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)

The ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.
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PMID:The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. 142 34

The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were met with COS monkey kidney cells that express a recombinant glutathione S-transferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony-forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20-22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection with DEAE-dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25-40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.
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PMID:Gene transfer by electroporation, lipofection, and DEAE-dextran transfection: compatibility with cell-sorting by flow cytometry. 154 55

Fatty acid ethyl ester synthases metabolize ethanol nonoxidatively in those extrahepatic organs most commonly damaged by alcohol abuse. This study was designed to isolate and purify human myocardial synthase-II, one of the enzymes responsible for catalyzing the formation of fatty acid ethyl esters. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase-I, synthase-II, and synthase-III activities, eluting at conductivities of 5, 7, and 11 mS, respectively. From this elution profile, fatty acid ethyl ester synthase-II accounts for up to 50% of total synthesis in the human heart. This enzyme species was purified over 2200-fold to homogeneity after chromatography over hydroxylapatite, CM-cellulose, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this homogeneous species showed a single band at 65 kDa which corresponded to its molecular weight determined by gel filtration. This molecular weight and its lack of glutathione transferase activity indicate that this species is not related to synthase-I and -III. Homogeneous synthase-II has a Vmax for palmitate, stearate, oleate, and linoleate of 70, 80, 140, and 120 nmol/mg/h, respectively. The Km for palmitate, stearate, oleate, and linoleate is 0.19, 0.12, 0.10, and 0.18 mM, respectively. The substrate specificity with respect to alcohol chain length was also investigated in the presence of 0.65 mM [14C]oleic acid. The Vmax for methanol, ethanol, propanol, and butanol was 180, 100, 280, and 410 nmol/mg/h, respectively. The Km for methanol, ethanol, propanol, and butanol was 1.16, 1.04, 0.58, and 0.33 M, respectively. The N-terminal 17-amino acid sequence of human synthase-II does not correspond to any known N-terminal amino acid sequence, indicating that this may be a novel protein. However, it has over 70% homology to a sequence close to the C terminus of rabbit cytochrome P-450IIC1 and over 50% homology to a sequence of human hemopexin starting at residue 16. Synthase-II does not cross-react with human hemopexin antibody and rat cytochrome P-450C antibody. Thus, this study provides evidence that synthase-II is a novel protein, distinct from synthase-I and -III, and it also provides a foundation for subsequent cloning and genetic studies of fatty acid ethyl ester synthase-II in man.
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PMID:Purification and characterization of fatty acid ethyl ester synthase-II from human myocardium. 161 26

Human muscle glutathione S-transferase isozyme, GST zeta (pI 5.2) has been purified by three different methods using immunoaffinity chromatography, DEAE cellulose chromatography, and isoelectric focusing. GST zeta prepared by any of the three methods does not recognize antibodies raised against the alpha, mu, or pi class glutathione S-transferases of human tissues. GST zeta has a blocked N-terminus and its peptide fingerprints also indicate it to be distinct from the alpha, mu, or pi class isozymes. As compared to GSTs of alpha, mu, and pi classes, GST zeta displays higher activities toward t-stilbene oxide and Leukotriene A4 methyl ester. GST zeta also expresses GSH-peroxidase activity toward hydrogen peroxide. The Kms of GST zeta for CDNB and GSH were comparable to those reported for other human GSTs but its Vmax for CDNB, 7620 mol/mol/min, was found to be considerably higher than that reported for other human GSTs. The kinetics of inhibition of GST zeta by hematin, bile acids, and other inhibitors also indicate that it was distinct from the three classes of GST isozymes. These studies suggest that GST zeta corresponds to a locus distinct from GST1, GST2, and GST3 and probably corresponds to the GST4 locus as suggested previously by Laisney et al. (1984, Human Genet. 68, 221-227). The results of peptide fingerprints and kinetic analysis indicate that as compared to the pi and alpha class isozymes, GST zeta has more structural and functional similarities with the mu class isozymes. Besides GST zeta several other GST isozymes belonging to pi and mu class have also been characterized in muscle. The pi class GST isozymes of muscle have considerable charge heterogeneity among them despite identical N-terminal sequences.
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PMID:Purification and characterization of human muscle glutathione S-transferases: evidence that glutathione S-transferase zeta corresponds to a locus distinct from GST1, GST2, and GST3. 184 34

