The isolation of a fetal rat liver glutathione S-transferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated from fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Yc (Mr 28,000) and a subunit (Mr 25,500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit 'Yfetus'. The enzyme which we term glutathione S-transferase YcYfetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Detection of S-glutathionylated proteins by glutathione S-transferase overlay

Oxidative and nitrosative stress lead to the S-glutathionylation of proteins and subsequent functional impairment. Glutathione S-transferase (GST) from Schistosoma japonicum was found to bind to the glutathione moiety of S-glutathionylated proteins, thus establishing a convenient method for detecting S-glutathionylated proteins by biotinylated GST. Applications of this method to proteins that were prepared from cultured cells and blotted onto a membrane exhibited numerous positive bands, which were abolished by treatment with dithiothreitol. Treatment of a cellular extract with nitrosoglutathione led to enhanced staining of the bands in a dose-dependent manner. The method was also applicable for the histochemical detection of S-glutathionylated proteins in situ. The positive staining by biotin-GST became faint in the presence of S-glutathionylated ovalbumin, suggesting that the reaction is specific to S-glutathionylated proteins. Collectively, these data indicate that the method established here is simple and useful for detecting S-glutathionylated proteins on blotted membrane and in situ.     

Conserved proteins as immunogens: glutathione S-transferase of Schistosoma

Although some immunogenic proteins of parasites evolve rapidly and seem to have no function other than the eliciting o f an immune response, others are proteins that are ordinarily conserved because of functional constraints. The 26 kDa and 28 kDa glutathione S-transferases (GSTs) of Schistosoma would seem to fall in the latter category. However, in the generally more conserved N-terminal portion o f the molecule, the 28 kDa GSTs have evolved over twice as fast at nor-synonymous nucleotide sites as have the 26 kDa GSTs. It is possible that this accelerated rate o f evolution results from natural selection by the host immune system, as discussed here by Austin Hughes.     

Support vector machine based prediction of glutathione S-transferase proteins

Glutathione S-transferase (GST) proteins play vital role in living organism that includes detoxification of exogenous and endogenous chemicals, survivability during stress condition. This paper describes a method developed for predicting GST proteins. We have used a dataset of 107 GST and 107 non-GST proteins for training and the performance of the method was evaluated with five-fold cross-validation technique. First a SVM based method has been developed using amino acid and dipeptide composition and achieved the maximum accuracy of 91.59% and 95.79% respectively. In addition we developed a SVM based method using tripeptide composition and achieved maximum accuracy 97.66% which is better than accuracy achieved by HMM based searching (96.26%). Based on above study a web-server GSTPred has been developed (http://www.imtech.res.in/raghava/gstpred/).     

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Summary Sequences encoding GST-fusion proteins were cloned into the Saccharomyces cerevisiae secretion vector, pYEX-S1, to direct secretion into the culture medium. GST and metallothionein fused to GST were secreted successfully and the fusion proteins purified. With several other GST-fusion proteins however, the proteins were retained inside the cell, indicating limitations to the types of proteins that can be secreted from yeast.     

Nitration/S-nitrosation of proteins by peroxynitrite-treatment and subsequent modification by glutathione S-transferase and glutathione peroxidase

In various peroxynitrite (PN)-treated proteins, the formations of stable 3-nitrotyrosine (nitration) and labile S-nitrosocysteine (S-nitrosation) were observed by employing rapid Western blot in 6 h. The steps of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and membrane-blotting were performed at 4°C. It was noted that the intensity of immunoreactive bands specific for anti-nitrotyrosine was stronger than that specific for anti-S-nitrosocysteine. Additionally, the intensity was in the manner of a dose-dependency of PN. Nitration/S-nitrosation were formed in the following treated proteins, including bovine serum albumin (BSA), DNase-1, ceruloplasmin, catalase and hemoglobin (Hb). The incubation of PN-pretreated hemoglobin with 1 mM reduced glutathione (GSH) did not change immunoreactivity significantly. However, the addition of glutathione S-transferase (GST) or glutathione peroxidase (GPX) to the above incubation mixture, resulted in decreased immunoreactivity, suggesting GSH may form a transition complex with PN-pretreated hemoglobin and/or partially reduce/modify the treated hemoglobin, thereby increasing the accessibility for the subsequent modification by GST or GPX. Such decreased immunoreactivity indicates that nitrotyrosine and S-nitrosocysteine of treated hemoglobin was, indeed, further modified via (a) converting –NO₂ to –NH₂ in tyrosine residues, (b) denitrating –NO₂ directly/indirectly in tyrosine residues, and/or (c) changing –S-NO to –SH in cysteine residues, or denitrosation. The findings imply similar enzymatic modifications of proteins may also occur in vivo, and therefore play a pivotal role in the NO-related cellular signaling cascade(s).     

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.     

Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility

We have previously described our strategy for high-throughput (HT) production of recombinant antigens for anti-mKIAA antibody generation, which involves using shotgun fragments generated during entire sequencing of mKIAA cDNAs. We applied this strategy to 1628 mouse KIAA (mKIAA) cDNA fragments, and 84.2% of the GST-mKIAA fusion proteins were successfully purified. The solubility of the proteins was predicted by a small-scale bacterial culture, and a large-scale culture was then performed according to the expected results. Among them, 43.8% of the proteins were purified as a soluble form and 56.2% as an insoluble form. The average yield of the soluble proteins was 0.15 nmol/mL of bacterial culture, and that of the insoluble proteins was 0.55 nmol/mL Statistical analysis of the data revealed a significant correlation between amino acid features of the recombinant proteins and their solubility. To achieve the most effective and feasible protein expression, we constructed a decision tree in which the analyzed data were reflected. The information described here may provide practical guidelines for HT production of recombinant proteins.     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Crystal structure of non-fused glutathione S-transferase from Schistosoma japonicum in complex with glutathione

Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.     

