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)

1. Neurones dissociated from Rana pipiens paravertebral sympathetic ganglia were studied by means of the whole-cell patch-clamp technique. Responses to agonists were best recorded when cyclic AMP was included in the patch pipette. 2. Two populations of cells were identified on the basis of size (input capacitance, Cin) and the presence or absence of a fast, transient outward current (A-current, IA). This current was usually present in the 'large' cells (Cin = 40.5 +/- 1.5 pF, n = 66) but absent from 'small' cells (Cin = 21.0 +/- 0.8 pF, n = 70). 3. Both cell types exhibited a slowly activating, non-inactivating K+ current (M-current, IM) which was suppressed by luteinizing hormone-releasing hormone (LHRH, 10-100 microM). Threshold for activation of IM was about -75 mV, half-maximal activation was at -50 mV and the M-conductance GM increased e-fold for at 7 mV change in membrane potential. The maximum value for IM studied in large cells by patch-clamp procedures was less than 0.2 nA. More M-channels were available per unit membrane area in the small cells (GM = 1495 microS cm-2) than in the large cells (GM = 1034 microS cm-2). Time constants for IM deactivation at -70 mV were faster in the large cells (37.2 +/- 4.6 ms, n = 16) than in the small cells (66.1 +/- 5.9 ms, n = 9). 4. Muscarine (10 microM) produced inward current in the large cells as a result of IM suppression. In 40% of the large cells, some of the M-channels were also sensitive to adrenaline (10-100 microM). In a few large cells (less than 10%) adrenaline produced outward current by increasing IM. 5. Muscarine failed to effect IM in the small cells and instead produced an inwardly rectifying K+ current which activated within 5 ms at -110 mV. The outward current produced in twenty out of thirty-seven small cells by adrenaline was occluded by that produced by muscarine, suggesting that both agonists affect the same K+ channels. 6. Inclusion of the protein kinase inhibitors, 1-(5-isoquinolinyl-sulphonyl)-2-methyl piperazine (H-7, 50 microM) or gold sodium thiomalate (GST, 50 microM) in the pipette solution failed to antagonize either muscarine-induced current. Both currents were prolonged when the 'internal solution' contained GTP-gamma-S (50 microM). 7. Phorbol-12-myristate-13-acetate (PMA, 2-5 microM) produced an inward current as a result of IM suppression in both small and large cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of muscarine and adrenaline on neurones from Rana pipiens sympathetic ganglia. 221 86

The adenovirus E1A243 protein can activate transcription of the mouse c-fos gene in a manner that depends on treatment of cells with inducers or analogs of cyclic AMP (cAMP). Activation requires conserved region 1 and the N-terminal domain of E1A243 and is mediated by a 22-bp E1A response element containing a cAMP response element (CRE) at -67 and a binding site for transcription factor YY1 at -54. In the absence of E1A243, YY1 represses CRE-dependent transcription of c-fos by physically interacting with ATF/CREB proteins bound to the -67 CRE. Here we present evidence that expression of E1A243 leads to relief of YY1-mediated repression by a disruption of the ATF/CREB-YY1 complex. Addition of E1A243 to in vitro binding assays prevented binding of ATF-2 to glutathione S-transferase-YY1. Similarly, expression of E1A243 in HeLa cells prevented the association of a YY1-VP16 fusion protein with endogenous ATF/CREB proteins bound to the -67 CRE of a transfected c-fosCAT reporter plasmid. In each case, the N-terminal domain of E1A243, which mediates a direct interaction with YY1, was responsible for disruption of the ATF/CREB-YY1 complex. On the basis of these and previously published results, we present a model for the synergistic transcriptional activation of the c-fos gene by E1A243 and cAMP.
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PMID:Adenovirus E1A243 disrupts the ATF/CREB-YY1 complex at the mouse c-fos promoter. 749 44

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.
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PMID:Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. 757 83

Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.
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PMID:Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica. 766 7

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocytes that binds to glutathione S-transferase fusion proteins corresponding to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fusion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little isoform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-terminal domain of Glut4. The GTBP70 binding to Glut4 was not affected by the presence of 2 mM EDTA, 2.4 mM Ca2+, or 150 mM K+. The binding was inhibited by ATP in a dose-dependent manner, with 50% inhibition at 10 mM ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenosine 5'-(beta, gamma-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. When these experiments were repeated using rat hepatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glucose transporter fusion proteins was evident, suggesting that either GTBP70 expression or its function is cell-specific. These findings strongly suggest the possibility that GTBP70 may play a key role in glucose transporter regulation in insulin target cells such as adipocytes.
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PMID:ATP-sensitive binding of a 70-kDa cytosolic protein to the glucose transporter in rat adipocytes. 771 80

