nitrophenols and 1-2-epoxy-3-(p-nitrophenoxy)propane

Glutathione transferase zeta 1 (GSTZ1), expressed in liver and several extrahepatic tissues, catalyzes dechlorination of dichloroacetate (DCA) to glyoxylate. DCA inactivates GSTZ1, leading to autoinhibition of its metabolism. DCA is an investigational drug for treating several congenital and acquired disorders of mitochondrial energy metabolism, including cancer. The main adverse effect of DCA, reversible peripheral neuropathy, is more common in adults treated long-term than in children, who metabolize DCA more quickly after multiple doses. One dose of DCA to Sprague Dawley rats reduced GSTZ1 expression and activity more in liver than in extrahepatic tissues; however, the effects of multiple doses of DCA that mimic its therapeutic use have not been studied. Here, we examined the expression and activity of GSTZ1 in cytosol and mitochondria of liver, kidney, heart, and brain 24 hours after completion of 8-day oral dosing of 100 mg/kg per day sodium DCA to juvenile and adult Sprague Dawley rats. Activity was measured with DCA and with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNPP), reported to be a GSTZ1-selective substrate. In DCA-treated rats, liver retained higher expression and activity of GSTZ1 with DCA than other tissues, irrespective of rodent age. DCA-treated juvenile rats retained more GSTZ1 activity with DCA than adults. Consistent with this finding, there was less measurable DCA in tissues of juvenile than adult rats. DCA-treated rats retained activity with EPNPP, despite losing over 98% of GSTZ1 protein. These data provide insight into the differences between children and adults in DCA elimination under a therapeutic regimen and confirm that the liver contributes more to DCA metabolism than other tissues. SIGNIFICANCE STATEMENT: Dichloroacetate (DCA) is one of few drugs exhibiting higher clearance from children than adults, after repeated doses, for reasons that are unclear. We hypothesized that juveniles retain more glutathione transferase zeta 1 (GSTZ1) than adults in tissues after multiple DCA doses and found this was the case for liver and kidney, with rat as a model to assess GSTZ1 protein expression and activity with DCA. Although 1,2-epoxy-3-(4-nitrophenoxy)propane was reported to be a selective GSTZ1 substrate, its activity was not reduced in concert with GSTZ1 protein.

Topics: Adult; Age Factors; Animals; Child; Dichloroacetic Acid; Dose-Response Relationship, Drug; Energy Metabolism; Epoxy Compounds; Female; Glutathione Transferase; Humans; Liver; Male; Mitochondrial Diseases; Models, Animal; Nitrophenols; Rats

The inhibition potencies of covalent inhibitors mainly result from the formation of a covalent bond to the enzyme during the inhibition mechanism. This class of inhibitors has essentially been ignored in previous target-directed drug discovery projects because of concerns about possible side effects. However, their advantages, such as higher binding energies and longer drug-target residence times moved them into the focus of recent investigations. While the rational design of non-covalent inhibitors became standard the corresponding design of covalent inhibitors is still in its early stages. Potent covalent inhibitors can be retrieved from large compound libraries by covalent docking approaches but protocols are missing that can reliably predict the influence of variations in the substitution pattern on the affinity and/or reactivity of a given covalent inhibitor. Hence, the wanted property profile can only be obtained from trial-and-error proceedings. This paper presents an appropriate protocol which is able to predict improved covalent inhibitors. It uses hybrid approaches, which mix quantum mechanical (QM) and molecular mechanical (MM) methods to predict variations in the reactivity of the inhibitor. They are also used to compute the required information about the non-covalent enzyme-inhibitor complex. Docking tools are employed to improve the inhibitor with respect to the non-covalent interactions formed in the binding site.

Topics: Catalytic Domain; Drug Design; Enzyme Inhibitors; Enzymes; Epoxy Compounds; HIV Protease; Humans; Molecular Docking Simulation; Nitrophenols; Quantum Theory

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.

Topics: Animals; Culicidae; Epoxy Compounds; Erythrocytes; Humans; Intracellular Membranes; Leucine; Microscopy, Electron; Nigericin; Nitrophenols; Plasmodium falciparum; Temperature; Tosyllysine Chloromethyl Ketone; Vacuoles; Xanthurenates

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore we investigated GST activity and the protein expression of glutathione S-transferases (GSTs) isoenzymes known to be involved in the metabolism of endogenous and exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. The interindividual variation in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and of 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) with glutathione (GSH) by cytosolic glutathione S-transferases (GSTs) were investigated in human breast matched normal and tumor samples. The GSTA, GSTM, GSTP and GSTT isoenzymes from the crude extracts of matched breast normal and tumor tissues in terms of their immunological properties using western blotting were compared. In most of the samples, the GST activities were higher in the tumor than in the normal cytosolic fractions against both CDNB and EPNP. In the western blotting analysis, it was proved statistically that in normal and tumor epithelial cells, there was difference between GST pi and theta isoenzymes expressions (p0.05). In normal epithelium there was a stronger GST theta expression than in invasive tumor tissues (p=0.013). However, the stronger GST pi expression was observed in tumor epithelium than in normal epithelium in human breast cancers (p=0.000). We found the GSTP protein level and GST activities were higher in the breast tumor than in the normal cytosolic fractions against both CDNB and EPNP, thus implicating a certain biological importance.

