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)

While glutathione S-transferase P form (GST-P), a reliable marker for preneoplastic lesions induced by mutagenic hepatocarcinogens, is generally not expressed in rat liver foci, hyperplastic nodules and hepatomas induced by peroxisome proliferators (PPs), such lesions can be detected due to their peroxisomal enzyme-negative nature. For comparative purposes we examined the inducibility of enoyl CoA hydratase (ECH), a key peroxisomal enzyme, in rat hepatic preneoplastic lesions induced by mutagenic carcinogens. Clofibrate (CF) was therefore administered for 2 or 4 weeks following performance of the Solt-Farber protocol using diethylnitrosamine and 2-acetylaminofluorene. Immunohistochemical examination revealed no or only very weak expression of ECH within the induced foci in clear contrast to the strong staining of surrounding parenchyma. ECH expression was thus diametrically opposed to that of GST-P which was found only in foci. Although ECH was completely lacking in GST-P-strongly positive foci, it was expressed in GST-P-negative hepatocytes inside some foci otherwise positive for GST-P. CF administration resulted in a significant decrease in the numbers and areas of foci exhibiting strongly positive or positive GST-P staining; this being reflected in a lowering of GST-P protein levels. Furthermore, in primary cultured rat hepatocytes, clofibric acid as well as dexamethasone suppressed the expression of both GST-P and the oncogene, c-jun. These results taken together suggest that possible interaction of the PP receptor with JUN might be involved in loss of ECH expression in GST-P-strongly positive foci.
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PMID:Lack of peroxisomal enzyme inducibility in rat hepatic preneoplastic lesions induced by mutagenic carcinogens: contrasted expression of glutathione S-transferase P form and enoyl CoA hydratase. 845 14

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).
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PMID:The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae. 864 87

Human arylamine N-acetyltransferase 1 (NAT1) has 290 amino acids and acetylates arylamines from acetyl coenzyme A. The acetyl group forms a thiolester with Cys 68 in the enzyme, and the acetyl group is then transferred to the arylamine. When NAT1 is expressed using the pGEX vector, the glutathione S-transferase (GST)-NAT1 fusion protein catalyses the acetylation of the NAT1 substrate p-aminobenzoic acid from acetyl CoA. Neither GST alone, nor a fusion protein of GST with the N-terminal 204 amino acids of NAT, catalyses the acetylation of p-aminobenzoic acid from acetyl CoA. Using [3H]acetyl CoA as substrate, it is shown that the full-length NAT1 and the N-terminal 204 amino acids of NAT1 each form an acetylated intermediate on reaction with acetyl CoA.
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PMID:A fragment consisting of the first 204 amino-terminal amino acids of human arylamine N-acetyltransferase one (NAT1) and the first transacetylation step of catalysis. 896 58

A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity. This study aims to define the binding site for gastrin and gastrin antagonists in greater detail. The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E. coli. Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-[Nle15]-gastrin binding in a covalent cross-linking assay. The N- and C-terminal fusion proteins bound gastrin with affinities of 9.9 +/- 6.1 and 71 +/- 48 microM, respectively (n = 3). These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin (0.23 +/- 0.15 microM). In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 +/- 13 and 10 +/- 4 mM, respectively) and benzotript (350 +/- 90 and 400 +/- 160 micro M, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 +/- 3.6 mM and 200 +/- 120 microM, respectively). It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin may bind simultaneously to both active sites. A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.
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PMID:Gastrin and gastrin receptor antagonists bind to both N- and C-terminal halves of the 78 kDa gastrin-binding protein. 902 82

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the beta-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.
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PMID:Characterization of S-hexylglutathione-binding proteins of human hepatocellular carcinoma: separation of enoyl-CoA isomerase from an Alpha class glutathione transferase form. 937 3

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin biosynthesis. This enzyme is of special biological interest because large changes in its activity drive the large night/day rhythm in circulating melatonin in vertebrates. In this study the kinetic mechanism of AANAT action was studied using bacterially expressed glutathione S-transferase (GST)-AANAT fusion protein. The enzymologic behavior of GST-AANAT and cleaved AANAT was essentially identical. Two-substrate kinetic analysis generated an intersecting line pattern characteristic of a ternary complex mechanism. The dead end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus tryptamine. Tryptophol was not an alternative substrate but was a dead end competitive inhibitor versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acetyl-CoA first. N-Acetyltryptamine, a reaction product, was a noncompetitive inhibitor versus tryptamine and uncompetitive with respect to acetyl-CoA. Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framework for inhibitor design.
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PMID:Kinetic analysis of the catalytic mechanism of serotonin N-acetyltransferase (EC 2.3.1.87). 944 20

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.
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PMID:Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose. 1032 81

While characterizing Eps15 partners, we identified a 48-kDa polypeptide (p48) which was precipitated by Eps15-derived glutathione S-transferase fusion proteins. A search in a murine expressed sequence tag data base with N-terminal microsequences of p48 led to the identification of two complete cDNA clones encoding two isoforms of a 439-amino acid protein sharing 95% nucleic and amino acid identity. Northern blot and immunoblotting studies showed that p48 was ubiquitously expressed. A significant homology (19% identity and 40% similarity) between p48 and rat brain cytosolic acyl-CoA thioesterase was observed in an 80-amino acid C-terminal domain, retrieved from proteins from human, nematode, and plants. The thioesterase function of p48 was further demonstrated against long chain acyl-CoAs in a spectrophotometric assay. Furthermore, data obtained from sequence analysis showed that p48 contained a mitochondrial targeting signal, cleaved in mature protein as assessed by microsequencing. The mitochondrial localization of both endogenous and transfected p48 was confirmed by confocal microscopy. These results indicate that p48, called MT-ACT48 (mitochondrial acyl-CoA thioesterase of 48 kDa), defines a novel family of mitochondrial long chain acyl-CoA thioesterases.
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PMID:Molecular cloning and characterization of MT-ACT48, a novel mitochondrial acyl-CoA thioesterase. 1038 25

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.
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PMID:High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity. 1054 59

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling.
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PMID:Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii. 1054 46


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