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PubMed1996

Glutathione, glutathione S-transferase and reactive oxygen species of human scalp sebaceous glands in male pattern baldness.

We investigated the contribution of reactive oxygen species to the development of sebaceous gland hyperplasia and the characteristics of the glutathione S-transferase/glutathione system in male pattern baldness. Glutathione S-transferase, glutathione, and thiobarbituric acid-reactive substances were determined in sebaceous gland-enriched scalp skin of men affected by male pattern baldness and were subjected to hair autotransplantation. In comparison with the hairy occipital-donor areas, the following results were obtained in alopecic frontoparietal samples: glutathione S-transferase-specific activity increased 7-fold (p < 0.001); enzyme affinity towards 1-chloro-2,4-dinitrobenzene decreased 2-fold (p = 0.009); glutathione content decreased 2.5-fold (p = 0.017); and thiobarbituric acid reactive substances increased 2-fold (p = 0.006). Chromatofocusing analysis, bromosulfophthalein IC50 values, enzyme-linked immunosorbent assay, and immunohistochemistry with polyclonal antibodies raised against glutathione S-transferases alpha, mu, and pi demonstrated the presence of alpha, pi, and probably the 5.8 alpha isoenzymes in the sebaceous gland. These results support the hypothesis that reactive oxygen species are involved in the pathogenesis of sebaceous gland hyperplasia in male pattern baldness.

Adult↗
PubMed1991

Characterization of glutathione S-transferase in cultured human keratinocytes.

The glutathione S-transferase activity and isozymic composition of cultured human keratinocytes were characterized. Keratinocytes were grown in culture and harvested at different stages of differentiation. Glutathione S-transferase activity was found in the soluble cell fraction but not in the microsomal cell fraction. The glutathione S-transferase specific activity of the soluble cell fraction was found to increase as the keratinocytes differentiated in culture. All of the enzymatic activity was found to reside with a single isozymic form that was concluded to be the pi form of the enzyme based on substrate specificity, sensitivity to inhibitors, molecular weight, and reactivity towards antibodies raised to alpha, mu, and pi forms of the enzyme. It is concluded that all of the isozymic forms of glutathione S-transferase noted in whole skin, with the exception of pi, are of extra-keratinocyte origin.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of a pi class glutathione S-transferase from human leukaemic cells.

The glutathione S-transferases are a group of enzymes involved in the detoxification of a wide range of xenobiotics. Elevation of the level of activity of glutathione S-transferases within the cytosol has been associated with the development of resistance to a number of cytotoxic drugs, including some commonly used in the treatment of leukaemia. In this paper we describe the purification and characterization of an anionic (p class) form of the enzyme from the peripheral blood of patients with acute myeloid leukemia, chronic myeloid leukaemia, and acute lymphocytic leukaemia and the spleen of a patient with chronic lymphocytic leukaemia. We present evidence that the form of enzyme purified closely resembles pi class glutathione S-transferase purified from human placenta. Immunoblotting performed on cytosol from the leukaemic cells from a range of cases of leukaemia at presentation, or on treatment, demonstrated that this form of glutathione S-transferase was the predominant isoenzyme expressed in all cases studied. However, in the limited number of cases studied there was no correlation between the level of expression and response to chemotherapy, suggesting that increased expression of pi class GST is not the sole cause of resistance to bifunctional alkylating agent in human leukaemias.

Aged↗
PubMed1980

Characterization of rat-liver microsomal glutathione S-transferase activity.

Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S-transferase can be activated up to eight fold by treatment with N-ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1-chloro-2,4-dinitrobenzene. Upon subcellular fractionation of the liver the N-ethylmaleimide-activateable glutathione S-transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug-metabolizing systems-i.e. phenobarbital, methylcholanthrene, and trans-stilbene oxide-all increase the activity of the cytoplasmic glutathione S-transferases, but they do not affect the microsomal activity. These and other considerations indicate that the microsomal glutathione S-transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.

Animals↗
PubMed1984

Glycosyl transferases of baby hamster kidney cells and ricin-resistant mutants. O-glycan biosynthesis.

