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PubMed1996

Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin.

1. Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP-ribosyl)transferase activity in receptor-dependent chemotaxis. 2. Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF-dependent actin polymerization and chemotaxis. The activity of cell surface Arg-specific mono(ADP-ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 +/- 1.46 microM, 2.0 +/- 0.1 microM and 4.7 +/- 0.1 microM respectively). 3. There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP-ribosyl)transferase, namely vitamin K1, novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA-BAG). 4. Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo-3 and Fura-Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP-ribosyl)transferase listed above had no effect on the magnitude of the response. 5. A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP-dependent polymerization of actin. 6. We conclude that eukaryotic Arg-specific mono(ADP-ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin-containing cytoskeletal microfilaments.

ADP Ribose Transferases↗
PubMedDate not supplied

Immunohistochemical localization of alpha and pi class glutathione transferases in normal human tissues.

The distribution of alpha and pi class glutathione transferases in autopsy and biopsy samples of normal human tissues was investigated by immunohistochemistry. The class alpha glutathione transferases exhibited restricted distribution. Intensive staining was visible in all hepatocytes, in kidney proximal tubular cells, in the zona reticularis of adrenal cortex and in Leydig cells of testis. Staining of lesser intensity could also be observed in the gastrointestinal epithelium, exocrine pancreas and some bile and pancreas ducts. In colon and gall bladder only nuclei were stained, but in the other tissues both nuclei and cytoplasm contained alpha class glutathione transferases. Glutathione transferase pi exhibited a more general distribution and could be observed in epithelia of the respiratory, gastrointestinal and urinary tracts, in all endocrine cells investigated, and also in the exocrine glands of prostate, in smooth muscle, adipocytes, blood vessel endothelium and placenta. It was also visible in the Schwann cells of peripheral nerves and in the choroid plexus. In gall bladder and colon only nuclei were stained, while in the intrahepatic bile ducts only cytoplasm was stained. All other positive cells exhibited glutathione transferase pi in both nuclei and cytoplasm.

Animals↗
PubMed1984

Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis.

Alleles↗
PubMed1993

Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene.

The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer.

Amino Acid Sequence↗
PubMed1985

Raised plasma glutathione S-transferase values in hyperthyroidism and in hypothyroid patients receiving thyroxine replacement: evidence for hepatic damage.

Using plasma glutathione S-transferase measurements hepatocellular integrity was assessed in groups of hyperthyroid and hypothyroid patients before and after treatment. Ten of 14 hyperthyroid patients had clearly raised plasma glutathione S-transferase values at presentation and in each patient treatment with either iodine-131 or carbimazole resulted in a significant fall in glutathione S-transferase. The eight hypothyroid patients had normal glutathione S-transferase values at presentation and all showed a significant increase in these after thyroxine replacement therapy. In three of these patients in whom standard doses of replacement therapy were associated with a raised free thyroxine (T4) concentration but normal total and free triiodothyronine (T3) values glutathione S-transferase was increased. Similar though less consistent changes were seen in the results of standard chemical tests of liver function. It is concluded that hyperthyroidism may produce subclinical liver damage in a high proportion of patients and that this resolves with effective treatment. More important, the data suggest that hypothyroid patients receiving thyroxine replacement therapy may have similar subclinical liver damage. Patients receiving thyroxine should be monitored by the measurement of free, not total hormone concentrations, and in those in whom free T4 is raised the dose of thyroxine should be reduced. It would also be expedient to include periodic biochemical assessment of liver function in patients receiving thyroxine.

Adult↗
PubMed1992

Glutathione S-transferase in chemotherapy resistance and in carcinogenesis.

Cytosolic glutathione S-transferases are composed of two monomeric subunits. These monomers are the products of different gene families designated alpha, mu, and pi. Dimerization yields either homodimeric or heterodimeric holoenzymes within the same family. The members of this complex group of proteins have been linked to the detoxification of environmental chemicals and carcinogens, and have been shown to be overexpressed in normal and tumor cells following exposure to cytotoxic drugs. They also are overexpressed in carcinogen-induced rat liver preneoplastic nodules in rat liver. In all of these cases, the changes in expression of glutathione S-transferases are paralleled by increased resistance to cytotoxic chemicals. The degree of resistance is related to the substrate specificity of the isozyme. The relationship of the glutathione S-transferase genes to drug resistance has been directly demonstrated by gene transfer studies, where cDNAs encoding the various subunits of glutathione S-transferase have been transfected into a variety of cell types. This review discusses the results of numerous studies that associate resistance to alkylating agents with overexpression of protective detoxifying glutathione S-transferase enzymes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Possible role of blood insulin levels on glutathione S-transferase activities from different tissues of male rats.

