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PubMed1986

Glutathione S-transferase in adult Dirofilaria immitis and Brugia pahangi.

Glutathione S-transferase (EC 2.5.1.18) was detected in the cytosolic and microsomal fractions of adult Dirofilaria immitis females at respective levels of 30 nmol and 3 nmol min-1 (mg protein)-1 activity with the substrate 1-chloro-2,4-dinitrobenzene (CDNB). The transferase activity in the cytosolic fraction of adult Brugia pahangi females was 10 nmol min-1 mg-1 with CDNB; determination of its activity in the microsomal fraction of this filariid was not attempted. These filarial glutathione S-transferases were further characterized after their purification by glutathione-affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytosolic transferase from D. immitis, molecular weight 47000, yielded a single subunit of around 28 kDa. The cytosolic and microsomal transferases from D. immitis differed in their activity with CDNB, 1,2-dichloro-4-nitrobenzene, 4-benzylchloride and ethacrynic acid. The cytosolic transferase from B. pahangi was distinguished by its high activity with ethacrynic acid. Both glutathione S-transferases from D. immitis also functioned as a glutathione peroxidase, strongly preferring cumene hydroperoxide as a substrate over hydrogen peroxide. Both were equiactive inhibitors of malonaldehyde formation in the NADPH-microsomal lipid peroxidation system. Thus, in addition to the ability of filarial glutathione S-transferases to detoxify electrophilic xenobiotics, at least those from D. immitis also exhibited selenium-independent glutathione peroxidase activity. Their glutathione S-transferase function suggests a potential role for these enzymes in the leukotriene synthetic pathway, if filariae can form such eicosanoids from arachidonate. Functioning as a glutathione peroxidase, they could serve to protect filarial membrane lipids from peroxidation.

Animals↗
PubMed2001

A new family of CoA-transferases.

CoA-transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well-known enzyme families, but recent work on unusual biochemical pathways of anaerobic bacteria has revealed the existence of a third family of CoA-transferases. The members of this enzyme family differ in sequence and reaction mechanism from CoA-transferases of the other families. Currently known enzymes of the new family are a formyl-CoA: oxalate CoA-transferase, a succinyl-CoA: (R)-benzylsuccinate CoA-transferase, an (E)-cinnamoyl-CoA: (R)-phenyllactate CoA-transferase, and a butyrobetainyl-CoA: (R)-carnitine CoA-transferase. In addition, a large number of proteins of unknown or differently annotated function from Bacteria, Archaea and Eukarya apparently belong to this enzyme family. Properties and reaction mechanisms of the CoA-transferases of family III are described and compared to those of the previously known CoA-transferases.

Bacteria, Anaerobic↗
PubMed1998

Relationship between glutathione S-transferase M1 deficiency and urothelial cancer in dye workers exposed to aromatic amines.

PURPOSE: It is speculated that the susceptibility to urothelial cancer in dye workers who are exposed to aromatic amines is affected not only by occupational environmental factors but by host specific factors. We evaluated the interaction between glutathione S-transferase M1 gene deficiency and the occupational environmental factors associated with urothelial cancer. MATERIALS AND METHODS: The study included 137 workers who had prior exposure to dyestuff intermediates, of whom 36 had urothelial cancer. The prevalence of a glutathione S-transferase M1 gene polymorphism was investigated using polymerase chain reaction. The relationship between the glutathione S-transferase M1 0/0 gene and occupational environmental factors in the onset of urothelial cancer was examined by multivariate analysis. RESULTS: The prevalence of glutathione S-transferase M1 gene deficiency did not differ significantly between the urothelial cancer (21 cases, 58.3%) group and the cancer-free (47, 46.3%) group. It was estimated that 29.6% of the urothelial cancers in these dye workers was attributable to the glutathione S-transferase M1 0/0 gene. Analysis using multiple logistic models showed low predictive ability for urothelial cancer due to glutathione S-transferase M1 gene deficiency (p = 0.084, odds ratio 2.260, 95% confidence interval [CI] 0.904 to 5.652). A history of working in small factories (p = 0.000, odds ratio 7.404, 95% CI 2.854 to 19.206) and a long period of exposure (p = 0.016, odds ratio 5.051, 95% CI 1.371 to 18.612) significantly predicted cancer. CONCLUSIONS: We demonstrated a strong trend using the multiple logistic analysis of the contribution of glutathione S-transferase M1 gene polymorphism and occupational environmental factors. Therefore, the glutathione S-transferase M1 enzyme might have an important role in the detoxification of aromatic amine derived carcinogens. Occupational environmental factors, however, might contribute more than a glutathione S-transferase M1 gene deficiency to the occurrence of urothelial cancer among individuals exposed to aromatic amines, because of the extremely potent carcinogenicity of some occupational environmental factors.

2-Naphthylamine↗
PubMed1999

Glutathione S-transferase isoenzymes in decidua and placenta of preeclamptic pregnancies.