Extracts of baby hamster kidney (BHK) cells catalyzed the incorporation of N-acetylgalactosamine from UDP-N-acetyl[14C]galactosamine into myelin basic protein and an acylated tetrapeptide, N-acetylthreonyl-triproline, based on the threonine residue 98, glycosylated in myelin basic protein. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to the peptide moieties. Several ricin-resistant BHK cell lines contained enhanced (approximately twofold) levels of the transferase activity. Apomucins obtained from bovine submaxillary gland mucin by chemical or enzymic degradation were relatively poor acceptors. Using asialomucin as acceptor, galactosyl, transferase activities and a weak sialyl transferase activity were detected in BHK cell extracts. Galactose transfer occurred at two sites: to peptide-linked N-acetylgalactosamine residues to form the linkage, galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine and to terminally linked N-acetylglucosamine residues that exist as a minor constituent in bovine submaxillary mucin O-glycans, to form a galactosyl N-acetylglucosamine linkage. This reaction was not inhibited by ovalbumin, an efficient acceptor of the beta 1 leads to 4 galactosyl transferase involved in N-glycan assembly. Incorporation of galactose and N-acetylgalactosamine into endogenous proteins of BHK cell extracts was also detected. Sialic acid, fucose and N-acetylglucosamine residues were not incorporated. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to polypeptide, and galactose incorporation represented synthesis of the galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine sequence linked to polypeptide. The major endogenous protein labelled by either sugar had a molecular weight of approximately 80 000. A BHK-cell-associated glycoprotein, analogous to the urinary Tamm-Horsfall glycoprotein of molecular weight similar to the major endogenous acceptor of glycosylation, was not glycosylated in the experiments in vitro.

Animals↗
PubMed1986

Heterogeneity of dog-liver glutathione S-transferases. Evidence for a unique temperature dependence of the catalytic process.

Dog liver glutathione S-transferase activities are associated with five cytosolic proteins and to approximately 1.5% with microsomal proteins determined on the basis of activity conjugating to 1-chloro-2,4-dinitrobenzene. The four major cytosolic enzymes were purified to apparent homogeneity by sequential use of ion-exchange, hydrophobic, hydroxyapatite and affinity chromatography. The isolated transferases are binary combinations of three classes of subunits: alpha (Mr = 26,000), beta (Mr = 27,000), gamma (Mr = 28,500). They were classified by roman numerals assigned in order of increasing isoelectric point as DI alpha gamma (pI 6.4), DII alpha alpha (pI 6.9), DIII beta gamma (pI 8.1), and DIV beta gamma (pI 8.7). Additionally, traces of conjugating activity may be attributed to a, beta monomeric or dimeric protein with cationic character. The differences in catalytic specificity, temperature and pH dependence of activity, and sensitivity and kinetic response to inhibitory ligands may reflect the intrinsic structural heterogeneity of the transferases. At physiological glutathione concentrations DI alpha gamma accounted for roughly 60% of the total 1-chloro-2,4-dinitrobenzene-conjugating activity, the rank order of activity being DI alpha gamma greater than DII alpha alpha greater than DIV beta gamma greater than DIII beta gamma. The glutathione-dependent denitration of organic nitrates seems to be restricted to the cationic enzymes, whereas 1,2-dichloro-4-nitrobenzene-conjugating activity is exclusively associated with the anionic transferases, DI alpha gamma much greater than DII alpha alpha. Arrhenius plots from initial rate experiments performed over a range of temperatures (15-40 degrees C) exhibit an upward bend for DI alpha gamma, an apparently constant slope for DII alpha alpha and DIII beta gamma, and a downward bend for DIV beta gamma.

Anilino Naphthalenesulfonates↗
PubMed1989

Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate.

The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit.

Animals↗
PubMed1990

Interaction of hemin with placental glutathione transferase.

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.

Bilirubin↗
PubMed1991

Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.

Centrifugation, Density Gradient↗
PubMed1991

Adrenocorticotropin-dependent regulation of glutathione transferase subunit 4 in cultured rat adrenal cells.

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down-regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3',5'-phosphate was administered to these cells, a down-regulation of this enzyme level was observed, in agreement with the in vivo situation. This down-regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol-side-chain-cleavage enzyme (cytochrome P-450scc) which is the rate-limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down-regulation of glutathione transferase subunit 4 by adrenocorticotropin.

Adrenal Glands↗
PubMed1995

UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase is amplified in tunicamycin-resistant soybean cells.