The activity of in vitro glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene was examined in liver, renal cortex, and small intestine (duodenum, jejunum, ileum) after the in vivo treatment of male Wistar rats with streptozotocin or alloxan. The studies were performed at 2, 10, 24, and 48 h and 7 and 15 days after streptozotocin treatment or 24 and 48 h after alloxan treatment. The results indicated that while the blood levels of insulin-glucose did not show variations, there were no alterations of the glutathione S-transferase activity in the tissues tested. On the other hand, when the treatments caused modifications on blood insulin-glucose levels, there were changes of glutathione S-transferase activity in all tissues (except in the ileum) in such a way that a direct relationship between plasma insulin levels and glutathione S-transferase activity could be demonstrated. These results were also confirmed through insulin administration to control and diabetic rats. The data demonstrate a possible regulation of glutathione S-transferase activity by blood insulin and (or) glucose levels in the tissues tested.

Animals↗
PubMed1993

Effect of inhibitors of glutathione S-transferase on glyceryl trinitrate activity in isolated rat aorta.

We investigated the role of glutathione S-transferases (enzymes known to biotransform organic nitrates) in the vascular action of glyceryl trinitrate (GTN). Relaxation of phenylephrine-contracted rat aortic strips was assessed in the presence or absence of the glutathione S-transferase inhibitors Basilen Blue, bromosulfophthalein, Rose Bengal, hematin, chlorotriphenyltin, and (octyloxy)benzoylvinylglutathione. Whereas none of the inhibitors increased the EC50 for GTN relaxation, glutathione S-transferase activity in the 100,000 x g supernatant fraction of rat aorta was inhibited markedly by most of the inhibitors. In addition, GTN-stimulated activation of aortic guanylyl cyclase in broken-cell preparations was attenuated by all of the glutathione S-transferase inhibitors, suggesting a direct inhibitory action on guanylyl cyclase. In other experiments using aortic strips preexposed to phenylephrine, the inhibitors had no effect on GTN-induced cyclic GMP accumulation or on vascular biotransformation of GTN. In contrast, both Basilen Blue and bromosulfophthalein significantly inhibited GTN-induced relaxation of K(+)-contracted aortic strips, and Basilen Blue significantly inhibited GTN biotransformation in aortic strips preexposed to 25 mM K+. This may be due to a more favourable electrochemical gradient for entry of the inhibitors into membrane-depolarized tissues. We conclude that vascular glutathione S-transferases play a role in mediating the vasodilator actions of GTN in intact tissues in vitro, but that this appears to depend upon the nature of the contractile agent used in such studies.

Animals↗
PubMed2005

Physical training reverses defect in 3-ketoacid CoA-transferase activity in skeletal muscle of diabetic rats.

To investigate one potential mechanism whereby physical training improves the plasma concentration of ketone bodies in experimental diabetes mellitus, we measured the activity of 3-ketoacid CoA-transferase, the key enzyme in the peripheral utilization of ketone bodies. Diabetes was induced with streptozotocin (50 mg/kg) and training carried out on a treadmill with a progressive 10-wk program. Diabetes resulted in an increase (P < 0.001) in plasma concentration of beta-hydroxybutyric acid in sedentary rats, which was partly reversed by training (P < 0.001). Diabetes was also associated with a decreased activity of 3-ketoacid CoA-transferase in gastrocnemius muscle. When expressed per total gastrocnemius, training increased the activity of 3-ketoacid CoA-transferase by 66% in nondiabetic rats (P < 0.001) and by 150% in diabetic rats (P < 0.001), the decrease present in diabetic rats being fully reversed by training. Simple linear regression between the log of 3-ketoacid CoA-transferase activity and the log of plasma beta-hydroxybutyric acid levels showed a statistically significant (r = 0.563, P < 0.001) negative correlation. The beneficial effects of training on plasma ketone bodies in diabetic rats are probably explained, at least in part, by an increase in ketone body utilization, mediated by an increase in skeletal muscle 3-ketoacid CoA-transferase activity.

