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 cytotoxicity of the disulfide n-butyl 2-imidazolyl disulfide (III-2) was determined to be the result of a disruption in the cellular redox state and inhibition of critical membrane enzymes. These events cause perturbations in Ca2+ homeostasis, which may affect the cell signalling machinery and cause the activation of catabolic enzymes. Exposure of EMT6 cells to III-2 resulted in depletion of nonprotein and protein thiols. Under hypoxic conditions, the depletion of reduced glutathione was less than that measured when cells were treated in air, whereas following an exposure to 500 microM III-2 for 2 h the enzymes glutathione S-transferase and glutathione reductase were inhibited to a greater extent under hypoxia. Ca2+ homeostasis was disrupted with an initial shift from the mitochondrial to the cytoplasmic pool. The inhibition of plasma membrane Ca(2+)-ATPase resulted in accumulation of Ca2+ in the cytoplasm. At higher concentrations, further disruption was seen as a net loss of Ca2+ of the cytoplasmic excess with no change in the mitochondrial levels, resulting in lower total cellular Ca2+. Neither the inhibition of Ca(2+)-ATPase nor the disruption of Ca2+ homeostasis were different under hypoxic vs. oxic conditions. Due to these observations, HL60 cells were used to measure whether III-2 stimulated apoptosis. Morphologic changes and DNA laddering were observed following exposure to the disulfide, with lower concentrations required to stimulate the cellular changes under hypoxia. These events may be the result of the disruption in Ca2+ homeostasis due to thiolation or alteration in redox status of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Disulfide cytotoxicity under hypoxia. 762 Feb 24

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

Replication of bovine papillomavirus type 1 (BPV-1) DNA has been shown to require two viral proteins known to interact in a molecular complex: E2, a transcription activator, and E1, another nuclear phosphoprotein, which binds to the replication origin and for which helicase/ATPase activities have previously been reported. Here we characterize the BPV-1 E1 ATPase activity. In contrast to Seo et al. (Proceedings of the National Academy of Sciences, USA, 90, 702-706, 1993), we were able to detect this activity in the absence of nucleic acid in partially purified preparations of either E1 protein or of E1-E2 protein complex. Measurements of specific activity and kinetic parameters gave similar values for preparations of various kinds. ATPase activity was quantitatively retained by immunoprecipitates obtained by using anti-E1 or, in the case of E1-E2 complex, anti-E2 antibodies. Significantly, preparations of bacterially expressed glutathione S-transferase-E1 fusion protein exhibited levels of DNA-independent ATPase activity comparable to those of baculovirus-expressed E1. The presence of nucleic acids of various types, including stoichiometric amounts of a BPV-1 ori DNA fragment containing E1 and E2 binding sites, did not grossly affect E1 ATPase activity, the most notable effect being a 2-fold stimulation by unspecific ssDNA. Altogether, our results indicate that BPV-1 E1 possesses an intrinsic ATPase activity which does not depend on the presence of nucleic acid; moreover, they render unlikely any modulation of E1 ATPase activity due to binding either E2 protein or target DNA sequences, or as a result of protein phosphorylation.
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PMID:Bovine papillomavirus type 1 E1 ATPase activity does not depend on binding to DNA nor to viral E2 protein. 773 Jul 98

Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical.
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PMID:Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein. 784 16

Recombinant hsc70, a purified glutathione S-transferase (GST) fusion protein containing the C-terminal domain of hsc70 (GST-Ct), and an internal 18-kDa polypeptide located immediately after the 44-kDa ATPase domain of hsc70 were investigated for their peptide binding properties. The dissociation constants of the S-peptide for native hsc70 (Kd = 5-8 microM), GST-Ct (Kd = 6.5 microM), and the 18-kDa fragment (Kd = 8.1 microM) are virtually identical. In addition, polylysine and (Pro-Pro-Gly)5 do not show high affinity toward hsc70, GST-Ct, and the 18-kDa fragment, whereas peptide GT4 and P3a show comparably high affinity toward these polypeptides. These observations indicate that the peptide binding domain of hsc70 is confined in the internal 18-kDa fragment.
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PMID:Identification of the peptide binding domain of hsc70. 18-Kilodalton fragment located immediately after ATPase domain is sufficient for high affinity binding. 825 14

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 two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli. The GST-L peptide contained the hydrophilic region from Ala340 to Ser660. The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E. coli culture. The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL. ATP competitively displaced the TNP-ATP binding. The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein. The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide. Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5.
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PMID:Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma membrane H(+)-ATPase. 840 44

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.
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PMID:Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini. 857 96

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.
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PMID:A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity. 867 Aug 31

Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum. The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies. The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity. The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C. The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES. The catalytic properties of a recombinant C. thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E. coli [Ciruela (1995) Ph.D. Thesis, University of Kent] were identical with those of the authentic C. thermocellum Cpn60. Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer. Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E. coli GroEL complexes.
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PMID:Thermostable chaperonin from Clostridium thermocellum. 868 8


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