Topics: Blotting, Western; Breast Neoplasms; Dinitrochlorobenzene; Epoxy Compounds; Female; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Nitrophenols; Substrate Specificity

We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.

Topics: Benzene Derivatives; Enzyme Stability; Epoxy Compounds; Genetic Variation; Glutathione Transferase; Humans; Hydrocarbons, Iodinated; Inactivation, Metabolic; Nitrobenzenes; Nitrophenols; Polymorphism, Single Nucleotide

The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs ( approximately 27.8- and approximately 25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.

Topics: Biofilms; Cytoplasm; Dinitrobenzenes; Dinitrochlorobenzene; Enzyme Induction; Epoxy Compounds; Fluorescent Antibody Technique; Fungal Proteins; Glutathione Transferase; Halogenated Diphenyl Ethers; In Situ Hybridization, Fluorescence; Microscopy, Confocal; Microscopy, Fluorescence; Microsomes; Molecular Weight; Mucor; Nitrobenzenes; Nitrophenols; Phenyl Ethers; Spores, Fungal; Thiosulfates; Water Microbiology

Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.

Topics: Animals; Aspartic Acid Endopeptidases; Bacterial Proteins; Bacterial Toxins; Binding Sites; Biological Factors; Catalysis; Cell Extracts; Cell Line; Clostridioides difficile; Epoxy Compounds; Nitrophenols; Phytic Acid; Protein Processing, Post-Translational; Protein Transport; Spleen; Swine; Virulence Factors

Glutathione S-transferase (GST) theta 1 (GSTT1) has been regarded as one of the key enzymes involved in phase II reactions because of its unique substrate specificity. In this study, we generated mice with the disrupted Gstt1 gene (Gstt1-null mice) by gene targeting and analyzed the metabolic properties in cytosolic and in vivo studies. The resulting Gstt1-null mice failed to express the Gstt1 mRNA and GSTT1 protein by reverse transcriptase-polymerase chain reaction analysis and two-dimensional fluorescence difference gel electrophoresis/mass spectrometry analysis, respectively. However, the Gstt1-null mice appeared to be normal and were fertile. In an enzymatic study using cytosolic samples from the liver and kidney, GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), dichloromethane (DCM), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was markedly lower in Gstt1-null mice than in the wild-type controls, despite there being no difference in GST activity toward 1-choloro-2,4-dinitrobenzene between Gstt1-null mice and the wild-type controls. Gstt1-null mice had GST activity of only 8.7 to 42.1% of the wild-type controls to EPNP, less than 2.2% of the wild-type controls to DCM, and 13.2 to 23.9% of the wild-type controls to BCNU. Plasma BCNU concentrations after a single i.p. administration of BCNU to Gstt1-null mice were significantly higher, and there was a larger area under the curve(5-60) min (male, 2.30 times; female, 2.28 times, versus the wild-type controls) based on the results. In conclusion, Gstt1-null mice would be useful as an animal model of humans with the GSTT1-null genotype.

Topics: Animals; Carmustine; Chromatography, Liquid; Cytosol; Dinitrochlorobenzene; Electrophoresis, Gel, Two-Dimensional; Epoxy Compounds; Female; Fertility; Fluorescence; Genotype; Glutathione Transferase; Injections, Intraperitoneal; Kidney; Kinetics; Liver; Male; Methylene Chloride; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrophenols; Phenotype; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.

Topics: Animals; Antibodies; Cytosol; Dinitrochlorobenzene; Epoxy Compounds; Escherichia coli; Glutathione Transferase; Humans; Hydrolases; Isoenzymes; Kinetics; Liver; Male; Nitrophenols; Rabbits; Rats; Rats, Wistar; Recombinant Proteins; Subcellular Fractions; Substrate Specificity

Chemical modification of spin-cast chitosan films has been performed. This modification involves the attachment of 1,2 Epoxy-3-phenoxy-propane, commonly known as glycidyl phenyl ether (GPE), to the amine group of the chitosan molecule. Optical properties of modified films have been determined in the infrared region of the spectrum using spectroscopic ellipsometry, and are reported in this paper. Special attention is paid to the infrared region where the index of refraction and extinction coefficients from 750 to 4000 cm(-1) were determined. Difference plots of IR optical data before and after chemical modification were generated to confirm that modification had occurred. Optical modeling of infrared spectroscopic ellipsometry (IRSE) data with respect to chemical bond vibrations has also been performed. This modeling involved curve fitting of resonant chemical bond absorptions using Lorentz oscillators. These oscillator models allow for comparison of modified chitosan to unmodified chitosan. The purpose of this research was to determine infrared optical constants of chemically modified chitosan films This work shows that surface chemistry of biomaterials can be studied quite sensitively with spectroscopy ellipsometry, detecting as little as 100 ng/cm(2) of GPE.

