The many faces of glutathione transferase pi

Glutathione transferase Pi (GST-pi, GSTP) is known to strongly affect human susceptibility to several cancers, asthma and neurodegenerative disorders. As with other glutathione transferases, it catalyses the addition of reduced glutathione to electrophilic species, and it is important in metabolite detoxification. It also was shown to bind proteins and compounds containing iron and nitric oxide. Some of these interactions have developed in the course of evolution into regulatory pathways that back up the GST's most ancient catalytic functions and provide precise and diverse responses to chemical and redox stresses. An aim of this review is to summarise recent knowledge on GSTP's complementary functions in crosstalking pathways of conventional glutathione transfer, nitric oxide and lipid metabolism and ASK1-dependent stress response. This review will describe how these complex interactions affect regulation of cell respiration, biosynthesis of lung surfactant, organism's immunity and circadian rhythms. Integration of the data leads to a new interpretation of the role of GSTP in normal human physiology, pathology and an organism's susceptibility to diseases.     

Studies on interactions between plant secondary metabolites and glutathione transferase using fluorescence quenching method

The interactions between plant secondary metabolites (tannic acid, rutin, cinnamic acid and catechin) and glutathione transferase (GST) were investigated by fluorescence and UV-Vis absorption spectroscopy. Intrinsic fluorescence of GST was measured by selectively exciting their tryptophan (Trp) residues and quenching constants were determined using the Stern-Volmer equation. The binding affinity was found to be strongest for tannic acid and ranked in the order tannic acid>rutin>cinnamic acid>catechin. The pH values in the range of 6.7-7.9, except for tannic acid, did not affect significantly the affinity of rutin, cinnamic acid and catechin with GST. Results showed that the fluorescence quenching of GST was a static_quenching. Fluorescence quenching and UV-Vis absorption spectroscopy suggested that only the tannic acid changed the microenvironment of the Trp residues. Furthermore, the number of binding sites and binding constants at different pH values showed that tannic acid had strongest affinity towards GST and hydrogen bonding played an important role in the affinity between GST and the metabolites.     

Specific recognition of human pi glutathione transferase by an antipeptide antibody

Antibodies were raised in a rabbit by using a 12-residue synthetic peptide, corresponding to fragment 2-13 of rat placental glutathione S-transferase, as the immunogen. The antiserum appeared to react with the fragment as well as with the corresponding human enzyme (GST-pi), which shares with the rat transferase a 92% sequence homology at the N terminus. In addition, the binding of the antibody to the protein was completely inhibited by small amounts of peptide. The enzymatic activity of glutathione transferase was not affected by the antibody. This might indicate that the N-terminal fragment is not involved in the catalytic activity of the enzyme. This antibody of predetermined specificity might thus find a useful application for the detection and approximate quantitation of this marker in human preneoplastic lesions.     

Investigation of the active site of human placenta glutathione transferase pi by means of a spin-labelled glutathione analogue.

A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.     

Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements.

The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins.     

Intrinsic tryptophan fluorescence of membranes of GH3 pituitary cells: quenching by thyrotropin-releasing hormone.

The intrinsic tryptophan fluorescence of membranes prepared from the GH₃ strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH₃ cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH₃ membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH₃ membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH₃ membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH₃ membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.     

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.     

Chromosomal localization of human glutathione transferase genes of classes alpha,mu and pi

Summary The numerous human glutathione transferases may be divided into three classes, mu, alpha and pi. Using a panel of human-rodent somatic cell hybrids and DNA probes specific for each of the three classes, we have mapped a class mu gene to chromosome 3, a class alpha gene to chromosome 6 and a class pi gene to chromosome 11. The two latter assignments confirm earlier reports, whereas the assignment of the class mu gene represents a new addition to the human gene map.     

Suppression of glutathione transferase P expression by glucocorticoid.

A strong enhancer element, GPEI, of the glutathione transferase P gene (GST-P) gene is composed of two phorbol 12-O-tetradecanoate 13-acetate (TPA) responsive element (TRE)-like sequences at opposite orientation. Unlike TRE sequences of other genes, GPEI exhibits a strong enhancer activity in F9 cells, which contains little AP-1. GPEI bound to AP-1 In vitro and GST-P expression was activated by TPA and exogenously introduced c-jun gene in a rat fibroblast cell line. Both the stimulated expression of GST-P gene by TPA and that by over-expressed c-Jun were suppressed to the basal level by dexamethasone, an inhibitor of AP-1. Basal expression of GST-P gene, however, was not inhibited by dexamethasone. Transfected chloramphenicol acetyltransferase (CAT) gene having GPEI also behaved as the endogenous GST-P gene. These results indicate that the GPEI is activated by AP-1 but constitutive activity of this enhancer in a rat fibroblast cell line 3Y1 cells is due to some unknown mechanism other than AP-1.     

Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione.

In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested. Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues. A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity. A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose. Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.     

Accessibility of adenine binding sites in dehydrogenases to small molecules studied by fluorescence quenching.