3-Hydroxybutyric Acid↗
PubMed1995

Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.

Animals↗
PubMed1983

Decomposition of hydroperoxides derived from microsomes or lipoprotein by glutathione peroxidase and glutathione S-transferase.

Protective effects of glutathione peroxidase, glutathione S-transferase purified from human liver and superoxide dismutase against lipid peroxidation were investigated. In the presence of glutathione, lipid hydroperoxides found in microsomal membrane were decomposed by glutathione peroxidase and cationic glutathione S-transferase, but anionic glutathione S-transferase had no effect on them. Superoxide dismutase exhibited antioxidation effect by preventing accumulation of lipid hydroperoxides. Serum lipid hydroperoxides existing in low density lipoprotein fraction were also decomposed by glutathione peroxidase and cationic glutathione S-transferase. These findings suggest that the hydroperoxide level, which has high toxicity, could be controlled by these glutathione-dependent glutathione peroxidase and cationic glutathione S-transferase.

Animals↗
PubMed1998

Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects.

Genotypes responsible for interindividual differences in ability to activate or detoxify genotoxic agents are recognized as biomarkers of susceptibility. Among the most studied genotypes are human glutathione transferases. The relationship of genetic susceptibility to biomarkers of exposure and effects was studied especially in relation to the genetic polymorphism of glutathione S-transferase M1 (GSTM1). For this review papers reporting the effect of GSTM1 genotype on DNA adducts, protein adducts, urine mutagenicity, Comet assay parameters, chromosomal aberrations, sister chromatid exchanges (SCE), micronuclei, and hypoxanthine-guanine phosphoribosyl transferase mutations were assessed. Subjects in groups occupationally exposed to polycyclic aromatic hydrocarbons, benzidine, pesticides, and 1,3-butadiene were included. As environmentally exposed populations, autopsy donors, coal tar-treated patients, smokers, nonsmokers, mothers, postal workers, and firefighters were followed. From all biomarkers the effect of GSTM1 and N-acetyl transferase 2 was seen in coke oven workers on mutagenicity of urine and of glutathione S-transferase T1 on the chromosomal aberrations in subjects from 1,3-butadiene monomer production units. Effects of genotypes on DNA adducts were found from lung tissue of autopsy donors and from placentas of mothers living in an air-polluted region. The GSTM1 genotype affected mutagenicity of urine in smokers and subjects from polluted regions, protein adducts in smokers, SCE in smokers and nonsmokers, and Comet assay parameters in postal workers. A review of all studies on GSTM1 polymorphisms suggests that research probably has not reached the stage where results can be interpreted to formulate preventive measures. The relationship between genotypes and biomarkers of exposure and effects may provide an important guide to the risk assessment of human exposure to mutagens and carcinogens.

Animals↗
PubMed1988

Human placental glutathione transferase: interactions with steroids.

Glutathione transferases exhibit both isomerase and transferase activity. The acceptance of steroids as substrates for or inhibitors of these activities was studied using a 350-fold enriched preparation of the enzyme from human placenta. As an isomerase, the enzyme preparation catalyzed the conversion of pregn-5-ene-3,20-dione (Km 0.03 mmol/l) and androst-5-ene-3,17-dione (Km 0.05 mmol/l) to the respective 4-ene-3-oxosteroids (specific activity 0.8 U/mg protein). This isomerase activity strictly depended on the presence of glutathione (Km 0.04 mmol/l). As a transferase, the enzyme preparation catalyzed the conjugation of glutathione (Km 0.5 mmol/l) with 1-chloro-2,4-dinitrobenzene (Km 1.0 mmol/l) (specific activity 100 U/mg protein). This transferase activity was inhibited by all phenolic (KI values 0.2-1.5 mmol/l) and some of the neutral steroids (KI values 1.4-3.5 mmol/l) tested. Phenolic steroids inhibited the enzyme activity competitively to 1-chloro-2,4-dinitrobenzene and non-competitively to both substrates. The results indicate that steroids can interact with the placental glutathione transferase in vitro both as substrates and as inhibitors. Since, however, the observed Km and KI values of the steroids are far above the values of their concentrations in the placenta, these interactions are of only minor physiological relevance.