Topics: Adsorption; Biocompatible Materials; Chitin; Chitosan; Epoxy Compounds; Membranes, Artificial; Microscopy, Atomic Force; Models, Statistical; Nitrophenols; Oscillometry; Polymers; Spectrophotometry; Spectrophotometry, Infrared; Surface Properties; Time Factors

The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 A using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 A, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters.

Topics: Anisotropy; Crystallization; Crystallography, X-Ray; Dimerization; Epoxy Compounds; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Ligands; Nitrophenols; Phenylmethylsulfonyl Fluoride; Protein Structure, Tertiary; Pyrococcus furiosus; Trans-Activators; Transcription, Genetic; X-Ray Diffraction

The correlation between sequence diversity and enzymatic function was studied in a library of Theta class glutathione transferases (GSTs) obtained by stochastic recombination of fragments of cDNA encoding human GST T1-1 and rat GST T2-2. In all, 94 randomly picked clones were characterized with respect to sequence, expression level, and catalytic activity in the conjugation reactions between glutathione and six alternative electrophilic substrates. Out of these six different compounds, dichloromethane is a selective substrate for human GST T1-1, whereas 1-menaphthyl sulfate and 1-chloro-2,4-dinitrobenzene are substrates for rat GST T2-2. The other three substances serve as substrates for both enzymes. Through this broad characterization, we have identified enzyme variants that have acquired novel activity profiles that differ substantially from those of the original GSTs. In addition, the expression levels of many clones were improved in comparison to the parental enzyme. A library of mutants can thus display a distribution of properties from which highly divergent evolutionary pathways may emerge, resembling natural evolutionary processes. From the GST library, a clone was identified that, by the point mutation N49D in the rat GST T2-2 sequence, has a 1700% increased activity with 1-menaphthyl sulfate and a 60% decreased activity with 4-nitrophenethyl bromide. Through the N49D mutation, the ratio of these activities has thus been altered 40-fold. An extensive characterization of a population of stochastically mutated enzymes can accordingly be used to find variants with novel substrate-activity profiles and altered catalytic properties. Recursive recombination of selected sequences displaying optimized properties is a strategy for the engineering of proteins for medical and biochemical applications. Such sequential design is combinatorial protein chemistry based on remodeling of existing structural scaffolds and has similarities to evolutionary processes in nature.

Topics: Animals; Catalysis; Clone Cells; Dinitrochlorobenzene; Epoxy Compounds; Evolution, Molecular; Gene Library; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Methylene Chloride; Models, Molecular; Mutation; Naphthalenes; Nitrobenzenes; Nitrophenols; Rats; Recombinant Proteins; Sequence Analysis, DNA; Structure-Activity Relationship; Substrate Specificity

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Topics: Animals; Callithrix; Chromatography, Affinity; Clofibric Acid; Cytosol; Dinitrochlorobenzene; Epoxy Compounds; Glutathione Transferase; Humans; Liver; Methyl Chloride; Methylene Chloride; Mice; Mice, Inbred BALB C; Nitrophenols; Phenobarbital; Polychlorinated Dibenzodioxins; Rats; Rats, Wistar; Rifampin; Species Specificity

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the alpha4 helix, Asp161 and Gln165 on the alpha6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) of the S209W mutant is 2. 5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested. We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting beta1 and alpha1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.

Topics: Amino Acid Substitution; Animals; Binding Sites; Chickens; Crystallography, X-Ray; Deuterium; Epoxy Compounds; Glutamine; Glutathione; Glutathione Transferase; Hydrogen Bonding; Kinetics; Models, Molecular; Mutation; Nitrophenols; Oxidoreductases; Protein Conformation; Protons; Recombinant Proteins; Structure-Activity Relationship; Temperature; Tryptophan; Tyrosine

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatial

Topics: Aspartic Acid; Binding Sites; Catalysis; Dimerization; Epoxy Compounds; HIV Protease; HIV Protease Inhibitors; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Nitrophenols; Oligopeptides; Protein Conformation; Stereoisomerism; Structure-Activity Relationship; Substrate Specificity; Thermodynamics

1. The circadian variation in glutathione S-transferase (GST) activity was studied in the hepatic cytosolic fraction of the male and female mouse. A circadian variation in GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the male, the activity being higher in the light phase (07:00-19:00 h) than in the dark phase (19:00-07:00 h) during a day under normal lighting conditions. 2. The circadian variation was only existed from June to October. The difference between the lowest activity (at 01:00 h) and the highest activity (at 13:00 h) was maximum in August. 3. In both the normal and reversed light/dark cycle (lights on 07:00 and 19:00 h, respectively), reduced glutathione (GSH) content was lowest in the middle of the light period and highest in the middle of the dark period and GST activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) exhibited opposite peaks and troughs. GST activities toward CDNB and 1,2-dichloro-4-nitrobenzene (DCNB) during the normal lighting schedule was higher at 13:00 h than at 01:00 h, but no differences were observed under reversed lighting conditions. 4. A circadian variation in GST activity for CDNB and DCNB was also observed in the female in a similar manner to the male, but the variation in the activity for EPNP was not observed in the female. 5. Thus, the circadian variation of hepatic GST activities in mouse were dependent on the enzyme substrates used, and seemed to be reflected by the difference in each isozyme levels. The daily change in the hepatic GSH levels is also thought involved, at least in part, in the regulation of GST activity.

