Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limited proteolysis of glutathione transferase P1-1 (GSTP1-1) by chymotrypsin performed at 20 degrees and 30 degrees C mainly generates two complementary peptides of 17 kDa and 6 kDa molecular mass with concomitant loss of catalytic capacity. Sequence analysis of these peptides showed that the peptide bond between Tyr47 and Gly48 was cleaved. The analysis of the recently resolved three-dimensional structure of GSTP1-1 [Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federici, G. & Parker, M. W. (1992) J. Mol. Biol. 227, 214-226] suggests that the proteolytically cleaved bond results located in a portion of the polypeptide chain lining the G-site which has been demonstrated to be part of an exposed and flexible region of the N-terminal domain (structural elements alpha B1 and alpha B2) [Aceto, A., Caccuri, A. M., Sacchetta, P., Bucciarelli, T., Dragani, B., Rosato, N., Federici, G. & Di Ilio, C. (1992) Biochem. J. 285, 241-245]. The fragments which are generated by proteolysis at 20 degrees C, remain linked by noncovalent interaction in a complex (nicked GSTP1-1) which is dissociated by incubation at higher temperatures. As shown by circular dichroic analysis, although inactive, nicked GSTP1-1 retains an overall secondary structure closely resembling that of the parent enzyme. However, the fluorescence data of the nicked GSTP1-1 indicate that the Trp38, which is near the chymotrypsin-cleavable bond, becomes exposed in a more polar environment. This indicates that, in the nicked enzyme, the polypeptide portion containing the structural elements alpha B1 and alpha B2 has more freedom of fluctuation. The fact that this polypeptide chain portion contains two essential amino acid residues of the G-site (Trp38 and Lys42) might account for the loss of ability to bind glutathione by the nicked enzyme which is consequently catalytically inactive. Proteolysis performed at 30 degrees C generated a homodimeric 17-kDa fragment. The structural analysis of this fragment suggests that the GSTP1-1 alpha C helix, which is located in the domain I and is thought to be involved in the inter-domain interaction, could exert a critical role in maintaining the native folding of domain II.
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PMID:Investigation of intra-domain and inter-domain interactions of glutathione transferase P1-1 by limited chymotryptic cleavage. 828 36

We have developed Salmonella typhimurium strains expressing human glutathione S-transferases (GSTs) to establish the role of these enzymes in chemical activation and deactivation. Alpha and pi class GSTs, GSTA1-1 and GSTP1-1, were expressed in Salmonella TA100 using a regulatable tac promoter expression system. The ability of these GST to modulate the mutagenicity of a range of mutagens including ethylene dibromide, ethylene dichloride and methylene dichloride was then investigated. Ethylene dibromide, ethylene dichloride and methylene dichloride were directly mutagenic in the control TA100 strain. The mutagenicity of ethylene dibromide and ethylene dichloride was increased in cells expressing GSTA1-1, but not in cells expressing GSTP1-1. In contrast, methylene dichloride mutagenicity was unaffected by the presence of either GST. The mutagenicity of 2-aminofluorene, was not altered by the presence of either GST isozyme, while that of N-hydroxy-2-acetylaminofluorene was slightly reduced with both isozymes. The mutagenicity of aflatoxin B1 (AFB1) was marginally decreased in strains expressing GSTP1-1. When GSTA1-1 expression was maximally induced, however, a more pronounced reduction was observed suggesting a role for GSTA1-1 in AFB1 deactivation. The tester strains described here should be valuable in establishing the specificity of human GST isozymes towards chemical toxins and carcinogens, especially for compounds whose reactive intermediates are short lived.
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PMID:Human glutathione S-transferase-expressing Salmonella typhimurium tester strains to study the activation/detoxification of mutagenic compounds: studies with halogenated compounds, aromatic amines and aflatoxin B1. 833 Mar 52

