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)

A 183-bp fragment encoding variable domain IV (VD IV) of Chlamydia trachomatis serovar B major outer membrane protein (MOMP) (amino acids 273 to 333) and containing the species-specific epitope was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-VD IV). The fusion protein was affinity purified under nondenaturing conditions and used to immunize rabbits. Antisera were characterized by microimmunofluorescence, immunoblot, dot blot, peptide enzyme-linked immunosorbent, and in vitro neutralization assays. Antisera recognized MOMP from all 12 tested serovars of C. trachomatis but not from Chlamydia psittaci. In a dot blot assay, antisera bound to elementary bodies of serovars B, D, E, L2, and K in a strong fashion and to elementary bodies of serovars F, G, A, and H in a weak fashion but not to elementary bodies of serovars C, J, and I. High-resolution peptide mapping with synthetic overlapping serovar B MOMP peptides in a solid-phase enzyme-linked immunosorbent assay showed that immunization with GST-VD IV produced a serologic response that closely mimicked the response produced with purified serovar B elementary bodies. Antipeptide antibodies with strong binding to species- and subspecies-specific epitopes were elicited. Antisera were able to neutralize only those C. trachomatis serovars that bound antibodies in the dot blot assay. These results suggest that antigenic fragments from VD IV containing the species-specific epitope may be useful in the construction of a chlamydial vaccine for some but not all C. trachomatis serovars.
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PMID:Immunologic characterization of a cloned fragment containing the species-specific epitope from the major outer membrane protein of Chlamydia trachomatis. 170 15

The protective immune response to infection with Chlamydia trachomatis is associated with antibody reactivity to serovar-specific determinants on the major outer membrane protein (MOMP). Because this immunity is T cell dependent, it is essential to define those Th cell determinants that promote natural boosting of the protective antibody response. The gene for MOMP of serovar B was separated into nine overlapping fragments that represent the five C and four V regions. These fragments were expressed as fusion peptides with GST and used to identify the regions of the MOMP that contain T cell determinants recognized in BALB/c mice. We identified peptides that elicit a T cell response to Chlamydia by immunizing mice with the fusion peptides and testing the proliferative response of T cells in vitro to intact organism. For analysis of determinants seen after infection, animals were inoculated with live organism and the T cell proliferative response to each fusion peptide was measured in vitro. In contrast to proliferative analysis in which several regions of the MOMP elicited T cell responses, functional analysis demonstrated that a single fusion peptide, containing V segment three, elicited T cell help in vivo for the production of high titered antisera, specific for protective determinants on the MOMP.
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PMID:A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants. 171 17

Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.
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PMID:Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein. 197 54

Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferase were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and Km's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.
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PMID:Identity of microsomal glutathione S-transferases. 714 47

The beta spectrin genes each produce two alternate transcripts the longer of which has a approximately 210 amino acid C-terminal extension including a pleckstrin homology (PH) domain and also an uncharacterized membrane binding site. GST constructs including the entire or the N-terminal segment of the beta I sigma II spectrin PH domain bind to crude and extracted brain membranes, to protein free brain lipid and to vesicles containing phosphatidylinositol-4,5-bisphosphate. This PH domain also binds radiolabelled inositol-1,4,5-trisphosphate (IP3) and preincubation with IP3 inhibits binding to extracted brain membranes. We conclude that membrane binding of the beta I sigma II spectrin C-terminal region is by means of a direct interaction between the N-terminal region of the PH domain and membrane lipids and does not require membrane protein. The PH domain of the beta-adrenergic receptor kinase showed different binding properties in every assay employed, showing that different PH domains may have different membrane binding specificity.
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PMID:The association of the C-terminal region of beta I sigma II spectrin to brain membranes is mediated by a PH domain, does not require membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site. 750 42

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complement-mediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that 5F2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.
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PMID:Mapping of bactericidal epitopes on the P2 porin protein of nontypeable Haemophilus influenzae. 752 Apr 20

The herpes simplex virus 1 UL10 gene encodes a hydrophobic membrane protein dispensable for viral replication in cell culture (J.D. Baines and B. Roizman, J. Virol. 65:938-944, 1991). We report the following. (i) A fusion protein consisting of glutathione S-transferase fused to the C-terminal 93 amino acids of the UL10 protein was used to produce a rabbit polyclonal antiserum. The antiserum reacted with infected-cell proteins which formed in denaturing polyacrylamide gels a sharp band (apparent M(r) of 50,000) and a very broad band (M(r) of 53,000 to 63,000). These bands were not formed by lysates of UL10- virus or by lysates of infected cells boiled in the presence of sodium dodecyl sulfate before electrophoresis. (ii) The proteins forming both bands were labeled by [3H]glucosamine, indicating that they were glycosylated. (iii) The UL10 protein in cells treated with tunicamycin formed a single band (apparent M(r) of 47,000) reactive with the anti-UL10 antibody, indicating that the 47,000-M(r) protein was a precursor of N-glycosylated, more slowly migrating forms of UL10. Treatment of the immunoprecipitate with endoglycosidase H increased the electrophoretic mobility of the 50,000-M(r) species to that of the 47,000-M(r) species, indicating that the 50,000-M(r) species contained high-mannose polysaccharide chains, whereas the proteins forming the 53,000- to 63,000-M(r) bands contained mature chains inasmuch as they were resistant to digestion by the enzyme. (iv) The UL10 protein of R7221 carrying a 20-amino-acid epitope formed only one band with an M(r) of 53,000. This band was sensitive to endoglycosidase H, suggesting that the epitope inserted in the R7221 UL10 protein may have interfered with glycosylation. (v) The UL10 protein does not contain a cleavable signal sequence inasmuch as the first UL10 methionine codon was reflected in the 50,000-M(r) protein. (vi) The UL10 protein is present in virions and plasma membranes of unfixed cells that were reacted with the polyclonal rabbit antibody. In accordance with the current nomenclature, the UL10 protein is designated glycoprotein M.
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PMID:The UL10 gene of herpes simplex virus 1 encodes a novel viral glycoprotein, gM, which is present in the virion and in the plasma membrane of infected cells. 767 47

