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 common substrate for glutathione S-transferases (GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth. This growth inhibition by CDNB is enhanced when E. coli expresses a functional GST. Cells under growth inhibition have reduced intracellular GSH levels and form filaments when they resume growth. Based on this differential growth inhibition by CDNB we have developed a simple procedure to select for null-mutants of a human GST in E. coli. Null mutations in the human GST gene from hydroxylamine mutagenesis or oligonucleotide-directed mutagenesis can be selected for on agar plates containing CDNB after transformation. The molecular nature of each mutation can be identified by DNA sequence analysis of the mutant GST gene. We have identified three essential amino acid residues in an alpha class human GST at Glu31, Glu96, and Gly97. Single substitution at each of these residues, E31K, E96K, G97D, resulted in mutant GST proteins with loss of CDNB conjugation activity and failure in binding to the S-hexyl GSH affinity matrix. In contrast, a mutant GST (Y8F) resulting from substitution of the conserved tyrosine near the N terminus has much reduced CDNB conjugation activity but was still capable of binding to the S-hexyl GSH-agarose. Additional mutant GSTs with substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S) resulted in changes in both Km and kcat to different extents. The in vitro CDNB conjugation activity of the purified mutant enzymes correlate negatively with the plating efficiencies of strains encoding them in the presence of CDNB. Based on the x-ray structure model of human GST 1-1, two of these residues are involved in salt bridges (Arg19-Glu31, Arg68-Glu96) and the third Gly97 is in the middle of the helix alpha 4. Our results provide evidence in vivo that Tyr8, Gly97, and the two salt bridges are important for GST structure-function. This molecular genetic approach for the identification of essential amino acids in GSTs should be applicable to any GSTs with CDNB conjugation activity. It should also complement the x-ray crystallographic approach in understanding the structure and function of GSTs.
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PMID:A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli. 781 27

A chimeric enzyme (GST121) of the human alpha-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P2(1)2(1)2(1), with cell dimensions a = 49.5, b = 92.9, c = 115.9 A, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution, resulting in a change of its solution properties.
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PMID:A surface mutant (G82R) of a human alpha-glutathione S-transferase shows decreased thermal stability and a new mode of molecular association in the crystal. 789 74

An oncogene product, p53, interacts with a simian virus 40-encoded T-antigen, which is an initiation protein for the viral DNA replication and also works as DNA helicase during elongation. Here we examine the interaction of p53 with cellular DNA helicase. A recombinant human wild type p53 fused with glutathione S-transferase was immobilized on glutathione-agarose as a ligand for affinity column. Hela cell extract was applied to the p53 column and the adsorbed proteins were eluted with buffers containing salt, 50% ethylene glycol, and glutathione. The ethylene glycol fraction contained a number of p53 binding proteins, and this fraction showed a DNA helicase activity measured by the displacement of DNA fragment from partially duplexed M13 DNA. The DNA helicase translocated in a 5'-to-3' direction on the single-stranded DNA using ATP as an energy source. The glutathione fraction that contained the p53 glutathione S-transferase fused protein also showed the same activity. The corresponding fractions from a control column carrying glutathione S-transferase showed only a trace amount of activity of DNA helicase. Therefore, the binding may be specific. Furthermore, an anti-p53 antibody column retained a p53-DNA helicase complex when the crude extracts of human placenta and of osteosarcoma cells were applied. These results indicate that p53 physically interacts with DNA helicase in vitro as well as in vivo.
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PMID:Anti-oncogene product p53 binds DNA helicase. 795 81

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0-200 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.
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PMID:Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography. 818 Jun 56

The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast. The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non-permissive temperature, it was not investigated further. Here, we report the characterization of the single allele of this gene, cdc5-120, in more detail. The mutant arrests with a 2N DNA content and a single interphase nucleus. Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle. We have cloned and sequenced the cdc5+ gene. The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors. Deletion mapping of the cdc5+ gene has shown that the N-terminal 232 amino acids of the protein, which contain the Myb-related region, are sufficient to complement the cdc5ts strain. A cdc5 null mutant was generated by homologous recombination. Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene. A fusion protein consisting of bacterial glutathione S-transferase joined in-frame to the N-terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations. This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2.
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PMID:The Schizosaccharomyces pombe cdc5+ gene encodes an essential protein with homology to c-Myb. 831 92