Previously, we characterized nucleotide sequences of two cDNAs encoding adenylate kinase from rice plants (Oryza sativa L.). Each cDNA (Adk-a or Adk-b) was cloned into the expression vector pET 11d-GST to produce GST-AK fusion proteins in Escherichia coli. Recombinant proteins were cleaved by thrombin, and GST-free adenylate kinase proteins were obtained. Enzyme activity profiles of different pH and inhibition effects to the enzyme by Ap5A (adenosine-5'-pentaphospho-5'-adenosine) indicates that both adenylate kinase proteins have similar biochemical characteristics. Among the nucleoside monophosphates (AMP, CMP, GMP and UMP) investigated, only AMP reacted with ATP. Furthermore, using the antiserum against the rice adenylate kinase proteins, the cellular location of adenylate kinase proteins was examined by immunomicroscopic analysis in combination with a subcellular fractionation method. The results indicated that adenylate kinase proteins were distributed largely in cytosol of rice cells.
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PMID:Biochemical properties of rice adenylate kinase and subcellular location in plant cells. 776 84

Three different forms of glutathione transferase (GST) have been resolved in the two mouse adrenal tumour cell lines Y1 and Kin 8. Two of these belong to the mu and pi classes respectively. The third form is so far unidentified. In the Y1 cells, the levels of the mu form (mGTmu1) and the unidentified form, are both down-regulated in the presence of adrenocorticotrophic hormone (ACTH) while the pi form is unaffected. The Kin 8 cell line is derived from Y1 cells and harbours a defect in the cyclic AMP (cAMP)-dependent protein kinase, making it refractory to cAMP-dependent regulation of several enzymes. The GST levels in this cell line were unaffected by ACTH. Also, the steady-state levels of mGTmu1 mRNA were much lower in Y1 cells treated with forskolin (which activates adenylate cyclase) compared with control cells, but there was no difference in mGTmu1 mRNA levels between control and forskolin-treated Kin 8 cells. This indicates that the ACTH-dependent regulation of the mu class GST is pre-translational and that a functional cAMP-dependent protein kinase is required for the regulation. We have further shown that the difference in mRNA steady-state levels between control and forskolin-treated Y1 cells is abolished when transcription is inhibited by actinomycin D. In light of the stability of mGTmu1 mRNA, it would appear most likely that actinomycin D inhibits the transcription of short-lived factors which regulate the turn-over of mGTmu1 transcripts in response to changes in intracellular cAMP levels.
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PMID:Adrenocorticotrophic-hormone-dependent regulation of a mu-class glutathione transferase in mouse adrenocortical cells. 782 17

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct regions in human T-cell lymphotropic virus type I tax mediate interactions with activator protein CREB and basal transcription factors. 785 24

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.
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PMID:Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element. 800 91

The Shope fibroma virus (SFV) DNA ligase gene has been cloned and sequenced, and the biochemical requirements of the gene product have been determined in vitro. The SFV ligase gene maps to the BamHI L1/L2 boundary and spans 1.7 kb. The gene is predicted to encode a 559-amino-acid protein of M(r) = 63,139 which shares 45% amino acid identity with Orthopoxvirus ligases. The C-terminal two-thirds of the protein appears to encode the catalytic domain and shares distant homology with many ligases. The N-terminal homology is shared between only Orthopoxviruses and Leporipoxviruses and suggests that DNA ligases may be composite structures consisting of two independently evolved protein domains. Although the the gene encodes features characteristic of both early and late poxviral genes, Northern analysis showed that SFV ligase is expressed as a late gene product. In order to prove the identity of the protein it was expressed as a glutathione S-transferase fusion in Escherichia coli, affinity purified, and shown to be a Mg2+.ATP-dependent ligase in vitro. The recombinant protein can also form a covalent ligase.AMP complex characteristic of ATP-dependent DNA ligases. The SFV ligase gene can be disrupted and is thus not essential for viral growth in culture. This was shown by recombining a PCR product, encoding a P7.5 promoter and E. coli guanine phosphoribosyltransferase gene (gpt) into the open reading frame, and selecting for gpt+ viruses. This work provides insights into the evolution of Orthopoxviruses and Leporipoxviruses and strains suitable for a detailed analysis of the role DNA ligases play in poxviral recombination.
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PMID:Characterization of the Shope fibroma virus DNA ligase gene. 803 Feb 29


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