OBJECTIVE: To investigate a possible involvement of glutathione S-transferases, major detoxificating enzymes, in the pathophysiology of preeclampsia. METHODS: Levels of glutathione S-transferase isoforms and enzyme activity were assessed in placental and decidual tissues in 22 preeclamptic and 21 normotensive women. Measured values were analyzed statistically using the Mann-Whitney U test for comparison between groups, and the signed-rank test for comparison within groups. RESULTS: Glutathione S-transferase pi is the main glutathione S-transferase isoform in normal placental and decidual tissue. Lower median placental and decidual glutathione S-transferase pi levels were found in preeclamptic women compared with controls: 1268 (range: 524-3925) and 2185 (range: 503-6578), P = .05, for placenta; 1543 (range: 681-2967) and 2169 (range: 893-3929), P = .02, for decidua. The total amount of glutathione S-transferases in control and preeclamptic pregnancies was higher in decidua than in placenta. CONCLUSION: Reduced levels of glutathione S-transferase class pi in preeclampsia might indicate a decreased capacity of the glutathione/glutathione S-transferase detoxification system. A higher total amount of glutathione S-transferases in decidual tissue might point to a more pronounced protective role of decidua compared with placenta.

Adult↗
PubMed2001

Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: mono- and vicinal dihaloethanes.

Glutathione (GSH) transferases are generally involved in the detoxication of xenobiotic chemicals. However, conjugation can also activate compounds and result in DNA modification. Activation of 1,2-dihaloethanes (BrCH(2)CH(2)Br, BrCH(2)CH(2)Cl, and ClCH(2)CH(2)Cl) was investigated using two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and a bacterial dichloromethane dehalogenase (DM11). Although the literature suggests that the bacterial dehalogenase does not catalyze reactions with CH(3)Cl, ClCH(2)CH(2)Cl, or CH(3)CHCl(2), we found a higher enzyme efficiency for DM11 than for the mammalian GSH transferases in conjugating CH(3)Cl, CH(3)CH(2)Cl, and CH(3)CH(2)Br. Enzymatic rates of activation of 1,2-dihaloethanes were determined in vitro by measuring S,S-ethylene-bis-GSH, the major product trapped by nonenzymatic reaction with the substrate GSH. Salmonella typhimurium TA 1535 systems expressing each of these GSH transferases were used to determine mutagenicity. Rates of formation of S,S-ethylene-bis-GSH by the GSH transferases correlated with the mutagenicity determined in the reversion assays for the three 1,2-dihaloethanes, consistent with the view that half-mustards are the mutagenic products of the GSH transferase reactions. Half-mustards [S-(2-haloethyl)GSH] containing either F, Cl, or Br (as the leaving group) were tested for their abilities to induce revertants in S. typhimurium, and rates of hydrolysis were also determined. GSH transferases do not appear to be involved in the breakdown of the half-mustard intermediates. A halide order (Br > Cl) was observed for both GSH transferase-catalyzed mutagenicity and S,S-ethylene-bis-GSH formation from 1,2-dihaloethanes, with the single exception (both assays) of BrCH(2)CH(2)Cl reaction with DM11, which was unexpectedly high. The lack of substrate saturation seen for conjugation of dihalomethanes with GSTs 5-5 and T1 was also observed with the mono- and 1,2-dihaloethanes [Wheeler, J. B., Stourman, N. V., Thier, R., Dommermuth, A., Vuilleumier, S., Rose, J. A., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1118-1127], indicative of an inherent difference in the catalytic mechanisms of the bacterial and mammalian GSH transferases.

Alkanes↗
PubMed1980

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.

Animals↗
PubMed1981

The hypothalamic--hypophyseal--gonadal regulation of hepatic glutathione S-transferases in the rat.

Hepatic glutathione S-transferase activities were determined with the substrates 1,2-dichloro-4-nitrobenzene and 1-chloro-2,4-dinitrobenzene. Sexual differentiation of glutathione S-transferase activities is not evident during the prepubertal period, but glutathione conjugation with 1,2-dichloro-4-nitrobenzene is 2-3-fold greater in adult males than in females. Glutathione conjugation with 1-chloro-2,4-dinitrobenzene is slightly higher in adult males than adult females. No change in activity was observed after postpubertal gonadectomy of males or females. Neonatal castration of males results in a significant decrease in glutathione conjugation with 1,2-dichloro-4-nitrobenzene. Hypophysectomy, or hypophysectomy followed by gonadectomy did result in significantly higher glutathione S-transferase activities in both sexes. These increases can be reversed by implanting an adult male or female pituitary or four prepubertal pituitaries under the kidney capsule. Postpubertal sexual differentiation of glutathione S-transferase activities is neither dependent on pituitary sexual differentiation nor pituitary maturation. Prolactin concentrations are inversely related to glutathione S-transferase activities in hypophysectomized rats with or without ectopic pituitaries. Somatotropin exogenously administered to hypophysectomized rats results in decreased glutathione S-transferase activities, whereas prolactin has no effect. Adult male rats treated neonatally with monosodium l-glutamate to induce arcuate nucleus lesions of the hypothalamus have decreased glutathione S-transferase activities towards 1,2-dichloro-4-nitrobenzene and decreased somatotropin concentrations. Our experiments suggests that sexual differentiation of hepatic glutathione S-transferase is a result of a hypothalamic inhibiting factor in the male (absent in the female). This postpubertally expressed inhibiting factor acts on the pituitary to prevent secretion of a pituitary inhibiting factor (autonomously secreted by the female), resulting in higher glutathione S-transferase activities in the adult male than the adult female.

