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)

SecA performs a critical function in the recognition, targeting, and transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. In this study we investigate the substrate specificity of SecA, including the influence of the early mature region of the preprotein on SecA interactions, and the extent to which SecA recognizes targeting signals from different transport pathways. A series of fusion proteins were generated which involved the tandem expression of GST, signal peptide, and the first 30 residues from alkaline phosphatase. These were purified and evaluated for their ability to promote SecA ATPase activity. No significant difference in the stimulation of SecA-lipid ATPase activity between the synthetic wild-type alkaline phosphatase signal peptide and a fusion that also contains the first 30 residues of alkaline phosphatase was observed. The incorporation of sequence motifs in the mature region, which confer SecB dependence in vivo, had no impact on SecA activation in vitro. These results suggest that the early mature region of alkaline phosphatase does not affect the interactions between SecA and the signal peptide. Sec, Tat, and YidC signal peptide fusions were also assayed for their ability to stimulate SecA ATPase activity in vitro and further analyzed in vivo for the Sec dependence of the transport of the corresponding signal peptide mutants of alkaline phosphatase. Our results demonstrate that E. coli Sec signals give the highest level of SecA activation; however, SecA-signal peptide interactions in vitro are not the only arbiter of whether the preprotein utilizes the Sec pathway in vivo.
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PMID:SecA specificity for different signal peptides. 1196 18

Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.
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PMID:A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins. 1218 6

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having single-stranded poly(A) overhangs.
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PMID:An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K. 1218 65

Glutathione-mediated free-radical-scavenging and plasma membrane ATPase activities increase as sinks for metabolic energy with advancing tuber age. Plasma membrane ATPase activity from 19-month-old tubers was 77% higher than that from 7-month-old tubers throughout sprouting. The higher activity was not attended by an increase in the amount of ATPase per unit plasma membrane protein. Concentrations of oxidized (GSSG) and reduced glutathione more than doubled as tuber age advanced from 6 to 30 months, but the proportion of GSSG to total glutathione remained constant with age. The activity of glutathione transferase, an enzyme that catabolizes lipid-hydroperoxides, increased by 44 and 205% on a fresh weight and protein basis, respectively, as tubers aged from 6 to 30 months. Glutathione reductase activity also increased with advancing age, by 90% on a fresh weight basis and 305% on a protein basis. Older tubers had more glutathione reductase per unit of soluble and mitochondrial protein. The age-induced increase in cytosolic glutathione transferase activity was likely due to increased availability of lipid-hydroperoxides and/or a positive effector. Synthesis of glutathione requires ATP, and the increased reduction of GSSG resulting from catalysis of lipid-hydroperoxides is NADPH-dependent. Thus, increased plasma membrane ATPase and glutathione-mediated free-radical-scavenging activities likely constitute substantial sinks for ATP in older tubers prior to and during sprouting. Increased oxidative stress and loss in membrane integrity and central features of aging that undoubtedly contribute to the enhanced respiration of sprouting older tubers.
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PMID:Oxidative Stress Results in Increased Sinks for Metabolic Energy during Aging and Sprouting of Potato Seed-Tubers. 1222 48

The natural increase of UV-B radiation levels due to depletion of the ozone layer in the atmosphere may impose additional stress for the survival of zooplanktons which serve as a major constituent of the aquatic food chain. To study the adverse effects of UV-B radiation on the aquatic biomass, studies were conducted using the aquatic organism Tubifex as a model, as UV-B radiation is known to penetrate into the natural waters. UV-B radiation induced mortality in tubifex and the production of activated oxygen species by these organisms. Alterations in DNA, RNA, protein, glutathione (GSH), hydrogen peroxide H(2)O(2), thiobarbituric acid-reactive substance (TBA-RS), ATPase, AChE, GST, and LDH activities in Tubifex at various doses (0-2.0 J) of UV-B radiation were found. LC(50) value for UV-B-induced mortality of Tubifex was 0.80+/-0.15 J and the threshold dose was 0.35+/-0.05 J; mortality began within 3h postirradiation. UV-B dose-dependent production of singlet oxygen, superoxide anion, and hydroxyl radicals by Tubifex was observed. DNA, RNA, protein, and GSH contents were found to decrease significantly (P<0.001) while H(2)O(2) and TBA-RS increased (P<0.01) under the influence of UV-B radiation. The activities of ATpase, AChE, and GST enzymes were inhibited (P<0.01) and LDH activity was significantly increased (P<0.001) in Tubifex exposed to UV-B radiation. The results suggest that an increase in UV-B radiation alters several biochemical processes, leading to the mortality of the organism. Tubifex could be useful as a sensitive alternate model for studying UV-B-induced phototoxicity and possible mechanisms of action.
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PMID:Tubifex: a sensitive model for UV-B-induced phototoxicity. 1229 92

The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.
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PMID:Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia. 1240 78

Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP.
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PMID:The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity. 1241 11

The plant plasma membrane H(+)-ATPase contains a C-terminal autoinhibitory domain whose displacement from the catalytic site is caused by binding of regulatory 14-3-3 proteins. Members of the highly conserved 14-3-3 family bind their individual target proteins in a sequence-specific and phosphorylation-dependent manner within a central groove, the latter characterized by the presence of highly invariant residues. However, an atypical binding site for 14-3-3s within the H(+)-ATPase has been identified that does not resemble any other 14-3-3 binding motif. Combination of site-directed mutagenesis with glutathione S-transferase pull-down assays points to the importance of the central 14-3-3 groove for the interaction with the apparently unique site of the H(+)-ATPase. Furthermore, a 14-3-3 dimer is essential for binding such unusual motif.
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PMID:Regulatory 14-3-3 proteins bind the atypical motif within the C terminus of the plant plasma membrane H(+)-ATPase via their typical amphipathic groove. 1243 22

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [gamma-(32)P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins. The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.
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PMID:Interactions between Na,K-ATPase alpha-subunit ATP-binding domains. 1251 76

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed.
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PMID:Protective role of L-methionine against free radical damage of rat brain synaptosomes. 1254 97


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