Topics: Animals; Circadian Rhythm; Darkness; Dinitrochlorobenzene; Epoxy Compounds; Ethacrynic Acid; Female; Glutathione; Glutathione Transferase; Light; Liver; Male; Mice; Mice, Inbred Strains; Nitrobenzenes; Nitrophenols; Protease Inhibitors; Seasons; Sex Factors

Acid protease inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) is commonly used in research as a substrate for glutathione-S-transferase activity (GST) and recently was found to inhibit human immunodeficiency virus 1 (HIV-1) protease. The question of DNA-adduct formation and mutagenicity was investigated and found that ENPP causes DNA damage and acts directly to induce mutagenicity in Salmonella. Using HPLC analysis, ENPP was shown to bind covalently to guanine residues. The Salmonella mutagenicity assay indicated that ENPP enhanced the mutation frequencies in the base-substitution strain TA00 by more than 20 times above the background. Its mutagenic potency was comparable to that of well-known carcinogens, N-methyl-N-nitrosourea (MNU) and aflatoxin B(1)-8,9-epoxide (AFB(1)-8,9-epoxide). The results suggest that ENPP should be classified as a mutagenic compound and a potential carcinogen.

Topics: Carcinogens; Chromatography, High Pressure Liquid; DNA Adducts; DNA Damage; Epoxy Compounds; Glutathione Transferase; Guanine; HIV Protease Inhibitors; HIV-1; Mutagenicity Tests; Mutagens; Nitrophenols; Salmonella

Topics: Adult; Allergens; Dermatitis, Allergic Contact; Dermatitis, Occupational; Epoxy Compounds; Female; Humans; Nitrophenols

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

Topics: Animals; Cricetinae; Cytosol; Epoxy Compounds; Erythrocytes; Female; Glutathione Transferase; Humans; In Vitro Techniques; Indicators and Reagents; Kidney; Liver; Male; Methyl Chloride; Methylene Chloride; Mice; Nitrophenols; Rats; Species Specificity

Recombinant human theta class glutathione transferase T1-1 has been heterologously expressed in Escherichia coli and a simple purification method involving immobilized ferric ion affinity chromatography and Orange A dye chromatography is described. The catalytic properties of the enzyme differ significantly from those of other glutathione transferases, also within the theta class, with respect to both substrate selectivity and kinetic parameters. In addition to 1,2-epoxy-3-(4-nitrophenoxy)propane, the substrate used previously to monitor the enzyme, human glutathione transferase T1-1 has activity with the naturally occurring phenethylisothiocyanate and also displays glutathione peroxidase activity with cumene hydroperoxide. Further, the enzyme is active with 4-nitrobenzyl chloride and 4-nitrophenethyl bromide, but shows no detectable activity with the more chemically reactive 1-chloro-2,4-dinitrobenzene. The Michaelis constant for glutathione, K(m)GSH, with 1,2-epoxy-3-(4-nitrophenoxy)propane as second substrate, is high at low pH values but decreases at higher pH values. This is mirrored in kcat/K(m)GSH which increases with an apparent pKa value of 9.0, reflecting the ionization of the thiol group of glutathione in solution. The same results are obtained with 4-nitrophenethyl bromide as electrophilic substrate, although the K(m)GSH value (0.72 mM at pH 7.5), as well as the pKa (8.1) derived from the pH dependence of kcat/K(m)GSH, are lower with this substrate. In contrast, kcat and kcat/K(m)electrophile display either a maximum or a plateau at pH 7.0-7.5, and an apparent pKa value of 5.7 was determined for the pH dependence of kcat with both 4-nitrophenethyl bromide and 1,2-epoxy-3-(4-nitrophenoxy)propane as electrophilic substrates. This pKa value reflects an ionization of enzyme-bound GSH, most probably involving the sulfhydryl group, whose pKa value thus is lowered by the enzyme. Three differences in the cDNA as compared to the sequence previously published were found. One of these differences causes a change in the deduced amino acid sequence and involves the nucleotide triplet encoding amino acid 126, which was determined as GAG (Glu), instead of the published GGG (Gly).

Topics: Chromatography, Affinity; Cloning, Molecular; Epoxy Compounds; Escherichia coli; Glutathione; Glutathione Transferase; Humans; Hydrogen-Ion Concentration; Kinetics; Nitrobenzenes; Nitrophenols; Recombinant Proteins; Sequence Analysis, DNA; Substrate Specificity; Sulfhydryl Compounds

Topics: Epoxy Compounds; Female; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Kidney Cortex; Kidney Neoplasms; Male; Naphthalenes; Nitrophenols; Sex Characteristics

Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one(t-PBO), delta 5-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Brain; Butanones; Coturnix; Cytosol; Dinitrochlorobenzene; Electrophoresis, Polyacrylamide Gel; Epoxy Compounds; Ethacrynic Acid; Etiocholanolone; Female; Glutathione Transferase; Isoelectric Focusing; Kidney; Liver; Lung; Male; Molecular Sequence Data; Nitrobenzenes; Nitrophenols; Protease Inhibitors; Sequence Alignment; Sequence Homology, Amino Acid; Stilbenes; Tissue Distribution

An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C). The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++. An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

Topics: Amino Acid Sequence; Amino Acids; Aspartic Acid Endopeptidases; Bacillus; Enzyme Stability; Epoxy Compounds; Hot Temperature; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Nitrophenols; Norleucine; Protease Inhibitors; Sequence Analysis; Sequence Homology, Amino Acid; Substrate Specificity

The acute combined effects of cadmium (Cd) and nickel (Ni) on hepatic monooxygenase activities (ethylmorphine N-demethylase, EMND; aminopyrine N-demethylase, AMND; aniline 4-hydroxylase, AH), cytochrome P-450, cytochrome b5, microsomal heme and reduced glutathione (GSH) levels and glutathione S-transferase (GST) activities toward several substrates (1-chloro-2,4-dinitrobenzene, CDNB; 1,2-dichloro-4-nitrobenzene, DCNB; ethacrynic acid, EAA; 1,2-epoxy-3-(p-nitrophenoxy)-propane, ENPP) were determined and compared with those of Cd or Ni alone in mice. Male adult mice (25-30 g) were administered either a single dose of Cd (3.58 mg CdCl2.H2O/kg, i.p.) 48 hr prior to killing or a single dose of Ni (59.5 mg NiCl2.H2O/kg, s.c.) 16 hr prior to killing. For the combined treatment, the animals received the single dose of Ni 32 hr after the single dose of Cd and were then killed 16 hr later. Cd treatment alone significantly decreased EMND, AMND, and AH activities and cytochrome P-450 and heme levels as compared with controls. Cytochrome b5 level was not altered by Cd treatment. Cd also inhibited GSH level and the GST activities toward CDNB, EAA and ENPP significantly. No significant change was observed in the GST activity for DCNB by Cd. Ni treatment alone, however, decreased the monooxygenase and GST activities studied, and cytochrome P-450, cytochrome b5, heme and GSH levels significantly. Combined treatment significantly depressed the monooxygenase activities and cytochromes and heme levels. GSH level was not significantly altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aniline Hydroxylase; Animals; Cadmium; Cytochrome P-450 Enzyme System; Cytochromes b5; Dinitrochlorobenzene; Drug Synergism; Epoxy Compounds; Ethacrynic Acid; Ethylmorphine-N-Demethylase; Glutathione; Glutathione Transferase; Liver; Male; Mice; Mixed Function Oxygenases; Nickel; Nitrobenzenes; Nitrophenols; Protease Inhibitors; Substrate Specificity

In this study a polymorphism in the conjugating activity of human erythrocyte cytosol towards the dihaloethane, ethylene dibromide (EDB; 1,2-dibromoethane) was found. Two out of 12 human erythrocyte cytosols did not catalyze the formation of glutathione (GSH) conjugates of [1,2-14C]EDB. Ten cytosols formed the S,S'-ethylenebis(GSH) conjugate at a rate ranging from 0.5 to 3.2 (mean 1.76 +/- 0.95) pmol min-1 (mg protein)-1. The activity of the cytosols towards EDB was compared with the activity towards 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) and 1-chloro-2,4-dinitrobenzene (CDNB). The GSH conjugates formed from EDB, EPNP and CDNB were all quantified by HPLC. Every cytosol was active with the classical GST substrate CDNB (2.04 +/- 0.74 nmol min-1 (mg protein)-1). The two samples not showing any detectable activity towards EDB were also inactive towards EPNP: The activity towards EDB correlated significantly with EPNP (rs = 0.90, P < 0.005; Spearman's rank correlation), but not with CDNB (rs = 0.36, P > 0.10). In the incubations with EPNP, the alpha-, mu-, and pi- class glutathione S-transferase (GST) inhibitor S-hexyl(GSH) was included, indicating that the class-theta GST is the principal GST class conjugating EDB in erythrocyte cytosol. The apparent polymorphism of GST-theta which has recently been recognized to be crucial for several mono- and dihalomethanes, will thus also have considerable implications for the risk assessment of EDB.

Topics: Chromatography, High Pressure Liquid; Cytosol; Dinitrochlorobenzene; Epoxy Compounds; Erythrocytes; Ethylene Dibromide; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Kinetics; Nitrophenols; Polymorphism, Genetic

Topics: Acetophenones; Amino Acid Sequence; Animals; Birds; Enzyme Stability; Epoxy Compounds; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Nitrophenols; Norleucine; Pepsin A; Pepstatins; Protease Inhibitors; Thermodynamics