Recently, Bora et al. (Bora, P. S., Bora, N. S., Wu, X., and Lange, L. G. (1991) J. Biol. Chem. 266, 16774-16777) reported the cloning and expression of a human fatty acid ethyl ester synthase III (FAEES-III) cDNA that has only four amino acid substitutions compared with human glutathione S-transferase (GST) GSTP1-1, and, when expressed in MCF-7 cells, the protein has both FAEES and GST activities. By site-directed mutagenesis of a GSTP1 cDNA, we have constructed a clone that encodes the FAEES-III protein described by Bora et al. (1991). The recombinant FAEES-III protein was expressed in Escherichia coli and has been shown to be devoid of FAEES and GST activities. The recombinant FAEES-III protein does not bind to a glutathione agarose affinity matrix, presumably because two of the substituted amino acids, Trp-39-->Cys and Gln-52-->Glu, are thought to contribute to the GST glutathione binding site. One of the base substitutions in the FAEES-III cDNA encodes an extra SacI site not found in the GSTPI cDNA. Polymerase chain reaction amplification of human genomic DNA has identified the GSTPI gene, but no DNA from the proposed FAEES gene with a diagnostic SacI site has been detected. Evaluation of the hybridization pattern of HindIII genomic restriction fragments has identified fragments that contain the GSTPI gene and a pseudogene (Board et al. 1992), and there do not appear to be any hybridizing fragments that could contain the FAEES-III gene. Our results do not provide any evidence in support of a relationship between FAEES-III and GST, and the cDNA reported by Bora et al. (1991) may have resulted from a cloning artifact.
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PMID:Evidence against a relationship between fatty acid ethyl ester synthase and the Pi class glutathione S-transferase in humans. 834 Mar 90

High levels of expression of GSTP1-1 are associated with cell proliferation, embryogenesis and malignancy. Given the role of glutathione S-transferase (GST) in detoxication, it is possible that GSTP1-1 evolved specifically to protect proliferating cells and share regulatory mechanisms with other cellular genes which are involved in cell division and tumorigenesis. We have previously shown that the expression of GSTP1 is suppressed by retinoic acid (RA) in the presence of the retinoic acid receptor (RAR) as a result of decreased transcription from its promoter. Through deletion analysis, we show here that the RA-RAR-dependent repression is mediated by the region -73 to +8. Further mutation analysis of this region indicates that the DNA sequence required for RA-RAR-dependent repression co-localizes with a consensus activator protein-1 (AP1) site essential for the promoter activity. The degree of repression correlates with the residual activity of the AP1 site. There are two adjacent G/C boxes. The one immediately downstream from the AP1 site is not essential for the promoter activity, but mutation of the second, further downstream, impairs the promoter. On the other hand, mutation of either of these two G/C boxes has little effect on RA-RAR suppression. We also show that the expression of GSTP1 is regulated by the redox status of the cell. Using the chloramphenicol acetyltransferase assay system, we have demonstrated that treatment with H2O2 induced transcription from the promoter and that this effect can be blocked by pre-incubation with N-acetylcysteine (NAC). It was shown that the induction by H2O2 is mediated by trans-acting factor NF-kappa B (nuclear factor kappa B), via a putative NF-kappa B site, 'GGGACCCTCC', located from -96 to -86. Co-transfection with an NF-kappa B (p65) expression construct increased the promoter activity, an effect which could be blocked by co-transfection with an I kappa B (MAD-3) expression construct. Deletion of the NF-kappa B site abolished the effect of both H2O2 and co-transfection of NF-kappa B. Interestingly, NAC is also an inducer for GSTP1. The effect of NAC was shown to be mediated largely by the AP1 site, since mutation of this site abolished the induction by NAC.
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PMID:The organization of the human GSTP1-1 gene promoter and its response to retinoic acid and cellular redox status. 854 77

Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.
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PMID:Interaction of glutathione transferase P1-1 with captan and captafol. 867 7

