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)

Mice of the strain WEHI 129/J are genetically resistant to chronic Schistosoma mansoni infection. Resistance is expressed in at least 50% of mice, with the remaining mice showing normal susceptibility to infection. The serum antibody specificities in the resistant proportion of WEHI 129/J were analyzed at various times after exposure to cercariae by using both Western blotting and immunoprecipitation. Comparisons with the susceptible proportion of WEHI 129/J and other permissive mouse strains revealed four antigens that were differentially recognized by resistant mice at various times of infection: Sm25, an Mr 25,000 integral membrane protein of adult worms that was better recognized by resistant mice 40 to 50 days after exposure; Sm67, an Mr 67,000 water-soluble antigen of adult worms that was better recognized by resistant mice at days 30 to 40; Sm120, an Mr 120,000 antigen expressed by cercariae and adult worms that was differentially recognized, although inconsistently, at days 20 to 40 postexposure; and Sm26, an Mr 26,000 glutathione S-transferase that was uniquely recognized by resistant mice at day 20 in two of three experiments. Analysis of antibody specificities in (BALB/c x WEHI 129/J)F1 x WEHI 129/J backcross mice indicated that high responsiveness to Sm25 at days 40 to 50 correlated with resistance. The candidacy of these four molecules as vaccines for schistosomiasis mansoni is discussed.
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PMID:Schistosoma mansoni antigens differentially recognized by resistant WEHI 129/J mice. 313 67

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.
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PMID:Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats. 618 97

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.
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PMID:Molecular cloning and characterization of human kinectin. 778 43

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.
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PMID:Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5. 813 57

Vaccination with radiation-attenuated cercariae confers the highest levels of resistance to challenge infection in experimental schistosomiasis and requires Ag-specific T cells. Therefore, this study aimed to identify specific Ag that stimulate the cellular immune response of mice vaccinated with irradiated cercariae of Schistosoma mansoni. Four experimental groups representing different levels of resistance in the vaccine model (C57BL/6J versus CBA/J mice vaccinated with 15- or 50-krad irradiated cercariae) were compared for in vitro lymphocyte proliferation and lymphokine production. Adult worm extracts fractionated by isoelectric focusing were used as Ag. Lymphocyte proliferation of all groups was limited to three consecutive isoelectric fractions (pH 4.6-6.3). Interestingly, the antibody response of these mice was directed to Ag in the same isoelectric fractions, three of which had previously been identified as paramyosin, heat shock protein 70, and the integral membrane protein Sm23. These Ag as well as two 28 kDa proteins, triosephosphate isomerase and glutathione S-transferase, in purified native or recombinant form or as a synthetic peptide, stimulated lymphocyte proliferation. Lymphocytes of vaccinated C57BL/6J mice generally showed higher levels of proliferation than did CBA/J mice. Interestingly, cells of once-vaccinated mice responded better than did cells of mice vaccinated three times. Lymphokine assays demonstrated that IL-2 and IL-4 was generally reduced after multiple vaccinations and varied qualitatively as well as quantitatively between mouse strains. This study substantiates that the five Ag, paramyosin, heat shock protein 70, triosephosphate isomerase, glutathione S-transferase, and the integral membrane protein Sm23, are important candidates for a defined antischistosomal vaccine.
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PMID:Candidate vaccine antigens that stimulate the cellular immune response of mice vaccinated with irradiated cercariae of Schistosoma mansoni. 832 27

In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, we examined the effect of (i) single versus multiple vaccinations with irradiated cercariae, (ii) the dose of irradiation (15 or 50 kilorads) administered to the cercariae, and (iii) the genetic background of mouse strains, high-responder (C57BL/6J) versus moderate-responder (CBA/J) mice. We found that the number of vaccinations did not alter antibody specificity but modified the relative antibody titers against particular antigens. The dose of irradiation used to attenuate the immunizing cercariae had a similar effect on antibody titers but in addition influenced antibody specificity. Only mice that had been vaccinated with moderately irradiated cercariae recognized cathepsin B (Sm31) and Sm32. Interestingly, when vaccinated mice of the two strains, C57BL/6J and CBA/J, were compared, differences in antibody responses to particular antigens were observed. Both strains recognized the integral membrane protein Sm23, glutathione S-transferase, and cathepsin B, whereas Sm32 and paramyosin were recognized only by CBA/J mice, and heat shock protein 70 was recognized exclusively by C57BL/6J mice. In this study, we conclusively identified six distinct antigens that are specifically recognized by the humoral immune response of vaccinated mice.
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PMID:Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni. 841 37

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.
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PMID:Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. 862 45

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.
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PMID:Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene. 877 32

The far-ranging distribution of genes for aromatic hydrocarbon catabolism, predominantly studied in soil pseudomonads, is extended to a marine oligobacterium by finding five homologous sequences in a 5.7-kb chromosomal DNA from a new isolate, Cycloclasticus oligotrophus RB1. RB1 is capable of growth in unamended seawater or mineral salts media supplemented with a variety of aromatic compounds, including toluene, o-, m-, or p-xylenes, as sole carbon sources. The five open reading frames, designated xylM, K, G, C1, and C2, are 57% A+T-rich. XylM is predicted to be an integral membrane protein; XylK and XylG possess glutathione S-transferase (GST) and 2-hydroxy-5methyl-6-oxohexa2,4-dienoate dehydrogenase activities, respectively; XylC1C2 are homologs of the large and small subunits of the iron sulfur protein component of the biphenyl dioxygenase (e.g., BphA1A2).
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PMID:A marine oligobacterium harboring genes known to be part of aromatic hydrocarbon degradation pathways of soil pseudomonads. 878 14

Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Galpha subunits, H-Ras, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Galpha subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein alpha-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.
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PMID:Src tyrosine kinases, Galpha subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. 891 May 75


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