Biomedical subjects

Philip G Board

Publications and source records attributed to Philip G Board.

34 records · Page 2Linked to original sources

PubMed2004

Mice deficient in glutathione transferase zeta/maleylacetoacetate isomerase exhibit a range of pathological changes and elevated expression of alpha, mu, and pi class glutathione transferases.

Glutathione transferase zeta (GSTZ1-1) is the major enzyme that catalyzes the metabolism of alpha-halo acids such as dichloroacetic acid, a carcinogenic contaminant of chlorinated water. GSTZ1-1 is identical with maleylacetoacetate isomerase, which catalyzes the penultimate step in the catabolic pathways for phenylalanine and tyrosine. In this study we have deleted the Gstz1 gene in BALB/c mice and characterized their phenotype. Gstz1(-/-) mice do not have demonstrable activity with maleylacetone and alpha-halo acid substrates, and other GSTs do not compensate for the loss of this enzyme. When fed a standard diet, the GSTZ1-1-deficient mice showed enlarged liver and kidneys as well as splenic atrophy. Light and electron microscopic examination revealed multifocal hepatitis and ultrastructural changes in the kidney. The addition of 3% (w/v) phenylalanine to the drinking water was lethal for young mice (<28 days old) and caused liver necrosis, macrovesicular steatosis, splenic atrophy, and a significant loss of circulating leukocytes in older surviving mice. GSTZ1-1-deficient mice showed constitutive induction of alpha, mu, and pi class GSTs as well as NAD(P)H:quinone oxidoreductase 1. The overall response is consistent with the chronic accumulation of a toxic metabolite(s). We detected the accumulation of succinylacetone in the serum of deficient mice but cannot exclude the possibility that maleylacetoacetate and maleylacetone may also accumulate.

Acetone↗

PubMed2004

Glutathione transferase zeta-catalyzed bioactivation of dichloroacetic acid: reaction of glyoxylate with amino acid nucleophiles.

Dichloroacetic acid (DCA) is a drinking water contaminant, a therapeutic agent, and a rodent carcinogen. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloalkanoates and the cis-trans isomerization of maleylacetoacetate. GSTZ1-1 catalyzes the bioactivation of fluorine-lacking dihaloacetates to S-(alpha-halocarboxymethyl)glutathione, a reactive intermediate that covalently modifies and inactivates the enzyme or is hydrolyzed to glyoxylate. The purpose of this study was to examine the GSTZ1-1-catalyzed bioactivation of DCA, including the reaction of DCA-derived glyoxylate with amino acid nucleophiles and the characterization of the structures and kinetics of adduct formation by LC/MS. The binding of [1-(14)C]DCA-derived label to bovine serum albumin required both GSTZ1-1 and GSH, whereas the binding to dialyzed rat liver cytosolic protein was increased in the presence of GSH. Studies with model peptides (antiflammin-2 and IL-8 inhibitor) indicated that glyoxylate, rather than S-(alpha-chlorocarboxymethyl)glutathione, was the reactive species that modified amino acid nucleophiles. Both addition (+74 Da) and addition-elimination (+56 Da) adducts of glyoxylic acid were observed. Addition adducts (+74 Da) could not be characterized completely by mass spectrometry, whereas addition-elimination adducts (+56 Da) were characterized as 2-carboxy-4-imidazolidinones. 2-Carboxy-4-imidazolidinones were formed by the rapid equilibrium reaction of glyoxylate with the N-terminal amino group of antiflammin-2 to give an intermediate carbinolamine (K(eq) = 0.63 mM(-1)), which slowly eliminated water to give an intermediate imine (k(2) = 0.067 hour(-1)), which rapidly cyclized to give the 2-carboxy-4-imidazolidinone. Glucose 6-phosphate dehydrogenase was inactivated partially by glyoxylate when reactants were reduced with sodium borodeuteride, which may indicate that glyoxylate reacts with selective lysine epsilon-amino groups. The results of the present study demonstrate that GSTZ1-1 catalyzes the bioactivation of DCA to the reactive metabolite glyoxylate. The reaction of glyoxylate with cellular macromolecules may be associated with the multiorgan toxicity of DCA.

Acetoacetates↗

PubMed2004

Functional polymorphism of human glutathione transferase A2.

OBJECTIVES: Single nucleotide polymorphisms that cause amino acid substitutions in enzymes involved in the metabolism of xenobiotics can potentially have a significant effect on the efficacy and safety of therapeutic drugs. METHODS: We have utilized a bioinformatic approach to identify new polymorphisms in the glutathione transferase super family. RESULTS AND CONCLUSIONS: In this report we describe a P110S polymorphism in GSTA2 that occurs at a low frequency in Africans, Chinese and Europeans. The serine containing isoform has significantly diminished activity with a range of substrates including, delta-Androsten-3,17-dione, 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. The activity with cumene hydroperoxide may reflect a diminished physiological function since the glutathione peroxidase activity of GSTA2-2 plays a role in prostaglandin synthesis. In contrast, activity with p-nitrophenol acetate was significantly elevated. The position of this polymorphism in the active site and its effects on model substrates suggest that further investigation of its capacity to conjugate alkylating drugs is warranted.

