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H W Dirr

Publications and source records attributed to H W Dirr.

17 recordsLinked to original sources
PubMed1996

Determination of a binding site for a non-substrate ligand in mammalian cytosolic glutathione S-transferases by means of fluorescence-resonance energy transfer.

To determine the location of the non-substrate-ligand-binding region in mammalian glutathione S-transferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-alpha glutathione S-transferase, and in a human Trp28-->Phe mutant class-pi glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-alpha and class-pi enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligand/protein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis [McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995) J. Mol. Biol. 246, 21-27]. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.

Binding Sites↗
PubMed1993

Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes.

The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Amino Acid Sequence↗
PubMed1992

Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi.

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.

Amino Acid Sequence↗
PubMed1991

Equilibrium unfolding of class pi glutathione S-transferase.

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.

Animals↗
PubMed1991

Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization.

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.

Amino Acid Sequence↗
PubMed1991

The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution.

The three-dimensional structure of class pi glutathione S-transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1-74) consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81-207) contains five alpha-helices. The dimeric molecule is globular with dimensions of about 55 A x 52 A x 45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S-transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.

Amino Acid Sequence↗
PubMed1991

Effect of aflatoxin B1 on human platelet protein kinase C.

1. Aflatoxin B1 serves as an activator for protein kinase C (PKC). 2. Following activation, the enzyme translocates from the cytosol to the particulate fraction. 3. Aflatoxin B1 leads to rapid and extensive phosphorylation of the known substrate of the enzyme. 4. Aflatoxin B1 causes rapid hydrolysis of phosphatidylinositol-4,5-biphosphate to diacylglycerol and inositol-1,4,5-triphosphate.

Aflatoxin B1↗
PubMed1988

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes.

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (Mr 48,000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of Mr 23,500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The Km values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I50) were as follows: 12 microM bromosulphophthalein, 34 microM S-hexylglutathione, 339 microM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (Ki = 8 microM) and glutathione (Kis = 4 microM; Kii = 11.5 microM) while S-hexylglutathione was competitive with glutathione (Ki = 5 microM).

Animals↗
PubMed1987

Characterization of the aflatoxin B1-binding site of rat albumin.

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.

Aflatoxin B1↗
PubMed1987

Solvent effects on the spectroscopic properties of aflatoxin B1.

1. Solvent-induced changes in the spectral properties of aflatoxin B1 were investigated using protic and aprotic solvents. 2. The absorption data were less sensitive to solvent effects than the fluorescence emission data. 3. Stokes shifts in protic solvents were greater than those in aprotic solvents indicating hydrogen bond formation between solvent and the excited state of aflatoxin B1. 4. From the Stokes shift data for aprotic solvents, the dipole moment of aflatoxin B1 was estimated to increase by 15.7 Debye units upon excitation to the excited singlet state.

Aflatoxin B1↗
PubMed1987

Intracellular aflatoxin B1-binding proteins in rat liver.

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.

Aflatoxin B1↗
PubMed1987

Effects of hydrogen ion and fatty acid concentrations on the binding of aflatoxin B1 to human albumin.

The influence of pH and long-chain fatty acids on the interaction between aflatoxin B1 and human albumin was investigated by fluorescence spectroscopy. Both the binding of aflatoxin B1 to albumin and the fluorescence of albumin-bound aflatoxin are pH-dependent over the pH range of 6-9.5. The data indicates that the carcinogen has a higher affinity for the basic(B) than for the neutral(N) conformation of human albumin. Palmitic, stearic and oleic acids up to a molar ratio of 2 over albumin, increases the binding strength of aflatoxin B1 by means of an allosteric mechanism. Furthermore, the pH-dependence of the aflatoxin-albumin interaction is affected by the presence of oleic acid by narrowing the pH range over which the dependence occurs. At molar ratios of oleic acid to albumin in excess of 4.25 at pH6, 3.1 at pH7.4 and 2.4 at pH9 cause a decrease in aflatoxin B1 fluorescence as a result of reduced binding to albumin.

Aflatoxin B1↗
PubMed1987

Glutathione S-transferase composition of rat erythrocytes.

With 1-chloro-2,4-dinitrobenzene as the electrophilic substrate, the specific activity of glutathione S-transferase in rat haemolysates was found to range from 0.002 to 0.013 mumol/min/mg haemoglobin at 30 degrees C. To establish the glutathione S-transferase composition, chromatofocusing was used which indicated the presence of a single soluble isoenzyme with an apparent pI of 6.1. A molecular weight of 48,000 was determined for the enzyme by gel filtration. The transferase enzyme in intact erythrocytes is shown to catalyze the formation of S-(2,4-dinitrophenyl)-glutathione from 1-chloro-2,4-dinitrobenzene and endogenous glutathione. Efflux of this conjugate from erythrocytes proceeded at a rate of 13 nmol/min/ml at 37 degrees C.

Animals↗
PubMed1986

Aflatoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction.

Binding of [3H]aflatoxin B1 to rat plasma was investigated in vivo and in vitro. Column chromatographic and polyacrylamide gel electrophoretic analyses clearly demonstrated that aflatoxin B1 bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B1 binds an apolar site with an association constant of 30 mM-1 at pH 7.4 and 20 degrees C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had any significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (delta H = -29.3 kJ X mol-1), with a delta S value of -13.8 J X mol-1 X K-1.

Aflatoxin B1↗
PubMed1986

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

Amino Acids↗