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)

Elimination of the products of xenobiotic metabolism is an important step in cellular detoxification and involves a specific transport system or "export pump". ATP-dependent transport of glutathione S-conjugates has previously been demonstrated in a variety of tissues, mainly from rat. However, the characteristics of this pump have not been fully explored in human cells. This study investigated transport of a glutathione S-conjugate, 2,4-dinitrophenyl glutathione (GS-DNP), by a variety of cultured human cell lines. GS-DNP was generated intracellularly after treatment of cells with 1-chloro-2,4-dinitrobenzene and subsequent transport of the conjugate into the extracellular medium was measured spectrophotometrically at 340 nm. Calculation of the initial transport rates at 37 degrees C revealed considerable variation in GS-DNP secretion between cell lines which was statistically significant in some cases. A 2-fold increase in GS-DNP efflux was observed between Jurkat and HL-60 cells (11.360 +/- 3.893 vs. 5.662 +/- 2.263 nmol/10(6) cells/h, P < 0.007). The highest rate of transport was found in HepG2 cells (14.171 +/- 4.790 nmol/10(6) cells/h) whereas the 5637 cell line had the lowest level with a transport rate of 1.475 +/- 0.631 nmol/10(6) cells/h. For each cell line, transport of GS-DNP was almost totally inhibited or markedly reduced on ice. Pre-incubation of cells at 42 degrees C also lowered the initial transport rates compared with cells maintained at 37 degrees C but these were not significantly different except in the case of HeLa cells. ATP levels ranged from 30.5 to 89.3 nmol/mg protein and there was variation in the glutathione content and glutathione S-transferase activities of the cells. This report demonstrates firstly that transport of glutathione conjugates is a feature of many cell types in vitro and secondly that the basal levels of GS-DNP secretion vary significantly between human cells.
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PMID:Glutathione S-conjugate transport by cultured human cells. 798 Dec 41

We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.
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PMID:Expression and characterization of a rac family protein kinase of Entamoeba histolytica. 798 73

CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.
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PMID:Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain. 800 90

We have determined the primary structure of a myosin I (called mammalian myosin I beta, MMI beta) from bovine brain and identified its functional domains. The protein was previously purified from brain and adrenal gland. Several constructs were generated and expressed in Escherichia coli as glutathione S-transferase fusion proteins and the recombinant proteins were recognized by monoclonal antibodies that recognize either "head" or "tail" domains of native myosin I. A gel overlay method was used to confirm that calmodulin binds to the consensus calmodulin-binding sequence in MMI beta. Binding assays were used to detect interaction with anionic phospholipid vesicles. We conclude that MMI beta consists of an amino-terminal 80.5-kDa domain that contains the ATP- and actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-binding sequences and a basic 30-kDa carboxyl-terminal tail segment that binds to anionic phospholipids and membranes.
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PMID:Domain structure of a mammalian myosin I beta. 802 85

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

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
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PMID:Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. 805 68

Mitogen activated protein kinases (MAP) or extracellular signal regulated protein kinases (ERK) are a family of protein serine/threonine kinases that are activated very rapidly in response to many extracellular stimuli. elk-1, an ets related gene codes for two transcriptional factors elk-1, which regulates c-fos transcription and delta elk-1, both of which are substrates for MAP kinases. A part of the C-terminal transcriptional activation domain (ETA-2) which is common to both the proteins was previously shown to function as an activator of MAP kinases. In this report, in an attempt to investigate the mechanism of activation of MAP kinases, purified preparations of recombinant elk-1 and P44mpk/ERK-1/ERK-2 proteins were used to show the association of elk-1 proteins with MAP kinases. The specific interactions of elk-1 proteins with MAP kinases were confirmed by co-immunoprecipitation studies. Thus elk-1 proteins appear to regulate the activity of MAP kinases by interacting with them ensuring a conformational change and stimulating their autophosphorylation and activation property. The activation was dependent on the presence of ATP and Mg2+. In vitro phosphorylation of elk-1 protein was not regulatory for autonomous DNA binding activity of elk-1 protein. Cells which were exposed to EGF showed a rapid stimulation of an elk-1 specific kinase activity, probably MAP kinase which phosphorylated MBP and was found to be associated with immobilized GST-elk-1. Furthermore, dephosphorylation studies indicate that elk-1 proteins can activate only tyrosine phosphorylated MAP kinase. These results demonstrate the presence of an alternative pathway/mechanism (other than MAP kinase kinase, MAPKK/Mek) for the activation of MAP kinases with tyrosine phosphorylation occurring before serine/threonine autophosphorylation and activation by elk-1 proteins.
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PMID:elk-1 proteins interact with MAP kinases. 820 31

The lipase gene from Pseudomonas aeruginosa TE3285 is followed by another gene, lipB. The lipase gene was expressed in Escherichia coli BL21(DE3)pLysS using the T7 RNA polymerase expression system. The mature lipase was accumulated as inclusion bodies at 42% of the total cell proteins. The inclusion bodies were solubilized with 8 M urea, but lipase activity was not detected in the solubilized preparation containing 85% lipase protein even after removing urea by dialysis. The lipB gene, positioned downstream of the lipase gene and thought to be necessary for the expression of the lipase gene, was expressed in Escherichia coli JM109 as a fusion with the glutathione transferase gene from Schistosoma japonicum. The fusion protein was partially purified on glutathione-agarose beads to 36% purity. Incubated with the fusion protein at a molar ratio of 1:1 at 4 degrees C for 24 h, the solubilized lipase showed lipase activity of about a tenth that of the purified lipase prepared from Pseudomonas aeruginosa TE3285. Magnesium ions and ATP were not essential but increased the activation. When the fusion protein was treated with thrombin to release the glutathione transferase part, it retained its activity. The lipase activation with lipB protein probably proceeds to form a 1:1 complex with the inactive, solubilized lipase protein but by a different mode from known chaperones.
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PMID:Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene. 834 92

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

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics. 839 10


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