Radiation inactivation of microsomal glutathione S-transferase

Radiation inactivation analysis was used to determine the target size of rat liver microsomal glutathione S-transferase both in situ and following purification. When Tris-HCl-washed microsomes were irradiated, there was a 1.5-2.0-fold increase in enzymatic activity over the first 3-6 megarads followed by a decrease in enzymatic activity. Above 48 megarads the radiation inactivation curve of the Tris-HCl-washed microsomes was described by a monoexponential function which gave a target size of 48 kDa. The enzymatic activity of the microsomal enzyme was selectively increased by treating the Tris-HCl-washed microsomes either with N-ethylmaleimide or washing the microsomes with small unilamellar vesicles made from phosphatidylcholine. The inactivation curves obtained with both types of treated microsomes were simple monoexponential decays in enzymatic activity with target sizes of 46 kDa (N-ethylmaleimide) and 44 kDa (unilamellar vesicles). The microsomal enzyme was detergent solubilized and purified. The Mr value of the purified protein was 15,500 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These data suggest that the functional unit of the microsomal form of glutathione S-transferase in situ is a trimer. The target size of the purified enzyme solubilized in Triton X-100 was 85 kDa, and no increase in activity was observed at the lower radiation doses. The increase in the target size of the purified enzyme could not be ascribed solely to the presence of the detergent. This result suggests that the microsomal form of this enzyme can exist as catalytically active oligomers of different sizes depending on its environment.     

Stereoselective product inhibition of glutathione S-transferase

Isozymes 3-3 and 4-4 of rat liver glutathione S-transferase are stereoselectively inhibited by the diastereomers of 9,10-dihydro-9-glutathionyl-10-hydroxyphenanthrene, 1. The conformation of the biphenyl moiety is the same in the enzyme -1 complex as in aqueous solution with the glutathionyl and hydroxy groups in the axial positions. Isozyme 4-4 is also inhibited by the four diastereomers of 1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane. The stereoselectivity of inhibition is modest in all cases and is manifest in both the type of inhibition as well as the magnitude of Ki.     

A ligand function of glutathione S-transferase

Glutathione S-transferases (GSTs) are ubiquitous enzymes and abundant in plants. They are intimately involved in plant metabolism and stress defense related to reactive oxygen species. Our project assigned particular reactions including novel ones to certain GST-isoforms. Transformed E. coli was used to express recombinant GST-isoforms from maize. An N-terminal His tag allowed their purification by affinity chromatography. Three GST-monomers had a molecular weight of 26, 27, 29 kDa, and aggregated to dimers when assayed for their enzymic properties. Four dimeric isoforms were used to study how they interact with tetrapyrroles (of the chlorophyll biosynthesis pathway). It was found that protoporphyrin IX (Proto IX), Mg-protoporphyrin and other tetrapyrroles are bound non-covalently ("liganded") to GSTs but not conjugated with reduced glutathione. This binding is non-covalent, and results in inhibition of conjugation activity, the degree depends on type of the porphyrin and GST-isoform. I50-values between 1-10 microM were measured for Proto IX, the inhibition by mesoporphyrin and Mg-protoporphyrin was 2- to 5-fold less. The ligand binding is noncompetitive for the substrate 1-chloro-2,4-dinitrobenzene and competitive for glutathione. The dimer GST 26/26 prevents the (non-enzymic) autoxidation of protoporphyrinogen to Proto IX, which produces phytotoxic reactive oxygen species in the light. GST 27/27 protects hemin against degradation. Protoporphyrinogen is formed in the plastid and then exported into the cytosol. Apparently binding by a suitable GST-isoform ensures that the highly autoxidizable protoporphyrinogen can safely reach the mitochondrium where it is processed to cytochrome.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Calcium-dependent association of glutathione S-transferase with the human erythrocyte membrane

Elevations in intracellular calcium increase the adsorption of a cytoplasmic protein to human red blood cell membrane. This protein migrates on SDS polyacrylamide gels at 23,000 daltons and has been called band 8. The association of this protein with the membrane is increased in sickle cell anemia. This protein is extracted from the membrane with EGTA, a calcium chelator. Enzymatic and immunological studies identify band 8 as a glutathione S-transferase.     

Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B.

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide 'mapping' revealed structural differences between the two proteins.     

alpha-Tocopherol inhibits human glutathione S-transferase pi

alpha-Tocopherol is the most important fat-soluble, chain-breaking antioxidant. It is known that interplay between different protective mechanisms occurs. GSTs can catalyze glutathione conjugation with various electrophiles, many of which are toxic. We studied the influence of alpha-tocopherol on the activity of the cytosolic pi isoform of GST. alpha-Tocopherol inhibits glutathione S-transferase pi in a concentration-dependent manner, with an IC(50)-value of 0.5 microM. At alpha-tocopherol additions above 3 microM there was no GST pi activity left. alpha-Tocopherol lowered the V(max) values, but did not affect the K(m) for either CDNB or GSH. This indicates that the GST pi enzyme is noncompetitively inhibited by alpha-tocopherol. An inhibition of GST pi by alpha-tocopherol may have far-reaching implications for the application of vitamin E.