Busulfan is eliminated by glutathione S-transferase (GST)-catalyzed conjugation with glutathione (GSH). We have characterized the busulfan-conjugating activity of purified human liver GSTA1-1, GSTA1-2, GSTA2-2, GSTM1-1, and placental GSTP1-1. Isoforms were purified from cytosol by GSH-affinity chromatography and chromatofocusing. In addition, the busulfan-conjugating activity of cDNA-expressed GTH1 and GTH2, corresponding to GSTA1-1 and GSTA2-2, were characterized. The major product of busulfan conjugation, a thiophenium ion (THT+), was assayed by GC/MS after conversion to tetrahydrothiophene (THT). THT+ formation rate increased linearly with busulfan concentration up to its solubility limit for all GST isoforms. Because Vmax and KM could not be determined separately, the slope of the velocity vs. substrate concentration plot, Vmax/KM was used to compare isoform activities. Vmax/KM for GSTA1-1 was 7.95 microliters/min/mg protein, the highest busulfan-conjugating activity of all human liver and placenta isoforms evaluated. GSTM1-1 and GSTP1-1, respectively, had 46% and 18% of the activity of GSTA1-1. Since the polymorphic mu-class GST catalyzed busulfan conjugation, we examined busulfan clearance in 50 patients undergoing high-dose busulfan before bone marrow transplantation. Busulfan clearance was normally distributed, suggesting that GSTM1-1 does not contribute significantly to the elimination of busulfan from the body. We conclude that GSTA1-1 is the major isoform catalyzing busulfan conjugation, whereas GSTM1-1 and GSTP1-1 may be important in the protection of specific cells.
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PMID:Busulfan conjugation by glutathione S-transferases alpha, mu, and pi. 888 13

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.
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PMID:Inhibition of glutathione S-transferase activity in human melanoma cells by alpha,beta-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal. 895 Feb 26

The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen-peroxide-mediated oxidation of class-pi glutathione S-transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non-substrate-ligand-binding properties. The overall hydrodynamic volume of the oxidised class-pi glutathione S-transferase type-1 homodimer (GSTP1-1) is decreased, and its tertiary-structural and secondary-structural elements are changed with respect to the native protein. Structural differences appear to be prominent in domain 1 of oxidised GSTP1-1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidised protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1-chloro-2,4-dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H-site) for electrophilic substrates is negatively affected in that the K(m) and catalytic-efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol-treated oxidised GSTP1-1 is able to regain its overall hydrodynamic volume; however, both its secondary-structural and tertiary-structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidised glutathione S-transferase and dithiothreitol-treated oxidised glutathione S-transferase are less thermostable (tM = 55.5 degrees C and 56.3 degrees C, respectively) than the native enzyme (tM = 59 degrees C). These results indicate that the class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation.
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PMID:Class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation by hydrogen peroxide. 897 47

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.
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PMID:Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions. 942 4

Deficiencies of the glutathione transferase isoenzymes GSTM1-1 and GSTT1-1 have been shown to be risk modifiers in a number of different cancers but there have been no similar studies with GSTP1-1, the only member of the Pi class of glutathione S-transferases expressed in humans. Over-expression of GSTP1-1 in tumours suggests that it may be a significant factor in acquired resistance to certain anticancer drugs. We previously identified a cDNA clone with two amino acid substitutions (I105V, A114V). This clone suggests that the GSTP1 gene is polymorphic and it is possible that the different genotypes may be associated with altered cancer risk or drug resistance. In the present study, we report methods for genotyping individuals at codons 105 and 114 of GSTP1 and demonstrate that these two loci are polymorphic in several different racial groups. We also detected significant linkage disequilibrium between these two loci. To determine if either of the alleles at these two loci were associated with altered cancer susceptibility, we genotyped individuals with colorectal cancer or lung cancer. A total of 131 colorectal and 184 lung cancer patients were compared with 199 control individuals. Overall, there were no significant associations between the GSTP1 polymorphisms and either form of cancer.
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PMID:Polymorphism of the Pi class glutathione S-transferase in normal populations and cancer patients. 951 Nov 78


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