Cytosol↗
PubMed1984

Purification and characterization of glutathione S-transferases in human retina.

Human retina has two forms of glutathione (GSH) S-transferases. These forms having pI 4.5 and greater than 10 have been purified and their kinetic, structural and immunological characteristics are described. Both the enzymes of human retina do not express glutathione peroxidase II activity. The anionic enzyme (pI 4.5) of retina cross reacts with the antibodies raised against the anionic GSH S-transferases of human lung and placenta but does not cross react with the antibodies raised against the cationic enzymes of human liver. On the other hand, the cationic enzyme (pI greater than 10) of human retina cross reacts with the antibodies raised against the cationic GSH S-transferases of human liver but not with antibodies raised against the anionic enzymes of lung and placenta. Differences in the kinetic characteristics of the two forms of human retinal GSH S-transferases are also indicated. Results of these studies suggest that the anionic enzyme of retina may be similar to the anionic enzymes of lung and placenta. However, the cationic form appears to be different from all other GSH S-transferases of human tissues characterized so far. Human retina has selenium dependent glutathione peroxidase I and in this respect is different from bovine retina which has no glutathione peroxidase I as demonstrated in earlier studies.

Cross Reactions↗
PubMed1988

Glutathione transferases--structure and catalytic activity.

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.

Catalysis↗
PubMed2002

Establishment and characterization of acquired resistance to the farnesyl protein transferase inhibitor R115777 in a human colon cancer cell line.

R115777 (Zarnestra) is a farnesyl protein transferase inhibitor currently undergoing worldwide clinical trials. As acquired drug resistance may limit the efficacy of the drug, a model of acquired resistance has been established in vitro by continuous drug exposure of the human colon cancer cell line KM12. A stably resistant cell line possessing 13-fold resistance to R115777 was generated. The resistant cells showed cross-resistance to another, structurally different farnesyl transferase inhibitor-277, but not to GGTI-298. A lack of cross-resistance was observed to a variety of other agents, which included clinically used drugs, such as doxorubicin, etoposide, cisplatin, and paclitaxel, as well as signal transduction blockers, such as the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor UO126, the phosphatidylinositol 3'-kinase inhibitor LY294002, and the epidermal growth factor receptor tyrosine kinase inhibitor PD153035. Resistance did not appear to be related to differences in drug efflux pumps, such as P-glycoprotein or in drug accumulation. Total levels of farnesyl transferase protein subunits were similar in the parent and resistant cells, but, notably, the enzyme activity was markedly reduced in the resistant cell line compared with the parent cells. This was not because of a mutation in the enzyme or a difference in activation of the alpha-subunit of farnesyl transferase by phosphorylation. Hence, resistance to R115777 was generated; the mechanism of resistance in this model may be associated with the enzyme target of the inhibitor. The results suggest that the development of clinical resistance may occur with farnesyl protein transferase inhibitors.

Alkyl and Aryl Transferases↗
PubMed1992

Bovine erythrocyte glutathione S-transferase: purification, inhibition, and complex formation.

Glutathione S-transferase has been purified from bovine erythrocytes by affinity chromatography. The enzyme has an isoelectric point of 7.2, behaves as a 48-kDa protein composed of two identical subunits, and has an N-terminal sequence of PPYTIVYFPVQGR?EAMRMLL. This sequence, the amino acid composition, and the kinetic parameters suggest that the enzyme belongs to the pi-class of transferases. Hemins, porphyrins, and fatty acids form complexes with the enzyme and serve as effective inhibitors. Treatment of the transferase with N-ethylmaleimide, 3-amino-1,2,4-triazole, diethyl pyrocarbonate, or 2,3-butanedione inhibits transferase activity without altering tetrapyrrole binding. The role of the complexation and inhibition of glutathione S-transferase in erythroid metabolism has yet to be elucidated.

Amino Acid Sequence↗
PubMed1992

Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.

The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.

Antibodies, Monoclonal↗