Quenching of the fluorescence of ethenoadenine derivatives by iodide ions and by methionine was studied in solution and when the nucleotides were bound to several dehydrogenases. The fluorescence of epsilonADPR in neutral aqueous solution is dynamically quenched by both quenching agents. The quenching of free epsilonNAD+ by methionine was found to be predominantly static and was satisfactorily described to result from complex formation between quencher and dinucleotide. The rat constant for quenching by iodide of epsilonNAD+ in the ternary complex with LADH and pyrazole is comparable to that of free epsilonADPR or epsilonADP. it is concluded that the bound epsilon-adenine ring is partially exposed to the solvent. The opening, to the solvent, of the adenine binding site is not large enough to allow free methionine diffusion since the rate constant for quenching of bound coenzyme by this quenching agent is relatively small. The difference between the rate constants for quenching of free and enzyme bound nucleotide was used to evaluate the binding constants of epsilonADPR to GPDH, epsilonNAD+ to LDH, and oxalate to the LDH:epsilonNAD+ complex. This technique may prove to be particularly useful when the binding of a fluorescent ligand to a protein is not accompanied by significant changes in its fluorescence.     

Inhibition of glutathione transferase pi from human placenta by 1-chloro-2,4-dinitrobenzene occurs because of covalent reaction with cysteine 47.

Human placenta glutathione transferase pi is irreversibly inhibited when incubated with 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the cosubstrate glutathione. The enzyme is protected against CDNB inactivation by the presence of S-methylglutathione and glutathione. The kinetics of inactivation is pseudo-first-order with k(obs) = 0.04 min-1 when 44 microM enzyme is incubated in presence of 1 mM CDNB at pH 6.5. The inhibition is saturable with respect to the CDNB concentration and the enzyme-CDNB complex shows a K(i) = 2.7 mM. Concomitant to the inhibition process is formation of an absorption band at 340 nm. After trypsin digestion and HPLC analysis, the CDNB-reacted enzyme gives a single peptide absorbing at 340 nm. Automated Edman degradation of this peptide indicates cysteine 47 to be the residue alkylated by CDNB.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Microsomal glutathione transferase. Primary structure

The primary structure of rat liver microsomal glutathione transferase has been determined. The 14C-carboxymethylated protein was fragmented with CNBr and proteolytic enzymes. The basis of the analysis was information from sequenator degradations of the intact protein, the largest CNBr fragment, and a large COOH-terminal fragment derived from a digest with Glu-specific staphylococcal protease. Remaining, smaller fragments were analyzed with the manual dimethylaminoazobenzene isothiocyanate method. Pepsin and limited acid hydrolysis were used to obtain peptides to confirm and overlap hydrophobic structures in the COOH-terminal half of the protein where trypsin and chymotrypsin failed to give any cleavage. Combined, these data permit the deduction of a 154-residue amino acid sequence. No evidence for micro-heterogeneity was obtained. The NH2-terminal alanine residue has a free alpha-amino group and the cysteine residue involved in activation of the enzymatic activity by sulfhydryl reagents is at position 49. The protein chain contains three regions with predictions for long beta strand secondary structures (positions 11-26, 103-120, and 131-145). Predictions may be inaccurate in membrane-associated proteins, but two of these regions also affect the three most hydrophobic segments. Thus, residues 11-35 form a long, largely hydrophobic part interrupted by only one charged residue (Lys-25), and residues 81-97 and 114-126 constitute the most hydrophobic segments directly noticeable from the hydrophilicity curve of the protein chain. These special parts of the molecule are of interest in relation to membrane interactions.     

Intrinsic fluorescence studies on saccharide binding toArtocarpus integrifolia lectin

The combining region ofArtocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The -anorner of galactose binds more strongly than its -counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin.The lectin binds to the Thomsen-Friedenreich antigen (Gal(13)GalNAc) more strongly than the other disaccharides studied, viz. Gal/ (14) Gal and Gal (13) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region ofArtocarpus lectin is complementary to that of T-antigen.Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.Abbreviations MeGal 1-O-methyl--glucopyranoside - MeGal 1-O-methyl--glucopyranoside - GalNAc 2-acetamido-2-deoxy-galactose - Gal Galactose     

Activation of rat liver microsomal glutathione transferase by limited proteolysis.

The activity of rat liver microsomal glutathione transferase is increased by limited tryptic proteolysis; the membrane-bound and purified forms of the enzyme are activated about 5- and 10-fold respectively. The cleavage sites that correlate with this activation were determined by amino acid sequence analysis to be located after Lys-4 and Lys-41. Differences in the relative extent of cleavage at these two sites did not consistently affect the degree of activation. Thus the data support the conclusion that cleavage at either site results in activation. The trypsin-activated enzyme was compared with the form activated with N-ethylmaleimide, which modifies Cys-49. These two differently activated forms were found to have similar kinetic parameters, which differ from those of the unactivated enzyme. The relatedness of the two types of activation is also demonstrated by the observation that microsomal glutathione transferase fully activated by N-ethylmaleimide is virtually resistant to further activation by trypsin. This is the case despite the fact that the N-ethylmaleimide-activated enzyme is much more susceptible to trypsin cleavage at Lys-41 than is the untreated enzyme. The latter observation indicates that activation with N-ethylmaleimide is accompanied by a conformational change involving Lys-41.     

Identification of tyrosine 79 in the tocopherol binding site of glutathione S-transferase pi

Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, k(obs), exhibits a nonlinear dependence on reagent concentration, with K(I) = 19 microM and k(max) = 0.158 min(-)(1). Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on k(obs). These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr(79) is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr(79) to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in K(I) values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.