We have identified both biochemically and genetically a protein domain within the simian virus 40 virion protein Vp3, and within Vp2 since its carboxyl two-thirds are identical to the full-length Vp3, that binds DNA in a sequence nonspecific manner. Both the Vp2 and Vp3 (Vp2/3) components of SV40 and mutant SV40(202T) bound either SV40 or pBR322 DNA equally well. Wild type and mutant Vp2/3 proteins, expressed as fusion proteins with glutathione S-transferase (GST), were tested for their ability to bind DNA. GST-Vp3 bound DNA at physiological salt concentrations with an apparent Kd of 2.5 x 10(-8) M and also bound RNA with 4-fold higher affinity. Over 90% of the nucleic acid binding, and all of the activity, was lost upon removal of the carboxyl-terminal 13 and 35 residues, respectively. The DNA binding domain was shown to be distinct and separable from the Vp2/3 nuclear transport signal since mutations within the nuclear transport signal that reduce or abolish nuclear localization of Vp2/3 had no effect on the DNA binding activity of mutant Vp2/3 fusion proteins. The carboxyl-terminal 40 residues of Vp2/3 in the form of a beta-galactosidase fusion protein, F6, are sufficient for DNA binding and may cause compaction of the DNA. The significance of this DNA binding and possible compaction are discussed in relation to the assembly of virion particles.
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PMID:Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3. 840 20

Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids. Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions. However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure. RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras. This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.
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PMID:p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues. 866 24

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.
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PMID:Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit). 875 77

Numbers of light-footed clapper rails Rallus longirostris levipes, an endangered bird inhabiting southern California salt marshes, have substantially declined from historic levels. RAPD (randomly amplified polymorphic DNA) analysis was employed to assess the genetic variability within and among four of the largest remaining light-footed clapper rail populations. A single, larger population of the endangered Yuma clapper rail Rallus longirostris yumanensis was used for comparison. A total of 325 RAPD primers were tested on DNA from a subset of five clapper rails composed of a single representative for each of the four light-footed clapper rail populations and a representative for the single Yuma clapper rail population. Of the 1338 amplified bands (loci) surveyed in these five representative birds, approximately 1% were polymorphic, indicating the level of differentiation across all loci is quite low. Nine primers yielding these 16 polymorphic bands were used to analyse 48 individuals from five populations. Five of these bands were polymorphic in both subspecies, six were polymorphic only within the light-footed clapper rails, and five were polymorphic only within the Yuma clapper rail samples. Considering the few bands that were polymorphic among the light-footed clapper rail populations, a surprisingly high level of population differentiation (GST = 0.28) was found. This is in accord with the results of AMOVA analyses which show that a fairly high percentage of the limited variability among the rails is due to either differences between subspecies or differences between the light-footed rail populations. Because inbreeding depression is suspected and overall genetic distances between populations are low, movement of light-footed clapper rails from larger populations into smaller ones might be considered as a management strategy. Employing RAPDs as one of a series of assays is useful in revealing the population structure of genetically depauperate species.
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PMID:RAPD analysis reveals low genetic variability in the endangered light-footed clapper rail. 879 57

In vivo effects of methyl nitrosourea (MNU) on rat liver glutathione, glutathione S transferase and glutathione reductase have been studied. MNU was injected (20 mg per kg i.r.) weekly for 10 weeks. The animals were killed by decapitation 6 months later, livers were removed, homogenized and the enzymes were studied. Twenty-five animals were treated with MNU. Physiological salt solution was given to ten control animals. It was shown that the cytotoxic effects of MNU cause a decrease of glutathione levels. There was no difference in activity of glutathione reductase in the MNU treated group when compared to control, but glutathione S-transferase activity of the MNU treated group was found to be increased. Glutathione protects the cells against alkylating agents, which may cause cell damage due to depletion of glutathione levels. Increased levels of glutathione S-transferase may be a reaction of the organism to the alkylating agents. Because of depletion of glutathione levels, glutathione reductase levels may be unaffected.
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PMID:Glutathione and related enzymes in rat liver treated with methyl nitrosourea. 908 Mar 8


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