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)

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
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PMID:Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB. 765 Nov 40

The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
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PMID:ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. 836 61

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

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP-binding cassette (ABC) drug efflux pumps through binding to cis-acting sites known as PDREs (PDR responsive elements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST-Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either deltapdr1, deltapdr3 and deltapdr1 deltapdr3 deletions or pdr1-3 and pdr3-2 gain-of-function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.
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PMID:The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators. 942 26

The genetic differences between praziquantel-resistant (R) and susceptible (S) strains of Schistosoma mansoni (Fallon & Doenhoff, 1994) were explored using RAPD and by cloning differentially expressed mRNAs by subtractive PCR. No differences between the 2 strains were detectable by RAPD using 41 different primers indicating that no major genomic rearrangements were present. Subtractive PCR generated a number of fragments, 1 of which was shown to correspond to an over-expressed mRNA in the R strain and to encode a fragment of the subunit 1 of cytochrome-c oxidase (SCOX1). In the absence of a complete sequence for this gene, we used EST sequences to compile a consensus sequence for the 904 bp at the 3' end that enabled us to choose primers for semi-quantitative RT-PCR. This technique showed that SCOX1 was indeed over-expressed about 5 to 10-fold in the R strain whereas the genes encoding the 28 kDa glutathione S-transferase, glutathione peroxidase, NADH dehydrogenase subunit 5 and the ATP-binding cassette family protein SMDR2 were not. In contrast, cytochrome-c oxidase enzyme activity was 4-fold lower in the R strain than in the S strain.
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PMID:Alterations in cytochrome-c oxidase expression between praziquantel-resistant and susceptible strains of Schistosoma mansoni. 969 1

Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate ATPase (GS-X pump) activity is a common feature of some ATP-binding cassette (ABC) transporters, such as the multidrug resistance-associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S-transferase catalyzed reaction and be eliminated through GS-X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S-(2,4-dinitrophenyl)-glutathione (DNP-SG) conjugate through the GS-X pump, using whole cells and inside-out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS-X pump activity. In particular, HEp-2 larynx carcinoma cells possess an elevated DNP-SG export rate through the GS-X pump compared with HeLa, K562, U937 or HL-60 cells, which exhibit the lowest activity. The differences in DNP-SG export rates are not due to decreased glutathione S-transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS-X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS-X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.
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PMID:Glutathione metabolism and glutathione S-conjugate export ATPase (MRP1/GS-X pump) activity in cancer. I. Differential expression in human cancer cell lines. 976 21

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
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PMID:Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein. 993 1

AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.
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PMID:AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein. 1093 72

The futA1 (slr1295) and futA2 (slr0513) genes encode periplasmic binding proteins of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. PCC 6803. FutA1 was expressed in Escherichia coli as a GST-tagged recombinant protein (rFutA1). Solution containing purified rFutA1 and ferric chloride showed an absorption spectrum with a peak at 453 nm. The absorbance at this wavelength rose linearly as the amount of iron bound to rFutA1 increased to reach a plateau when the molar ratio of iron to rFutA1 became unity. The association constant of rFutA1 for iron in vitro was about 1 x 10(19). These results demonstrate that the FutA1 binds the ferric ion with high affinity. The activity of iron uptake in the Delta futA1 and Delta futA2 mutants was 37 and 84%, respectively, of that in the wild-type and the activity was less than 5% in the Delta futA1/Delta futA2 double mutant, suggesting their redundant role for binding iron. High concentrations of citrate inhibited ferric iron uptake. These results suggest that the natural iron source transported by the Fut system is not ferric citrate.
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PMID:Iron-binding activity of FutA1 subunit of an ABC-type iron transporter in the cyanobacterium Synechocystis sp. Strain PCC 6803. 1152 7

1. Genetically altered mice increasingly are being used in toxicology and pharmaceutical development. As such, knowledge of the compensatory activity of enzymes is critical when interpreting the results of studies using these animals. 2. The present study examined alterations in hepatic phase I and II enzyme activity, and alterations in phase III (transporter) RNA expression, between FVB mice and mice lacking the multidrug resistance-associated protein 1 (mrp1) gene (FVB/mrp1-/- mice). It was hypothesized that other transporters and phase I and II enzymes would be increased in the FVB/mrp1-/- mice, presumably as a compensatory mechanism. 3. No differences was found in hepatic cytochrome P450 activity between FVB and FVB/mrp1-/- mice, nor were there differences in the amount of total hepatic glutathione or in glutathione S-transferase enzyme activity. 4. However, sulfotransferase activity towards 2-naphthol was significantly increased by 2.6-fold in the FVB/mrp1-/- mice, whereas glucuronosyltransferase activity towards both 4-nitrophenol and testosterone was significantly reduced 1.5-fold. In addition, mrp2 RNA expression was significantly increased by 3.4-fold and mrp5 expression was significantly increased by 1.6-fold in the FVB/mrp1-/- mice. 5. Mice lacking mrp1 have significantly increased hepatic transcription of at least two other ATP-binding cassette transporters, as well as increased 2-naphthol sulfotransferase activity, presumably to compensate for the lack of mrp1.
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PMID:Altered expression of sulfotransferases, glucuronosyltransferases and mrp transporters in FVB/mrp1-/- mice. 1474 40


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