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)

Opening of the mitochondrial permeability transition pore (MPTP) is sensitized to [Ca(2+)] by oxidative stress (diamide) and phenylarsine oxide (PAO). We have proposed that both agents cross-link two thiol groups on the adenine nucleotide translocase (ANT) involved in ADP and cyclophilin-D (CyP-D) binding. Here, we demonstrate that blocking Cys(160) with 80 microM eosin 5-maleimide (EMA) or 500 microM N-ethylmaleimide (NEM) greatly decreased ADP inhibition of the MPTP. The ability of diamide, but not PAO, to block ADP inhibition of the MPTP was antagonized by treatment of mitochondria with 50 microM NEM to alkylate matrix glutathione. Binding of detergent-solubilized ANT to a PAO-affinity matrix was prevented by pre-treatment of mitochondria with diamide, EMA or PAO, but not NEM. EMA binding to the ANT in submitochondrial particles (SMPs) was prevented by pre-treatment of mitochondria with either PAO or diamide, implying that both agents modify Cys(160). Diamide and PAO pre-treatments also inhibited binding of solubilized ANT to a glutathione S-transferase-CyP-D affinity column, both effects being blocked by 100 microM EMA. Intermolecular cross-linking of adjacent ANT molecules via Cys(57) by copper phenanthroline treatment of SMPs was abolished by pre-treatment of mitochondria with diamide and PAO, but not with EMA. Our data suggest that PAO and diamide cause intramolecular cross-linking between Cys(160) and Cys(257) directly (not antagonized by 50 microM NEM) or using glutathione (antagonized by 50 microM NEM) respectively. This cross-linking stabilizes the "c" conformation of the ANT, reducing the reactivity of Cys(57), while enhancing CyP-D binding to the ANT and antagonizing ADP binding. The two effects together greatly sensitize the MPTP to [Ca(2+)].
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PMID:Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. 1214 99

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
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PMID:[Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists]. 1251 16

The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.
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PMID:Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase. 1281 49

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.
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PMID:Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11. 1453 Mar 82

Expression of glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. Curcumin, a natural compound extracted from Curcuma longa, has shown strong antioxidant and anticancer properties and also the ability to regulate a wide variety of genes that require activating protein 1 and nuclear factor kappaB (NF-kappaB) activation. In the present study, we examined the inhibitory effect of curcumin on the expression of GSTP1-1 mRNA as well as protein, and we correlated this inhibition with the apoptotic effect of curcumin on K562 leukemia cells. Curcumin efficiently inhibited the tumour necrosis factor alpha- and phorbol ester-induced binding of AP-1 and NF-kappaB transcription factors to sites located on the GSTP1-1 gene promoter. TNFalpha-induced GSTP1-1 promoter activity was also inhibited by curcumin as shown by reporter gene assay. In parallel, curcumin induced pro-caspases 8 and 9 as well as poly ADP ribose polymerase cleavage and thus leading to apoptosis in K562 cells. Our results overall add a novel role for curcumin as this chemoprotective compound could contribute to induce apoptosis by its ability to inhibit the GSTP1-1 expression at the level of transcription.
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PMID:Induction of apoptosis by curcumin: mediation by glutathione S-transferase P1-1 inhibition. 1455 24

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.
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PMID:Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3. 1538 45

The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.
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PMID:Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity. 1550 8

In cancer chemotherapy, it is necessary to design an agent that suppresses or inhibits the targets that influence cell growth and apoptosis. We focus on the apoptotic pathway via mitochondria in this article. In this pathway, c-Jun N-terminal kinase (JNK), one of the stress activated protein kinases, is predominantly activated by apoptotic stimuli. JNK activity is inhibited by the binding of glutathione S-transferase P1-1 (GST P1-1) through protein-protein interactions. It has been noted that GST P1-1 overexpression plays an important role in carcinogenesis and in part in the MDR phenotype. We show several useful modifications of an anticancer agent that suppress the enzyme activity and expression of GST P1-1. The release of cytochrome c from mitochondria to the cytosol during apoptosis is mediated by the mitochondrial permeability transition pore, which is a protein complex formed by the voltage-dependent anion channel, members of the pro- and anti- apoptotic Bax-Bcl-2 protein family, cyclophilin D, and adenine nucleotide (ADP/ATP) translocators. We propose some drugs, including a proteasome inhibitor that can triger the permeability transition.
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PMID:Chemotherapeutic agents that induce mitochondrial apoptosis. 1557 15

Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.
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PMID:Adenylate kinase of Escherichia coli, a component of the phage T4 dNTP synthetase complex. 1594 17

The purpose of this study was to investigate effects of some antibiotics on glucose-6-phosphate dehydrogenase (G6PD), antioxidant enzymes, and malondialdehyde (MDA). Initially, for in vitro studies, G6PD was purified from human erythrocyte, 9811-fold in a yield of 42.4% by using ammonium sulfate precipitation and 2',5' ADP-Sepharose 4B affinity gel. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of four different antibiotics (isepamicin sulfate, meropenem, chloramphenicol, and thiamphenicol glisinat hydrochloride) were investigated on the purified enzyme. K(i) value and type of inhibition were determined by means of Lineweaver-Burk graphs and regression analysis graphs. Isepamicin sulfate inhibited the enzyme activity (I(50) value, 2.1 mM; K(i) value, 1.7 mM), whereas thiamphenicol glisinat hydrochloride activated the G6PD dose dependently. Other drugs showed no inhibition and activation effect. In addition, the effects of isepamicin sulfate on the activities of G6PD, glutathione reductase (GR), superoxide dismutases (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferase (GST) and MDA concentrations were examined in Sprague-Dawley rat erythrocytes in vivo. A marked alteration in the activities of these enzymes and MDA levels may be the result of oxidative stress in the rats receiving isepamicin sulfate.
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PMID:Effects of some antibiotics on activity of glucose-6-phosphate dehydrogenase from human erythrocytes in vitro and effect of isepamicin sulfate on activities of antioxidant enzymes in rat erythrocytes. 1629 74


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