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)

The oxidized glutathione inhibited the activity of glutathione S-transferase purified from human placenta just through competitive inhibition. On the other hand, cystine and cystamine inactivated the activity by pseudo first-order in low concentrations, accompanying the stoichiometric incorporation of the radioactivity of [14C]-cystine to the enzyme protein until a half mole per one subunit. This and the protective effect of glutathione analogues suggested that the SH/SS exchange reaction occurred between the disulfide and the SH group near the glutathione binding site of the enzyme to form a mixed disulfide.
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PMID:Inactivation of human placenta glutathione S-transferase by SH/SS exchange reaction with biological disulfides. 199 55

The occurrence of glutathione transferase in human malignant melanoma cell lines and solid tumor material has been analyzed and compared with the enzyme composition in fibroblasts and naevus samples. All cells and tissues investigated contained essentially only the acidic class Pi glutathione transferase as demonstrated by SDS-PAGE and immunoblotting. The enzyme was purified from tumor material and characterized. Its intracellular concentration was significantly higher in all the melanoma cell preparations analyzed than in the non-malignant cells, supporting the view that the class Pi glutathione transferase may contribute to the drug resistance that is characteristic of malignant melanoma.
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PMID:Expression of class Pi glutathione transferase in human malignant melanoma cells. 311 48

The inhibition mechanism of the dimeric human placenta glutathione transferase (GST P1-1) by the antibiotic p-carboxyphenylazoxycyanide (calvatic acid) has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different calvatic acid/GST P1-1 molar ratios indicate that one mole of calvatic acid inactivates one mole of the homodimeric enzyme molecule, containing two catalytically equivalent active sites. The apparent second order rate constant for GST P1-1 inactivation is 2.4 +/- 0.3 M-1 s-1. The recovery of all the 5,5'-dithio-bis(2-nitro-benzoic acid)-titratable thiol groups as well as the original catalytic activity of GST P1-1 after treatment of the inhibited enzyme with dithiothreitol indicates that two disulfide bridges per dimer, likely between Cys47 and Cys101, have been formed during the reaction with calvatic acid. To the best of the authors knowledge, calvatic acid represents a unique case of enzyme inhibitor acting also throughout its reaction product(s).
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PMID:Inhibition of human placenta glutathione transferase P1-1 by calvatic acid. 806 31

The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.
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PMID:Relationship between zinc content and DNA-binding activity of the DNA-binding motif of the transcription factor ALCR in Aspergillus nidulans. 827 45

The pH-Vmax/KmGSH plot of glutathione S-transferase P (GST-P) showed a bell-shaped profile, indicating bifunctional catalysis for glutathione (GSH) conjugation. The ionization constant (Ke) and the heat of ionization (delta He) of the essential ionizable group in the GSH binding site were measured and the value of pKe1 was 5.9 and that of pKe2, 8.4, while the delta He1 and delta He2 were -0.2 and 7.9 kcal/mole, respectively. In a solvent containing 25% ethanol, pKe1 and pKe2 shifted to the alkaline side by 0.47 and 0.2, respectively. These kinetic results indicated that carboxyl and phenolic groups were ionizable groups essential for the GSH conjugation. Chemical modifications using aminomethane sulfonic acid and N-acetylimidazole supported the results of the kinetic studies.
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PMID:Identification of ionizable groups essential for the enzyme catalysis on glutathione S-transferase P. 835 5

The expression vector pGEX-2T under the control of the IPTG-inducible tac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin, HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.
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PMID:Matrilysin: expression, purification, and characterization. 856 47

GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-D-mannose to GDP-L-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion protein has a Km of 0.22 +/- 0.04 mM and a specific activity of 2.3 +/- 0.2 micromol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP-L-beta-fucose, the end-product of the pathway, inhibits it specifically. The GST-GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.
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PMID:Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli. 925 4

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
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PMID:Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli. 983 23

The compound 4-(fluorosulfonyl)benzoic acid (4-FSB) functions as an affinity label of the dimeric pig lung pi class glutathione S-transferase yielding a completely inactive enzyme. Protection against inactivation is provided by glutathione-based ligands, suggesting that the reaction target is near or part of the glutathione binding site. Radioactive 4-FSB is incorporated to the extent of 1 mol per mole of enzyme subunit. Peptide mapping revealed that 4-FSB reacts with two tyrosine residues in the ratio 69% Tyr7 and 31% Tyr106. The ratio is not changed by the addition of ligands. The results suggest that only one of the tyrosine residues can be labeled in the active site of a given subunit; i.e., reactions with Tyr7 and Tyr106 are mutually exclusive. We propose that the difference in labeling of these tyrosine residues is related to their pKa values, with Tyr7 exhibiting the lower pKa. The modified enzyme no longer binds to a S-hexylglutathione-agarose affinity column, even when only one of the active sites contains 4-FSB; these results may reflect interaction between the subunits. We conclude that Tyr7 and Tyr106 of the pig lung class pi glutathione S-transferase are important for function and are located at or close to the substrate binding site of the enzyme.
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PMID:Affinity labeling of pig lung glutathione S-transferase pi by 4-(fluorosulfonyl)benzoic acid. 1008 71

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.
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PMID:Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor. 1081 8


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