Triadin is a major membrane protein that is specifically localized in the junctional sarcoplasmic reticulum of skeletal muscle and is thought to play an important role in muscle excitation-contraction coupling. In order to identify the proteins in the skeletal muscle that interact with triadin, the cytoplasmic and luminal domains of triadin were expressed as glutathione S-transferase fusion proteins and immobilized to glutathione-Sepharose to form affinity columns. Using these affinity columns, we find that triadin binds specifically to the ryanodine receptor/Ca2+ release channel and the Ca(2+)-binding protein calsequestrin from CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid)-solubilized skeletal muscle homogenates. The luminal but not the cytoplasmic domain of triadin-glutathione S-transferase fusion protein binds [3H]ryanodine receptor, whereas neither the cytoplasmic nor the luminal portion of triadin binds [3H]PN-200-100-labeled dihydropyridine receptor. In addition, the luminal domain of triadin interacts with calsequestrin in a Ca(2+)-dependent manner and is capable of inhibiting the reassociation of calsequestrin to the junctional face membrane. These results suggest that triadin is the previously unidentified transmembrane protein that anchors calsequestrin to the junctional region of the sarcoplasmic reticulum, and is involved in the functional coupling between calsequestrin and the ryanodine receptor/Ca2+ release channel.
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PMID:Association of triadin with the ryanodine receptor and calsequestrin in the lumen of the sarcoplasmic reticulum. 772 13

The ppk gene, which codes for the enzyme polyphosphate kinase in Neisseria meningitidis strain BNCV, is preceded by an open reading frame coding for a protein with a predicted size of 19.2 kDa with a typical lipoprotein signal sequence of 21 amino acids. The protein has significant homology to the N-terminal portion of an outer membrane protein from Haemophilus somnus (J. Won and R. W. Griffith, Infect. Immun. 61:2813-2821, 1993). Sequencing of the same open reading frame from meningococcus strain M1080 predicted an almost identical protein. Antisera were raised against the lipoprotein, expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The antisera reacted with meningococcal membrane fractions on a Western blot (immunoblot) but did not elicit complement-dependent bactericidal activity. Restriction enzyme digestion demonstrated conservation of this portion of the meningococcal and gonococcal chromosomes. However, antisera raised to the recombinant protein showed that the protein was absent from all strains of gonococcus tested. The sequences of the gene from several strains of Neisseria gonorrhoeae and N. meningitidis were compared and found to be almost identical, except that the coding sequences from all of the gonococcal strains were terminated prematurely as a result of a frameshift mutation. The significance of the remarkable conservation of these gonococcal genes is discussed.
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PMID:Novel lipoprotein expressed by Neisseria meningitidis but not by Neisseria gonorrhoeae. 772 66

Four human breast cancer cell lines with or without estrogen and progesterone receptors were adapted to growth in the continuous presence of doxorubicin (Dox) at 10 (Zr-75-1), 15 (HTB-122), or 50 (MDA-MB-231 and Hs578T) ng/ml. The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines maintained or slightly increased their cis-platinum (CDDP) sensitivity. The sublines of HTB-122 showed resistance only to VP16 combined with a paradoxical increased sensitivity to VCR. The phenotypic alteration in the sublines with respect to Dox sensitivity was maintained for at least two months in the absence of Dox. The glutathione depletor buthionine sulfoximine (BSO) and the calcium channel blocker verapamil (Ver) increased the Dox sensitivity slightly only in the MDA-MB-231 and Hs578T sublines, respectively. Ver also tended to protect some of the sublines from CDDP. The sublines of Zr-75-1 and Hs578T showed increased expression of the 170-kDa permeability glycoprotein (P-gp), whereas expression of a 85-kDa membrane protein determined by the MRK20 antibody was increased in the sublines of Zr-75-1, and HTB-122. Class pi glutathione transferase (GST) levels varied greatly between the cell lines but increased during Dox selection only in the subline of Zr-75-1. Class mu GST was detectable in the MDA-MB-231, Hs578T and HTB-122 cell lines, whereas class alpha GST was detectable in these sublines but undetectable in their parental cell lines. The Zr-75-1 subline showed a 5-fold increase in the class alpha concentration. Except for a correlation between increased P-gp expression and resistance to Dox, VCR and VP16, no obvious correlations between receptor status, increased P-gp expression, membrane proteins, GST levels and acquired drug resistance were found. Thus, except for a possible role for P-gp in multidrug-resistance, these findings indicate a pronounced mechanistic heterogeneity responsible for cytotoxic drug sensitivity also in cells with a common histologic origin and exposed to the same drug.
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PMID:Characterization of four doxorubicin adapted human breast cancer cell lines with respect to chemotherapeutic drug sensitivity, drug resistance associated membrane proteins and glutathione transferases. 790 62


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