The irreversible inhibition of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and eight haloperidol derivatives has been studied. EPNP specifically inhibits HIV-1 and HIV-2 proteases with a stoichiometry of one EPNP molecule/dimeric enzyme. The site of modification of HIV-2 protease by EPNP has been unambiguously identified as Asp-25 using high performance tandem mass spectrometry. The haloperidol derivatives assayed consist of epoxides, ynones, and alpha,beta-unsaturated ketones. The Kinact values for these haloperidol derivatives range from 10.7 to 521 microM for HIV-1 protease and from 8.6 to 283 microM for the HIV-2 enzyme, being in some cases approximately 1000-fold more potent irreverisble inhibitors of HIV proteases than EPNP. This potency results from the haloperidol character of the compounds and the chemical reactivity of the groups capable of forming a covalent bond with the enzyme. Covalent modification of HIV-2 protease by a radiolabeled epoxide derivative of haloperidol, UCSF 84, is prevented by EPNP and the peptidomimetic transition state analog U-85548. In similar experiments, incorporation of UCSF 84 into HIV-1 protease is partially prevented by these active-site inhibitors. In contrast, a mutant HIV-1 protease, HIV-1 PR C95M, in which Cys-95 has been replaced by Met, is labeled 50% less than HIV-1 protease and is fully protected by EPNP and U-85548. These results indicate the presence of 2 reactive residues in HIV-1 protease: Cys-95 and another located in the active site of the enzyme. The alpha,beta-unsaturated ketone derivative of haloperidol, UCSF 191, which is stable over a broad pH range, was used to study the pH profile of inactivation of HIV-1 and HIV-2 proteases. Comparison of the profiles of inactivation of wild-type HIV-1 protease, HIV-1 PR C95M, and HIV-1 PR C67L as well as HIV-2 protease (which has no cysteine residues) reveals the contribution of Cys-95 to the reactivity of these irreversible inhibitors. The inhibitors UCSF 70, UCSF 84, UCSF 115, UCSF 142, and UCSF 191 reduce p55gag polyprotein processing when assayed in a mammalian cell line that produces HIV-1 viral particles lacking the envelope.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Epoxy Compounds; Haloperidol; HIV Protease Inhibitors; HIV-1; HIV-2; Humans; Molecular Sequence Data; Nitrophenols; Oligodeoxyribonucleotides

The detoxication of the enantiomers of glycidyl 4-nitrophenyl ether (GNPE), (-)-(R)- and (+)-(S)-GNPE, and glycidyl 1-naphthyl ether (GNE), (-)-(R)- and (+)-(S)-GNE, by rat liver glutathione transferase and epoxide hydrolase was studied. Enantioselectivity was observed with both enzymes favoring the (R)-isomers as determined by the formation of conjugate, diol, and remaining substrate measured by HPLC. Enantiomers of GNE were detoxified by cytosolic epoxide hydrolase but those of GNPE were not. Substantial nonenzymatically formed conjugates of enantiomers of GNPE were detected showing (S)-GNPE the more reactive of the pair.

Topics: Animals; Epoxide Hydrolases; Epoxy Compounds; Glutathione Transferase; Inactivation, Metabolic; Liver; Magnetic Resonance Spectroscopy; Male; Naphthols; Nitrophenols; Rats; Rats, Sprague-Dawley; Stereoisomerism; Structure-Activity Relationship

A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the alpha-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptide-based irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs.

Topics: Aspartic Acid; Aspartic Acid Endopeptidases; Binding Sites; Crystallography, X-Ray; Epoxy Compounds; Nitrophenols; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Simian Immunodeficiency Virus; Water

The effect of carbon monoxide (CO) on pulmonary glutathione-S-transferase (GST) activity was investigated using the isolated perfused rabbit lung (IPRL) preparation. CO at a concentration of 7.5% did not alter the conjugation of glutathione (GSH) with 1,2-epoxy-(p-nitrophenoxy)propane (ENP) in the IPRL. The rate of GSH conjugate formation with ENP was not altered when the lung was exposed to a selective inhibitor of gamma-glutamylcysteine synthetase, buthionine sulfoximine (0.1 mM), but was significantly diminished (P less than 0.01) by co-administration of GST inhibitor cibacron blue (1 microM) with ENP. These results indicate that the rabbit lung actively conjugates GSH with ENP and that this reaction may be decreased by specific inhibitors of GST. However, the rate of GSH conjugation is not affected by CO ventilation in the IPRL.

Topics: Administration, Inhalation; Animals; Antimetabolites; Buthionine Sulfoximine; Carbon Monoxide; Epoxy Compounds; Glutathione; Glutathione Transferase; Isoenzymes; Lung; Male; Methionine Sulfoximine; Nitrophenols; Perfusion; Protein Synthesis Inhibitors; Rabbits; Substrate Specificity; Triazines

The induction of pulmonary glutathione-S-transferase (GST) by ethanol was investigated using the isolated perfused rabbit lung (IPRL) preparation with particular attention paid to the duration and route of ethanol administration. For perfusion with buffer containing 0.2% ethanol or acute ethanol treatment (4 g/kg by gastric intubation) 4 h before the IPRL preparation, there were no differences in the rate of glutathione (GSH) conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) at low substrate concentrations (100-400 microM) but a decrease was observed in the rate at high substrate concentrations (500-1000 microM). Lungs from rabbits treated acutely showed the lowest maximal rate of GSH conjugation in the IPRL. Prolonged treatment with ethanol (10% in drinking water for 3 weeks) increased GSH conjugation (CDNB concentration of 300-750 microM). None of these ethanol treatments altered GSH conjugation with 1,2-epoxy(p-nitrophenoxy)propane (ENP). Upon termination of perfusion, there were no differences in pulmonary GSH concentration between control and ethanol-treated groups. Therefore, the effect of altered GSH level as a co-substrate on GST activity in lung might be excluded as an explanation for the effects of ethanol. These data suggest that ethanol has differential effects on GST activity depending upon the substrate and duration of ethanol administration.