Africa↗

PubMed2004

Functional polymorphism of human glutathione transferase A3: effects on xenobiotic metabolism and steroid biosynthesis.

The alpha class glutathione transferase GSTA3-3 is involved in steroid biosynthesis and the metabolism of some xenobiotics. A bioinformatics approach was utilized to identify novel coding region polymorphisms in the glutathione transferase A3 gene (GSTA3). We describe an I71L polymorphism in GSTA3 that occurs at a low frequency in African populations. The activity of the leucine containing isoform was significantly reduced in a range of glutathione-conjugating reactions due to a diminished affinity for reduced glutathione, indicating that this allele could be implicated in disease caused by oxidative stress in steroidogenic tissue. By contrast, the delta(5)-androsten-3,17-dione isomerase activity of GSTA3-3 was not affected by this substitution, indicating that there is no direct effect on steroid synthesis. However, the L71 isoform displayed diminished stability at 45 degrees C. If this relative instability is mirrored in vivo, testosterone and progesterone synthesis may be affected in individuals carrying this allele.

Africa↗

PubMed2004

An efficient system for high-level expression and easy purification of authentic recombinant proteins.

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.

Base Sequence↗

PubMed2003

Clarification of the role of key active site residues of glutathione transferase zeta/maleylacetoacetate isomerase by a new spectrophotometric technique.

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(+/-)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16-->Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the K(m) for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of alpha-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the alpha-halo acid substrates.

Amino Acids↗

PubMed2003

Characterization of the human Omega class glutathione transferase genes and associated polymorphisms.

The Omega class glutathione transferases (GSTs) have been identified in many organisms, including human, mouse, rat, pig, Caenorhabditis eglands and Drosophila melanogaster. These GSTs have poor activity with common GST substrates, but exhibit novel glutathione-dependent thioltransferase, dehydroascorbate reductase and monomethylarsonate reductase activities, and modulate Ca release by ryanodine receptors. An investigation of the genomic organization of human GSTO1 identified a second actively transcribed member of the Omega class (GSTO1). Both GSTO1 and GSTO2 are composed of six exons and are separated by 7.5 kb on chromosome 10q24.3. A third sequence that appears to be a reverse-transcribed pseudogene (GSTO3p) has been identified on chromosome 3. GSTO2 has 64% amino acid identity with GSTO1 and conserves the cysteine residue at position 32, which is thought to be important in the active site of GSTO1. Expression of GSTO2 mRNA was seen in a range of tissues, including the liver, kidney, skeletal muscle and prostate. The strongest GSTO2 expression was in the testis, which also expresses a larger transcript than other tissues. Characterization of recombinant GSTO2 has been limited by its poor solubility. Two functional polymorphisms of GSTO1 have been identified. One alters a splice junction and causes the deletion of E155 and another results in an A140D substitution. Characterization of these variants revealed that the A140D substitution affects neither heat stability, nor activity towards 1-chloro-2,4-dinitrobenzene or hydroxyethyl disulphide. In contrast, deletion of residue E155 appears to contribute towards both a loss of heat stability and increased enzymatic activity.

Amino Acid Sequence↗

PubMed2002

Alphavirus 6K proteins form ion channels.

Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40-800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.

Alphavirus↗

PubMed2002

Identification and characterization of GSTT3, a third murine Theta class glutathione transferase.

A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate.

Amino Acid Sequence↗

PubMed2002

From glutathione transferase to pore in a CLIC.

Many plasma membrane chloride channels have been cloned and characterized in great detail. In contrast, very little is known about intracellular chloride channels. Members of a novel class of such channels, called the CLICs (chloride intracellular channels), have been identified over the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water-soluble state as well as a membrane-bound state. A major step forward in understanding the functioning of these channels has been the recent crystal structure determination of one family member, CLIC1. The structure confirms that CLICs are members of the glutathione S-transferase superfamily and provides clues as to how CLICs can insert into membranes to form chloride channels.

Amino Acid Sequence↗

PubMed2002

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians.

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Nonrandom associations were found between the BamHI, EcoRI and BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the individuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms should prove useful for other population and forensic studies.

Alleles↗

PubMed2002

A semisynthetic glutathione peroxidase with high catalytic efficiency. Selenoglutathione transferase.

