scholarly journals Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress

2000 ◽  
Vol 346 (2) ◽  
pp. 553-559 ◽  
Author(s):  
Bartolo FAVALORO ◽  
Antonio TAMBURRO ◽  
Massimo A. TROFINO ◽  
Luciano BOLOGNA ◽  
Domenico ROTILIO ◽  
...  
Keyword(s):  
Mrna Level ◽  
Membrane Damage ◽  
Amino Acid Residues ◽  
Ochrobactrum Anthropi ◽  
Adaptive Responses ◽  
Glutathione S Transferase ◽  
Aromatic Substrates ◽  
Hydrophobic Compounds ◽  
Degrading Bacterium ◽  
Single Subunit

The gluthathione S-transferase gene of the atrazine-degrading bacterium Ochrobactrum anthropi (OaGST) encodes a single-subunit polypeptide of 201 amino acid residues (Favaloro et al. 1998, Biochem. J. 335, 573-579). RNA blot analysis showed that the gene is transcribed into an mRNA of about 800 nucleotides, indicating a monocistronic transcription of the OaGST gene. The modulation of OaGST in this bacterium, in the presence of different stimulants, was investigated. The level of expression of OaGST was detected both by measuring the mRNA level and by immunoblotting experiments. OaGST is a constitutive enzyme which is also inducible by several stimulants. In fact, atrazine caused an increase in the expression of OaGST even at concentrations which had no effect on growth rates of the bacteria. Moreover, the presence of other aromatic substrates of this bacterium, such as phenol and chlorophenols, leads to a marked enhancement in OaGST expression. In this case, the expression of OaGST was related to growth inhibition and membrane damage caused by these hydrophobic compounds, and to the adaptive responses of the cell membranes. On the other hand, toluene and xylene, two aromatic compounds not degradable by this bacterium, did not induce the OaGST expression. The same was observed for other stress conditions such as low pH, heat shock, hydrogen peroxide, osmotic stress, starvation, the presence of aliphatic alcohols or heavy metals. These results suggest a co-regulation of the OaGST gene by the catabolic pathways of phenols and chlorophenols in this bacterium. Therefore, OaGST could function as a detoxifying agent within the catabolism of these xenobiotics.

Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions

2001 ◽  
Vol 360 (3) ◽  
pp. 675-681 ◽  
Author(s):  
Antonio TAMBURRO ◽  
Nerino ALLOCATI ◽  
Michele MASULLI ◽  
Domenico ROTILIO ◽  
Carmine DI ILIO ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Mrna Level ◽  
Chemical Compounds ◽  
Stress Conditions ◽  
Detoxification Enzymes ◽  
Chemical Stress ◽  
Ochrobactrum Anthropi ◽  
Glutathione S Transferase ◽  
Cellular Protection ◽  
Glutathione S Transferases

Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573–579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553–559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

scholarly journals Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

2010 ◽  
Vol 76 (17) ◽  
pp. 6001-6005 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Ai-Sheng Xiong ◽  
Jing Xu ◽  
Wei Zhao ◽  
Feng Gao ◽  
...  
Keyword(s):  
Genomic Library ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Construction Process ◽  
Ochrobactrum Anthropi ◽  
Gene Encoding ◽  
Phosphate Synthase

ABSTRACT Applying the genomic library construction process and colony screening, a novel aro A gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.

scholarly journals Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog

2005 ◽  
Vol 37 (11) ◽  
pp. 784-787 ◽  
Author(s):  
Fen Guo ◽  
Yue-Qin Li ◽  
Shi-Qian Li ◽  
Zhi-Wen Luo ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Homeobox Gene ◽  
Interaction Model ◽  
Cell Division Cycle ◽  
Amino Acid Residues ◽  
Yeast Two Hybrid ◽  
Glutathione S Transferase ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

Abstract Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem cDNA. Fifty-two colonies were obtained after 1.57×108 colonies were screened by nutrition limitation and β-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37 homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.

Pyruvate-induced Long-term Maintenance of Glutathione S-Transferase in Rat Hepatocyte Cultures

2001 ◽  
Vol 29 (3) ◽  
pp. 335-346 ◽  
Author(s):  
Tamara Vanhaecke ◽  
André Foriers ◽  
Albert Geerts ◽  
Elizabeth A. Shephard ◽  
Antoine Vercruysse ◽  
...  
Keyword(s):  
Mrna Level ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Strong Increase ◽  
Mrna Levels ◽  
Glutathione S Transferase ◽  
Albumin Secretion ◽  
Rat Hepatocyte ◽  
Hepatocyte Cultures ◽  
Transmission Electron

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, μ and α class GST activities were well maintained, whereas GST π activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: μ class proteins remained relatively constant, whereas a decrease in the a class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, π class GST activity was increased, but total, μ, and α class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

Design and Characterization of a Labeling Reagent for Covalent Immobilization of Glutathione-S-Transferase

2019 ◽  
Vol 11 (11) ◽  
pp. 1547-1560
Author(s):  
Chao Xia ◽  
Jinpeng Lu ◽  
Bangtian Xu ◽  
Xiaolei Hu ◽  
Yixian Jing ◽  
...  
Keyword(s):  
Active Site ◽  
Covalent Immobilization ◽  
Sulfhydryl Groups ◽  
Submicron Particles ◽  
Primary Amines ◽  
Glutathione S Transferase ◽  
Irreversible Inhibitor ◽  
Hydrophobic Compounds ◽  
Hydrophobic Moiety

A labeling reagent against S. japonicum glutathione-S-transferase (sjGST), denoted as Br-I, was designed, prepared and characterized for covalent immobilization of sjGST on magnetic submicron particles (MSP). Br-I had a large hydrophobic moiety for binding to one active site of sjGST, an extended flexible bromoacetylamide moiety for covalent linkage to any of the accessible amino/sulfhydryl groups through nucleophilic substitution. In addition, Br-I had an extended carboxyl group for conjugation with aliphatic primary amines on the MSP, besides a flexible sketch to link those moieties together. Free Br-I was both a substrate/pro-inhibitor and a monovalent irreversible inhibitor of sjGST. There was >75% inactivation of sjGST after half an hour with free Br-I in excess to the sjGST active site, but only sulfhydryl groups far away from the active site were modified when their quantities were comparable. After conjugation to the MSP, Br-I selectively immobilized sjGST in the presence of alkaline phosphatase as a competitor. The treatment of immobilized sjGST with the mixture of free Br-I and GSH reduced unfavorable adsorption of small hydrophobic compounds. Therefore, after conjugation to biomaterials, Br-I showed promise for covalent site-specific immobilization of sjGST-fused targeted proteins.

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