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 Km values of 0.25 and 1.5 mM, respectively. Optima of pH and temperature were 7.5 and 35 degrees C. The activity was stimulated (30%) by ethylenediaminetetraacetic acid. The N-terminal amino acid sequence was: Met-Leu-Leu-Phe-Ile-Leu-Pro-Gly-Ala.
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PMID:Purification and study of a bacterial glutathione S-transferase. 218 38

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.
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PMID:Purification and characterization of glutathione S-transferases from guinea pig liver. 233 12

A novel, alpha-class glutathione S-transferase (GST) isozyme has been isolated from human liver using glutathione (GSH) affinity chromatography, DEAE-cellulose ion-exchange chromatography, and immunoaffinity chromatography. The isozyme is a dimer of approximately 25,000 Mr with blocked N termini. Structural, kinetic, and immunological properties of this enzyme indicate that it belongs to the alpha class of GSTs. Noticeable differences between the properties of this enzyme and the other alpha-class GSTs of human liver are its anionic nature (pI 5.0), GSH peroxidase activity toward hydrogen peroxide, and relatively higher GSH conjugating activities toward CDNB and epoxide substrates as compared to other alpha-class GSTs. Results of these studies indicate that anionic GST omega characterized previously (Y. C. Awasthi, D. D. Dao, and R. P. Saneto, 1980, Biochem. J. 191, 1-10) from human liver is a mixture of GST pi and a novel alpha-class GST. We have, therefore, reassigned the name GST omega to this new alpha-class anionic GST of human liver.
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PMID:Characterization of a novel alpha-class anionic glutathione S-transferase isozyme from human liver. 233 54

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.
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PMID:Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase. 308 77

This report describes the isolation of the major calmodulin-stimulated methyl acceptor protein of adult rat liver cytosol. This Mr 29,000 methyl acceptor protein (MeAP29) has been purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and Sephadex G-75. Affinity chromatography on glutathione-Sepharose and assays of enzyme activity indicate that MeAP29 is a member of the glutathione S-transferase family. We further show that glutathione can act as an inhibitor of calmodulin-stimulated in vitro methylation of MeAP29 and that MeAP29 methylation is enhanced in non-dialyzed liver cytosol from rats with lowered glutathione levels.
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PMID:Identification of glutathione S-transferase as a substrate and glutathione as an inhibitor of in vitro calmodulin-stimulated protein methylation in rat liver cytosol. 317 41

The anionic glutathione transferase of human heart has been purified to homogeneity by using DEAE-cellulose, affinity chromatography, and FPLC. The enzyme has an isoelectric point at pH 4.75 and has an electrophoretic mobility on SDS-PAGE identical to placental transferase pi, indicating that the heart enzyme is formed by two similar subunits of 23,000 Mr. Upon isoelectric focusing on ampholine PAG plates the enzyme recovered from FPLC gave two bands of activity at pH 4.75 and 4.9 which were reduced to essentially a single band at pH 4.75 after incubation with dithiothreitol. In the immunodiffusion experiment, the heart enzyme gave a positive precipitin line with the antibodies against transferase pi but not with antibodies prepared against the "basic" transferase of human skin or against the "near-neutral" transferase of human uterus. The substrate specificities, the sensitivities to characteristic inhibitors, the amino acid composition, together with the immunological studies, strongly indicate that the anionic enzyme of human heart is closely related to the transferase pi of human placenta. The N-terminal amino acid sequence of the first 48 residues was determined and compared with the N-terminal region of other reported human glutathione transferase sequences. The heart enzyme differs from the placental enzyme in a single residue (Trp instead of Arg in the 28th position) further supporting their similarity.
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PMID:Acidic glutathione transferase from human heart. Characterization and N-terminal sequence determination. 319 Sep 21


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