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)

Recently, a mouse glutathione S-transferase (GST) isozyme, mGSTA4-4, which belongs to a distinct group of GSTs has been characterized in our laboratory. During the present studies, Western blot analyses of bovine ocular tissues using the antibodies raised against the recombinant mGSTA4-4 obtained by expression in Escherichia coli revealed that the orthologs of mGSTA4-4 were present in cornea, retina, iris-ciliary body and sclera, but absent in lens. These novel GST isozymes of bovine ocular tissues were purified by immunoaffinity chromatography using the antibodies against rec-mGSTA4-4 and were designated as bGST 5.8 (their pI value being 5.8). Amino acid sequences of CNBr fragments of bGST 5.8 from cornea, sclera, retina and iris-ciliary body showed high degree of primary structure homologies with the corresponding regions of mGSTA4-4 indicating these bovine GST isozymes were distinct from the alpha. mu and pi group GSTs and were the newest members of the group of GSTs to which mGSTA4-4 belongs. There were significant differences among the amino acid sequences of bGST 5.8 of cornea and iris-ciliary body and retina suggesting presence of at least two closely related genes at bGST 5.8 locus. bGST 5.8 isozymes showed high activity toward 4-HNE (four-to-five-fold higher than that towards 1-chloro-2,4-dinitrobenzene), expressed GSH-peroxidase activity towards fatty acid hydroperoxides and phospholipid hydroperoxides, and showed GSH-conjugating activity towards fatty acid epoxides suggesting that these isozymes may play an important role in protection mechanism against the endogenous toxicants formed during lipid peroxidation.
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PMID:A group of novel glutathione S-transferase isozymes showing high activity towards 4-hydroxy-2-nonenal are present in bovine ocular tissues. 783 4

We have previously isolated a cDNA clone for a unique mouse lung glutathione S-transferase, mGSTA4-4 (GST 5.7) (Zimniak, P., Eckles, M. A., Saxena, M., and Awasthi, Y. C. (1992) FEBS Lett. 313, 173-176). By genomic Southern blotting and polymerase chain reaction single strand conformation polymorphism analysis we have now demonstrated the presence of at least two mGSTA4-related genes in the mouse. The heterogeneity of mGSTA4-4 was further examined by comparing the structural and kinetic properties of mGSTA4-4 isolated from mouse lung with those of recombinant rec-mGSTA4-4 expressed in Escherichia coli. Except for the isoelectric point, the physical properties of the two proteins were indistinguishable. Western blots using antibodies against rec-mGSTA4-4 have shown selective expression of the enzyme in mouse tissues. Even though the substrate specificity profiles of the tissue-isolated and recombinant enzymes, which point to a role of mGSTA4-4 in the detoxification of lipid peroxidation products, were generally similar, significant differences were observed with selected substrates. The existence of functionally distinct forms of mGSTA4-4 and the presence of more than one gene strongly suggest that the previously observed differences in properties of mGSTA4-4 isolated from various mouse tissues (Awasthi, S., Singhal, S. S., Srivastava, S. K., and Awasthi, Y. C. (1993) Arch. Biochem. Biophys. 301, 143-150) may be due to tissue-specific expression of mGSTA4-related genes.
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PMID:Estimation of genomic complexity, heterologous expression, and enzymatic characterization of mouse glutathione S-transferase mGSTA4-4 (GST 5.7). 790 5

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the alpha-class isozymes of GST. It is characterized by kinetic properties intermediate between the alpha- and pi-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P., et al. (1992) FEBS Lett., 313, 173-176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other alpha-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M(r) values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxides suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.
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PMID:A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues. 814 70

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and beta-migrating very-low-density lipoprotein (beta-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated alpha 2-macroglobulin and beta-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated alpha 2-macroglobulin (alpha 2M-T) was cleared rapidly by the liver (maximal uptake of 80.8 +/- 1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)/kg rat reduced the liver uptake to 62.2 +/- 2.3%, 59.3 +/- 1.1%, or 2.9 +/- 0.1% of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of 125I-beta-VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I-alpha 2M-T was 98% inhibited by GST-RAP with an IC50 of 0.3 microgram/ml (4.2 nM), whereas the binding of 125I-beta-VLDL and 125I-beta-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 micrograms/ml (700 nM). Also, the cell association and degradation of alpha 2M-T was blocked by RAP, while the association and degradation of beta-VLDL and beta-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of alpha 2M-T lasted for 1-2 h of incubation at 37 degrees C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (the alpha 2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of beta-VLDL by rat liver parenchymal cells is not mediated by the alpha 2Mr/LRP. The properties of binding of beta-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the alpha 2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.
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PMID:Blockade of the alpha 2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein on rat liver parenchymal cells by the 39-kDa receptor-associated protein leaves the interaction of beta-migrating very-low-density lipoprotein with the lipoprotein remnant receptor unaffected. 902

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.
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PMID:The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall. 977 3

Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a cloned thrombin consensus sequence (. LVPRGS. ) located between glutathione S-transferase and mMRP14. Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/Western blotting revealed two immunoreactive cleavage products with molecular weights around 13, 000. Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da. The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da). Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence (. NNPRGH. ) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) with a calculated mass of 11,918.6 Da. Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14. Native mMRP14 contains one intramolecular disulfide bond between Cys79 and Cys90. The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding.
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PMID:Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9). 1004 80

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.
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PMID:Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals. 1021 39

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
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PMID:Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase. 1067 18

In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation.
Anat Rec 2002 Dec 01
PMID:Enhanced immunocytochemical expression of antioxidant enzymes in rat submandibular gland after normobaric oxygenation. 1242 Feb 85

To elucidate the role of some viral and cellular proteins in the occurrence and development of HERV-K-associated germ-cell tumors (GCT), reverse-transcription polymerase chain reaction using specific primers has been employed to study the transcription of the protein Rec HERV-K and the possible interaction of the protein Rec(cORF), that has transforming properties, and the cellular protein PLZF, that is a negative regulator of cell division, in human GCT tissues, in the testicular parenchyma adjacent to a tumor, and in the normal testicular tissues. It was shown that there was expression of Rec(cORF) of mRNA, rather than cellular PLZF in all malignant GCT tissues, this led to the conclusion that no interaction occured between the Rec HERV-K and PLZF proteins in the GCT cells. At the same time co-expression of Rec and PLZF protein was first revealed at the level of transcription in the testicular parenchyma adjacent to a tumor that exhibited carcinoma in situ cells. By taking into account that the protein Rec HERV-K has transforming activity and it is presumed to be Implicated in the development of GCT, the authors discuss a possible role in the Rec HERV-K/HTDV and cellular PLZF interaction in the pathogenesis of GST at the early stages of its genesis.
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PMID:[HERV-K-associated carcinogenesis: co-expression of viral and cellular proteins in the development of human germ-cell tumors]. 1945 8


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