A tunicamycin-resistant soybean cell line was developed by gradually increasing the concentration of tunicamycin in the growth medium. At the final stage, the resistant cells could survive in media containing 60 micrograms/ml of tunicamycin, whereas normal cells show a greatly retarded growth rate at 0.5 microgram/ml of antibiotic. The tunicamycin-resistant cells had a greater than 40-fold increase in the activity of the enzyme UDP-GlcNAc:dolichyl-P GlcNAc1P transferase, a 2-3-fold increase in the activity of dolichyl-P-mannose synthase, but no increase in the activities of other enzymes of the lipid-linked saccharide pathway such as dolichyl-P-glucose synthase or mannosyl transferases. There was also no change in the activities of the glycoprotein-processing enzymes, glucosidase I or glucosidase II, as compared to wild-type cells. The increase in GlcNAc1P transferase was due to an increased production of enzyme, as seen by a dramatic increase in the amount of a 39-kDa protein, which is presumed to be this enzyme protein. The GlcNAc1P transferase from tunicamycin-resistant cells was equally sensitive to tunicamycin as was the wild-type enzyme, but was considerably more labile to temperatures above 30 degrees C. The activity in tunicamycin-resistant cells was greatly stimulated by exogenous dolichyl-P. The spectrum of oligosaccharides from labeled lipid-linked oligosaccharides was similar in wild-type and tunicamycin-resistant soybean cells, but the resistant cells had significantly greater amounts of the shorter and much lower amounts of the larger-sized oligosaccharides.

Anti-Bacterial Agents↗
PubMed1989

Changes in hepatic cytosolic glutathione S-transferase enzymes induced by clotrimazole treatment in rats.

1. The influence of the antifungal agent clotrimazole on cytosolic glutathione S-transferase activities was studied in male Wistar rats. 2. Animals received clotrimazole by gastric lavage for 3 days (75 mg/kg per day). Hepatic glutathione S-transferase activity was determined with five different substrates: 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), p-nitro-benzyl chloride (PNBC), ethacrynic acid (EA) and trans-4-phenyl-3-buten-2-one (TPBO). 3. The largest increases in glutathione S-transferase activity were found with CDNB, DCNB and PNBC (+61%, +50% and +50%, respectively, when expressed per mg of cytosolic protein). Enzyme activity toward EA was induced to a lower extent (+33%). Changes in the formation of the conjugate of TPBO were relatively small (+22%). 4. These data indicate a differential induction of glutathione S-transferase isoenzymes and suggest that clotrimazole is a phenobarbital-type inducer of enzyme activity.

Animals↗
PubMed1981

Glutathione-S-transferase activity in the brain: species, sex, regional, and age differences.

Glutathione-S-transferase activity in the brain of male mammals (rat and mouse) was found to be relatively lower than in that of females. In contrast, the male aves (pigeon, kite, vulture, and crow) exhibited comparatively higher activity in brain glutathione-S-transferase than the corresponding females. Postnatal development of cytosolic glutathione-S-transferase activity in the rat brain was also investigated. The day-7 rats showed a low activity of 48 nmol/min/mg protein that gradually increased 3.2-fold over the age of 28 days. No striking differences in brain enzyme activities were observed between the 35- and 90-day-old rats. Discrete brain regions of immature rats were found to possess considerable but lower quantities of glutathione-S-transferase activity than those of the adults. The activity increased with the onset of development and attained a steady state after 21 days of age.

Aging↗
PubMed1991

Nitroglycerin relaxes coronary artery of the pig with no change in glutathione content or glutathione S-transferase activity.

1. The role of glutathione content and glutathione S-transferase activity in vascular relaxant responses to nitroglycerin was evaluated in potassium (30 mM)-contracted coronary artery strips of the pig by measuring changes in tension, glutathione content and glutathione S-transferase activity. 2. Prior exposure of coronary artery strips to nitroglycerin (10(-5)M or 10(-4)M for 20 min) resulted in tachyphylaxis to subsequent relaxation to nitroglycerin (10(-8)-10(-5)M). 3. The glutathione content and glutathione S-transferase activity of the arterial strips rendered tachyphylactic by prior exposure to nitroglycerin (10(-5)M for 20 min or 10(-3)M for 120 min) were not significantly different from those of control strips. 4. Treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min) markedly depleted arterial glutathione content in a concentration-dependent manner with no change in glutathione S-transferase activity. 5. The relaxant response of coronary artery strips to nitroglycerin (10(-8)-10(-5)M) was completely unaffected following treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min). 6. The results suggest that vascular glutathione content does not play an important role in vascular relaxation or tolerance development to nitroglycerin, at least in pig isolated coronary artery.