Glutathione peroxidase (GPX) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides by glutathione (GSH). GPX therefore has potential therapeutic value as an antioxidant, but its pharmacological development has been limited because GPX uses a selenocysteine as its catalytic group and it is difficult to generate selenium-containing proteins with traditional recombinant DNA technology. Here, we show that naturally occurring proteins can be modified to generate GPX activity. The rat theta-class glutathione transferase T2-2 (rGST T2-2) presents an ideal scaffold for the design of a novel GPX catalyst because it already binds GSH and contains a serine close to the substrate binding site, which can be chemically modified to bind selenium. The modified Se-rGST T2-2 efficiently catalyzes the reduction of hydrogen peroxide, and the GPX activity surpasses the activities of some natural GPXs.

Antibodies, Monoclonal↗

PubMed2002

Kinetics of the biotransformation of maleylacetone and chlorofluoroacetic acid by polymorphic variants of human glutathione transferase zeta (hGSTZ1-1).

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate and the biotransformation of a range of alpha-haloacids. The objective of this study was to determine the kinetics of the biotransformation of maleylacetone (MA), an analogue of the natural substrate maleylacetoacetate, and chlorofluoroacetic acid (CFA) by polymorphic variants of recombinant hGSTZ1-1. The k(cat) of the four variants of hGSTZ1-1 with MA as the substrate followed the order: 1c-1c > 1b-1b > 1d-1d > 1a-1a whereas the k(cat) for the biotransformation of CFA followed the order: 1a-1a > 1b-1b approximately 1c-1c approximately 1d-1d. The turnover rates of MA were much higher than those of CFA for each variant and ranged from 22-fold (1a-1a) to 980-fold differences (1c-1c). The catalytic efficiencies of hGSTZ1-1 variants with MA as the substrate were much greater than those with CFA as the substrate, but little difference among the polymorphic variants was observed. MA was a mixed inhibitor of all variants with CFA as substrate: the mean competitive inhibition constant (K(ic)(MA)) for all variants was about 100 microM, and the mean uncompetitive inhibition constant (K(iu)(MA)) was about 201 microM. Hence, MA and alpha-haloacids apparently compete for the same active site on the enzyme. DCA-induced inactivation of the four variants showed that the inactivated enzymes show markedly reduced isomerase activities. The residual activities were different for each variant: 1a-1a (12%) > 1b-1b approximately 1c-1c approximately 1d-1d (<5%). This is the first kinetic analysis of polymorphic variants of hGSTZ1-1, and the similarity of the kinetic constants for hGSTZ1-1 variants with either MA or CFA as substrates indicates that few differences in DCA-induced perturbations of tyrosine metabolism would likely be observed in humans.

Acetates↗

PubMed2002

Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone.

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.

Acetone↗

PubMed2002

Mass spectral characterization of dichloroacetic acid-modified human glutathione transferase zeta.

Glutathione transferase zeta (GSTZ1-1) is widely expressed in eukaryotic species, and four human allelic variants of hGSTZ1-1 have been described. GSTZ1-1 catalyzes the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate and the biotransformation of a range of alpha-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking alpha,alpha-dihaloalkanoic acids, including dichloroacetic acid (DCA), results in the mechanism-based inactivation and covalent modification of the enzyme. The objective of this study was to investigate further the DCA-induced inactivation of hGSTZ1c-1c and to explore the mechanism of inactivation by characterization of the sites and types of DCA-induced covalent modifications. The partition ratio for the DCA-induced, mechanism-based inactivation of hGSTZ1c-1c was (5.7 +/- 0.5) x 10(2), and the k(cat) for the biotransformation of DCA was 39 min(-)(1). Inactivation of hGSTZ1c-1c in vitro was limited at high enzyme concentrations and was inhibited by glyoxylate. The stoichiometry of DCA binding to hGSTZ1c-1c was approximately 0.5 mol of DCA/mol of enzyme monomer. A single DCA-derived adduct was observed and was assigned to cysteine-16 by a combination of matrix-assisted laser-desorption-ionization time-of-flight and electrospray-ionization quadrupole ion-trap mass spectrometry and by analysis of [1-(14)C]DCA binding to C16A hGSTZ1c-1c. The DCA-derived adduct contained both glutathione and the carbon skeleton of DCA, presumably in a dithioacetal linkage. Also, cysteine-16 formed a mixed disulfide bond with glutathione. These data support a mechanism of inactivation whereby glutathione displaces a chlorine atom from DCA, and cysteine-16 in the enzyme active site displaces the second chlorine atom to result in a covalently modified and inactivated enzyme. These findings explain the DCA-induced inactivation of GSTZ1-1 observed in humans and rats.

Binding Sites↗

PubMed2002

Immunohistochemical localization and activity of glutathione transferase zeta (GSTZ1-1) in rat tissues.

Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 +/- 17 (liver) to 1.1 +/- 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 +/- 0.89 (liver) to 0.09 +/- 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism.

Acetates↗