytosol↗

PubMed1974

The identity of glutathione S-transferase B with ligandin, a major binding protein of liver.

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody produced against ligandin, bear entirely similar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was shown to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.

Animals↗

PubMed1991

Cellular mechanism of stimulation of renin secretion by the mercurial diuretic mersalyl.

The aim of the present study was to elucidate the cellular mechanism by which the mercurial diuretic mersalyl stimulates renin secretion in rabbit renal cortical slices in vitro. The stimulatory effect of mersalyl on renin secretion was rapid, reversible and concentration dependent. The stimulation was not dependent on the presence of ions such as Na+, Cl- and Ca++, and it was unaffected by inhibitors of Na+/K+/2Cl- cotransport, such as bumetanide and furosemide. However, the stimulation was blocked and reversed by thiols, such as L-cysteine and dithiothreitol. Furthermore, the maximal stimulatory effect of mersalyl on renin secretion was not additive to that produced by the non-diuretic mercurial sulfhydryl reagent P-chloromercuriphenylsulfonate nor to that produced by the non-mercurial diuretic sulfhydryl reagent, ethacrynic acid. These results support the hypothesis that mersalyl stimulates renin secretion by forming a reversible mercaptide bond with sulfhydryl groups, located perhaps on the plasma membrane of juxtaglomerular cells. These particular sulfhydryl groups appear to have no functional role in the diuretic action of mersalyl.

4-Chloromercuribenzenesulfonate↗

PubMed2005

Evolution of highly active enzymes by homology-independent recombination.

The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."

Animals↗