Animals↗
PubMed1998

Purification and characterization of a novel glutathione transferase from Ochrobactrum anthropi.

Glutathione transferase was purified from Ochrobactrum anthropi and its N-terminal sequence was determined to be MKLYYKVGACSLAPHIILSEAGLPY. The apparent molecular mass of the protein (24 kDa) was determined by SDS-polyacrylamide gel electrophoresis analysis. The amino acid sequence obtained showed similarities with known bacterial glutathione transferases in the range of 72-64%. Immunoblotting experiments performed with antisera raised against glutathione transferase from O. anthropi did not show cross-reactivity with two bacterial glutathione transferases belonging to Serratia marcescens and Proteus mirabilis.

Antibody Specificity↗
PubMed2006

Molecular cloning of AbGst1 encoding a glutathione transferase differentially expressed during exposure of Alternaria brassicicola to isothiocyanates.

The AbGst1 gene encoding a glutathione transferase from the necrotrophic pathogen Alternaria brassicicola was cloned from a benzyl isothiocyanate-treated conidial culture using differential display reverse transcription. The deduced amino-acid sequence of AbGst1p showed a significant degree of similarity to glutathione transferase-I from Saccharomyces cerevisiae and glutathione transferase-III from Schizosaccharomyces pombe. The transcription of AbGst1 was significantly enhanced by isothiocyanates, heavy metals and 1-chloro-2,4-dinitrobenzene. However, no significant transcript response was obtained with superoxide-generating menadione and paraquat. Recombinant AbGst1p expressed in Escherichia coli exhibited high transferase activity with allyl and benzyl isothiocyanates as substrate compared with 1-chloro-2,4-dinitrobenzene, but no peroxidase activity was detected. AbGst1 was upregulated in planta during the first day postinfection, suggesting the potential involvement of this enzyme in isothiocyanate detoxification mechanisms during host plant infection.

Alternaria↗
PubMed1984

Polysaccharide production by cell free transferases in saliva in relation to salivary microflora.

The supernatant of centrifuged whole saliva was incubated with radiolabeled sucrose to measure polysaccharide production by cell free transferases and to examine whether water-insoluble polysaccharides were produced. Amounts of polysaccharides were considered to reflect the level of cell free transferases in saliva. Plating samples on blood agar, MS and MSB plates gave salivary counts of total CFU, total streptococci, S. salivarius and S. mutans. The capability of the cell free portion of saliva to produce polymers was confirmed and it appeared that the cell free transferases were able to produce water-insoluble polysaccharides. Significant correlations were found between the total and the insoluble polysaccharides from 14C-G'-sucrose and total CFU, total streptococci, S. salivarius and S. mutans, respectively. Heavily S. mutans infected subjects seemed to produce particularly large amounts of water-insoluble polysaccharides from 14C-G'-sucrose. The apparently water-insoluble 14C-F'-polysaccharides correlated significantly to the number of S. salivarius. It was thus concluded that the constitution of the oral microflora and particularly the levels of S. mutans and S. salivarius were of importance for the level of cell free transferase activity.

Cell-Free System↗
PubMed1980

Sialylation of Thomsen-Friedenreich (T) and blood type NM antigens by transferases in human sera measured by [14C]NAN uptake.

Human erythrocyte T & NN antigens were exposed to fresh sera from single human donors and CMP-[14C]sialic acid without activators in a single 24 hr incubation at 37 degrees C. There was similar sialic acid (NAN) uptake, on the average 2.7 mol/mol T antigen subunit (mol wt 40,000), with various NN- and MM-derived T antigens and transferase sera in all different combinations. Sera from donors with the M gene but not from those lacking it incorporated NAN into NN antigen (c. 1 mol NAN/mol NN antigen subunit, mol wt 50,000). The profound difference in sialyltransferase action between sera from donors with the M gene and those lacking it was substantiated by repeated incubations of NN-derived T antigen with CMP-NAN and MM donor transferase serum, which incorporated 32% more NAN than did repeated incubations of the same antigen with CMP-NAN and NN donor transferase serum. The greater NAN incorporation by sera of donors with the M gene is due to an additional transferase-modifier substance present only in persons possessing the M gene.

Blood Group Antigens↗