Topics: Animals; Chromatography, High Pressure Liquid; Dinitrochlorobenzene; Enzyme Activation; Epoxy Compounds; Ethanol; Glutathione; Glutathione Transferase; Lung; Male; Nitrophenols; Rabbits; Substrate Specificity; Trypsin Inhibitors

Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease.

Topics: Aspartic Acid Endopeptidases; Binding Sites; Centrifugation, Density Gradient; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Epoxy Compounds; Escherichia coli; Gene Products, gag; HIV; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Molecular Weight; Nitrophenols; Oligopeptides; Peptide Fragments; Protease Inhibitors; Recombinant Proteins; Retroviridae Proteins; Substrate Specificity

Administration of phenobarbital, a known inducer of glutathione S-transferase activity in rat liver, failed to stimulate sulfobromophthalein (BSP) conjugation by liver cytosol in hamsters. The latter displayed poor ability to conjugate this substrate, despite very high glutathione-conjugating activity with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB). Of the six substrates tested, in this species, 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) was the only one whose conjugation was greatly enhanced by phenobarbital (+172%). Nevertheless, hamsters proved as responsive to phenobarbital induction as rats, since it increased their relative liver weight and microsomal enzyme activity. The deficient induction of liver BSP-conjugating activity observed with phenobarbital is consistent with the finding that it did not affect the hepatic transport of this substrate in hamsters.

Topics: Animals; Cricetinae; Dinitrochlorobenzene; Enzyme Induction; Epoxy Compounds; Glutathione; Glutathione Disulfide; Glutathione Transferase; Liver; Male; Mesocricetus; Nitrophenols; Phenobarbital; Sulfobromophthalein

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Topics: Blood Platelets; Dinitrochlorobenzene; Epoxy Compounds; Glutathione; Glutathione Transferase; Humans; Indomethacin; Isoelectric Point; Kinetics; Nitrobenzenes; Nitroglycerin; Nitrophenols

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.

Topics: Amino Acid Sequence; Aspartic Acid Endopeptidases; Binding Sites; Chromatography, High Pressure Liquid; Endopeptidases; Epoxy Compounds; Hydrolysis; Mitosporic Fungi; Nitrophenols; Peptide Fragments; Thermolysin

Female C57Bl/6J mice were given drinking water containing 0.05% propylthiouracil to induce a hypothyroid condition. Mitochondrial glycerol-3-phosphate dehydrogenase activity, used as an index of hypothyroidism, was 57.1 +/- 4.5 and 29.4 +/- 3.8 nmol/min per mg of protein for control and propylthiouracil-treated animals respectively. Administration of tri-iodothyronine resulted in an approx. 4.5-fold increase in dehydrogenase activity in propylthiouracil-treated animals. A dose-dependent increase in hepatic GSH S-transferase activity in propylthiouracil-treated animals was observed at tri-iodothyronine concentrations ranging from 2 to 200 micrograms/100 g body wt. This increase in transferase activity was seen only when 1,2-epoxy-3-(p-nitrophenoxy)propane was used as substrate for the transferase. Transferase activity with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrate was decreased by tri-iodothyronine. Administration of actinomycin D (75 micrograms/100 g body wt.) inhibited the tri-iodothyronine induction of transferase activity. Results of these studies strongly suggest that tri-iodothyronine administration markedly affected the activities of GSH S-transferase by inducing a specific isoenzyme of GSH S-transferase and suppressing other isoenzymic activities.

Topics: Animals; Dactinomycin; Enzyme Induction; Epoxy Compounds; Female; Glutathione Transferase; Hypothyroidism; Liver; Mice; Mice, Inbred C57BL; Nitrophenols; Propylthiouracil; Triiodothyronine

Glutathione S-transferase (GST) activity was measured in Daphnia magna and Ceriodaphnia reticulata using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as conjugation substrates. Levels of GST activity were comparable between species with CDNB; however, D. magna had nearly twice the GST activity with EA as compared to C. reticulata. GST activity with CDNB was elevated from exposure of daphnids to either CDNB or sodium pentachlorophenate (PCP), but not from exposure to EA. GST activity with EA could not be modulated from exposure to CDNB or EA. GST activity towards CDNB and EA was biochemically separated into different protein fractions suggesting the existence of two distinct isozymes. Preexposure of daphnids to CDNB or PCP increased the organisms' tolerance to the toxic effects of PCP, but not CDNB.

