Bacterial Glutathione S-transferase Research Articles

The Epstein--Barr virus open reading frame BDLF3 codes for a 100-150 kDa glycoprotein

The Epstein-Barr virus (EBV) open reading frame BDLF3 is predicted to code for a glycoprotein on the basis that it contains sequences with signal peptide and transdomain characteristics and nine potential N-linked glycosylation sites. No sequential or positional homologues of BDLF3 have been located in other herpesviruses. A bacterial glutathione S-transferase (GST)-BDLF3 fusion protein was used to demonstrate that over one-third of EBV-immune human sera tested recognized the fusion protein but not GST alone on Western blots. The fusion protein was used to raise polyclonal sera in rabbits. A BDLF3 recombinant baculovirus was constructed using the full-length BDLF3 sequence (AcBDLF3). Rabbit anti-fusion protein sera and some human EBV-immune sera recognized products of approximately 30 and 55 kDa from AcBDLF3-infected insect cells by Western blotting. A peptide representing the carboxy-terminal amino acids 215-234 of the BDLF3 sequence was used to raise anti-peptide sera in rabbits. Anti-peptide serum detected a product by indirect immunofluorescence in acetone-fixed EBV-infected B cells from all cell lines tested. A diffuse band with a molecular mass of 100-150 kDa was detected by Western blot in B95-8 cell lysates, partially purified B95-8 virus and B95-8-infected cell membranes after probing with anti-BDLF3 peptide serum. This product was shown to be glycosylated after enzymatic deglycosylation of a B95-8 virus preparation using neuraminidase, O-glycosidase or N-glycosidase F. The BDLF3 protein products have no known function.

Microbes, enzymes and genes involved in dichloromethane utilization

Dichloromethane (DCM) is efficiently utilized as a carbon and energy source by aerobic, Gram-negative, facultative methylotrophic bacteria. It also serves as a sole carbon and energy source for a nitrate-respiring Hyphomicrobium sp. and for a strictly anaerobic co-culture of a DCM-fermenting bacterium and an acetogen. The first step of DCM utilization by methylotrophs is catalyzed by DCM dehalogenase which, in a glutathione-dependent substitution reaction, forms inorganic chloride and S-chloromethyl glutathione. This unstable intermediate decomposes to glutathione, inorganic chloride and formaldehyde, a central metabolite of methylotrophic growth. Genetic studies on DCM utilization are beginning to shed some light on questions pertaining to the evolution of DCM dehalogenases and on the regulation of DCM dehalogenase expression. DCM dehalogenase belongs to the glutathione S-transferase supergene family. Analysis of the amino acid sequences of two bacterial DCM dehalogenases reveals 56% identity, and comparison of these sequences to those of glutathione S-transferases indicates a closer relationship to class Theta eukaryotic glutathione S-transferases than to a number of bacterial glutathione S-transferases whose sequences have recently become available. dcmA, the structural gene of the highly substrate-inducible DCM dehalogenase, is carried in most DCM utilizing methylotrophs on large plasmids. In Methylobacterium sp. DM4 its expression is governed by dcmR, a regulatory gene located upstream of dcmA, dcmR encodes a trans-acting factor which negatively controls DCM dehalogenase formation at the transcriptional level. Our working model thus assumes that the dcmR product is a repressor which, in the absence of DCM, binds to the promoter region of dcmA and thereby inhibits initiation of transcription.

Immunogold localization of glutathione transferase B1-1 in Proteus mirabilis

By using the immunolabelling technique, the cellular localization of glutathione transferase in Proteus mirabilis was investigated. Evidence was obtained indicating a significant higher content of glutathione transferase in the periplasmic than cytoplasmic space. This result further support the idea that bacterial glutathione transferase is involved in xenobiotic detoxication.

The Schizosaccharomyces pombe cdc5+ gene encodes an essential protein with homology to c-Myb.

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.

Sequence specificity in recognition of the epidermal growth factor receptor by protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases all contain a conserved cysteine that forms an intermediate thiophosphate ester bond during tyrosine phosphate hydrolysis. A bacterial glutathione S-transferase fusion protein containing rat brain phosphatase PTP1b was constructed in which this conserved cysteine was mutated to serine. The resulting catalytically inactive enzyme was labeled in vivo to high specific activity with 35S, and the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealed a nonlinear Scatchard plot, with a Kd for high affinity binding of approximately 100 nM. A number of glutathione S-transferase fusion proteins containing src homology 2 (SH2) domains attenuated phosphatase binding in a concentration-dependent manner. Phospholipase C (PLC) gamma and the GTPase-activating protein of ras were the most potent inhibitors. Tyrosine-phosphorylated EGF receptor peptide fragments were evaluated for specific inhibition of PTP1b and PLC gamma SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine 992, blocked the binding of both fusion proteins. Another phosphopeptide, modeled on tyrosine 1148, inhibited the binding of PTP1b but not the PLC gamma fusion protein. This site specificity was confirmed by analysis of equilibrium binding of the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence specificity in the binding of proteins involved in the regulation of intracellular signaling by receptor tyrosine kinases.

Characterization of Glutathione Transferase from Xanthomonas campestris

A single form of glutathione transferase (Xc-GST-4.5) having an isoelectric point at pH 4.5 was resolved from Xanthomonas campestris cytosol by affinity chromatography and isoelectric focusing. HPLC, N-terminal amino acid sequence, and SDS-PAGE analyses indicate that Xc-GST-4.5 is composed of two identical subunits, each with a molecular mass of 22 kDa. As indicated by its substrate specificity, immunological reactivity, and CD spectra, as well as by its N-terminal amino acid sequence, Xc-GST-4.5 appears to be distinct from the other bacterial glutathione transferases, Pm-GST-6.0 and Sm-GST-7.3, previously purified from the cytosolic fraction of Proteus mirabilis and Serratia marcescens. Xc-GST-4.5 also appears to be distinct from the GST so far purified from other sources.

Recombinant human pim-1 protein exhibits serine/threonine kinase activity

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.

Purification and study of a bacterial glutathione S-transferase

A glutathione S-transferase from Escherichia coli has been purified approximately 800-fold with an 11% activity yield by passage through DEAE Sephacel and glutathione-agarose affinity columns. Its functional form is a homodimer of two 24 000 Da polypeptides that catalyzes the binding of glutathione and 1-chloro-2,4-dinitrobenzene with K m values of 0.25 and 1.5 mM, respectively. Optima of pH and temperature were 7.5 and 35°C. The activity was stimulated (30%) by ethylenediaminetetraacetic add. The N-tenninal amino acid sequence was: Met-Leu-Leu-Phe-Ile-Leu-Pro-Gly-Ala.