Topics: Animals; Daphnia; Dinitrochlorobenzene; Enzyme Induction; Epoxy Compounds; Ethacrynic Acid; Glutathione Transferase; Nitrobenzenes; Nitrophenols; Pentachlorophenol; Substrate Specificity

Two active site inhibitors of acid proteinases were tested for their effects on human neutrophil chemotaxis and lysosomal enzyme release. Both bromphenacyl bromide (BPAB) and epoxy-p-nitrophenoxypropane (EPNP) inhibited chemotaxis and chemotaxin-induced enzyme release elicited by pepstatin and formylmethionyl peptides, which share membrane receptors, and also by zymosan-activated serum, the major active component of which (C5a) occupies a different receptor. In contrast to the previously tested acid protease inhibitor diazoacetylnorleucine methyl ester, neither BPAB nor EPNP blocked binding of [3H]fMLP to neutrophils. Thus BPAB and EPNP inhibit chemotaxin-mediated neutrophil functions, but not by interaction with the chemotaxin receptor.

Topics: Acetophenones; Acid Phosphatase; Chemotaxis, Leukocyte; Dipeptides; Epoxy Compounds; Humans; Lysosomes; Muramidase; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitrophenols; Oligopeptides; Pepstatins; Protease Inhibitors

Continuous cell cultures derived from various tissues of rat, mouse, hamster and man were assayed for their glutathione (GSH) content and glutathione S-transferase activities. GSH S-transferase activities were monitored toward the substrates 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (PO). All cell lines tested contained appreciable amounts of GSH ranging from 10 to 65 nmol/mg cellular protein. Likewise, all cell lines expressed GSH S-transferase activities. However, the various cell lines differed considerably in their relative transferase activities exhibiting some degree of species-specificity.

Topics: Animals; Cell Line; Cricetinae; Dinitrochlorobenzene; Epoxy Compounds; Glutathione; Glutathione Transferase; Humans; Mice; Nitrobenzenes; Nitrophenols; Rats; Species Specificity; Substrate Specificity

The activity of glutathione-S-transferases for five electrophilic substrates was measured in ten organs of the rat. Highest activity was found in the liver and steroidogenic organs. The activity in the heart, brain, spleen and lung was relatively low or nil for most substrates.

Topics: Adrenal Glands; Animals; Brain; Dinitrobenzenes; Epoxy Compounds; Female; Glutathione Transferase; Hexachlorocyclohexane; Inactivation, Metabolic; Intestine, Small; Iodides; Kidney; Liver; Lung; Male; Nitrophenols; Ovary; Rats; Spleen; Sulfobromophthalein; Testis; Tissue Distribution

Topics: Amino Acids; Animals; Azo Compounds; Binding Sites; Epoxy Compounds; Kinetics; Mice; Nitrophenols; Pepstatins; Protein Binding; Renin; Submandibular Gland; Tetranitromethane

1. Rabbits and rats dosed with 1,2-epoxy-3-phenoxypropane excrete 2-hydroxy-3-phenoxypropionic acid and N-acetyl-S-(2-hydroxy-3-phenoxypropyl)-L-cysteine. 2. Rabbits and rats dosed with 1,2-epoxy-3-(p-nitrophenoxy)propane excrete 2-hydroxy-3-(p-nitrophenoxy)propionic acid, N-acetyl-S-[2-hydroxy-3-(p-nitrophenoxy/propyl)propyl]-L-cysteine and p-nitrophenol. 3. The administration of either epoxide to the rat produces a marked fall in hepatic GSH level. 4. The biliary excretion of metabolites of 1,2-epoxy-3-(p-nitrophenoxy)-propane is described.

Topics: Animals; Bile; Biotransformation; Chromatography; Epoxy Compounds; Ethers, Cyclic; Female; Glutathione; Liver; Nitrophenols; Phenols; Rabbits; Rats

Mucor miehei protease (EC 3.4.21 -- ), an acid protease of fungal origin, was rapidly inhibited at pH 5.0 and 10 degrees C by a 78-fold molar excess of diazoacetyl norleucine methyl ester (N2Ac-Nle-OMe) when simultaneously added with a 78-fold molar excess of Cu(II). Preincubation with Cu(II) before the addition of N2Ac-Nle-OMe reduced the initial rate of activity loss presumably due to a copper-induced structural change as deduced from an examination of CD spectra. Cof norleucine and 1.02 +/- 0.041 mol of copper. The conformation of the N2Ac-Nle-OMe-inhibited enzyme appeared to be somewhat altered since the rate of H-3H exchange determined for the slowest exchanging class of hydrogens was reduced by more than 10-fold although the estimated number of hydrogens in this class remained constant. Mucor miehei protease was also inhibited by pepstatin; complete inactivation required a 6-fold molar excess of inhibitor and was associated with a major conformational change as determined from CD spectra. Loss of activity also occurred in the presence of 1,2-epoxy-3-(p-nitrophenyoxy)propane (EPNP).

Topics: Aminocaproates; Azo Compounds; Copper; Epoxy Compounds; Kinetics; Mucor; Nitrophenols; Norleucine; Oligopeptides; Osmolar Concentration; Pepstatins; Protease Inhibitors; Spectrophotometry, Ultraviolet