Age Factors↗
PubMed1984

Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase B (YaYc) and two other transferases that are homodimers of the individual Ya and Yc subunits were purified from rat liver. Inhibition of these three enzymes by Indocyanine Green, biliverdin and several bile acids was investigated at different values of pH (range 6.0-8.0). Indocyanine Green, biliverdin and chenodeoxycholate were found to be effective inhibitors of transferases YaYc and YcYc at low (pH 6.0) but not high (pH 8.0) values of pH. Between these extremes of pH intermediate degrees of inhibition were observed. Cholate and taurochenodeoxycholate, however, were ineffective inhibitors of transferase YcYc at all values of pH. The observed differences in bile acids appeared to be due, in part, to differences in their state of ionization. In contrast with the above results, transferase YaYa was inhibited by at least 80% by the non-substrate ligands at all values of pH. These effects of pH on the three transferases could not be accounted for by pH-induced changes in the enzyme's affinity for the inhibitor. Thus those glutathione S-transferases that contain the Yc subunit are able to act simultaneously as both enzymes and binding proteins. In addition to enzyme structure, the state of ionization of the non-substrate ligands may also influence whether the transferases can perform both functions simultaneously.

Bile Acids and Salts↗
PubMed1989

The distribution, induction and isoenzyme profile of glutathione S-transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells.

The distribution and inducibility of cytosolic glutathione S-transferase (EC 2.5.1.18) and glutathione peroxidase (EC 1.11.1.19) activities in rat liver parenchymal, Kupffer and endothelial cells were studied. In untreated rats glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene and 4-hydroxynon-2-trans-enal as substrates was 1.7-2.2-fold higher in parenchymal cells than in Kupffer and endothelial cells, whereas total, selenium-dependent and non-selenium-dependent glutathione peroxidase activities were similar in all three cell types. Glutathione S-transferase isoenzymes in parenchymal and non-parenchymal cells isolated from untreated rats were separated by chromatofocusing in an f.p.l.c. system: all glutathione S-transferase isoenzymes observed in the sinusoidal lining cells were also detected in the parenchymal cells, whereas Kupffer and endothelial cells lacked several glutathione S-transferase isoenzymes present in parenchymal cells. At 5 days after administration of Arocolor 1254 glutathione S-transferase activity was only enhanced in parenchymal cells; furthermore, selenium-dependent glutathione peroxidase activity decreased in parenchymal and non-parenchymal cells. At 13 days after a single injection of Aroclor 1254 a strong induction of glutathione S-transferase had taken place in all three cell types, whereas selenium-dependent glutathione peroxidase activity remained unchanged (endothelial cells) or was depressed (parenchymal and Kupffer cells). Hence these results clearly establish that glutathione S-transferase and glutathione peroxidase are differentially regulated in rat liver parenchymal as well as non-parenchymal cells. The presence of glutathione peroxidase and several glutathione S-transferase isoenzymes capable of detoxifying a variety of compounds in Kupffer and endothelial cells might be crucial to protect the liver from damage by potentially hepatotoxic substances.

Animals↗
PubMed1993

Nitroglycerin metabolism in vascular tissue: role of glutathione S-transferases and relationship between NO. and NO2- formation.

Nitroglycerin is a commonly employed pharmacological agent which produces vasodilatation by release of nitric oxide (NO.). The mechanism by which nitroglycerin releases NO. remains undefined. Recently, glutathione S-transferases have been implicated as important contributors to this process. They are known to release NO2- from nitroglycerin, but have not been shown to release NO.. The present studies were designed to examine the role of endogenous glutathione S-transferases in this metabolic process. Homogenates of dog carotid artery were incubated anaerobically with nitroglycerin, and NO. and NO2- production was determined by chemiluminescence. The role of glutathione S-transferases was studied by incubating homogenates with nitroglycerin in the presence of 1 mM GSH or 1 mM S-hexyl-glutathione, a potent inhibitor of glutathione S-transferases. Homogenates released 163 pmol of NO./h per mg of protein from nitroglycerin, and 2370 pmol of NO2-/h per mg. Adding GSH decreased NO. production by 82% and increased NO2- production by 98%. S-Hexylglutathione inhibited glutathione S-transferase activity by 96% and decreased NO2- production by 78%, but had no effect on NO. release. A linear relationship between glutathione S-transferase activity and NO2- production was observed, whereas glutathione S-transferase activity and NO. release were unrelated. Western-blot analysis demonstrated that dog carotid vascular smooth muscle contained Pi and Mu forms of glutathione S-transferases, with a predominance of the former. Purified preparations of human Pi and rat Mu isoforms metabolized nitroglycerin only to NO2- and not to NO.. On the basis of these findings, we conclude that (1) glutathione S-transferases do not contribute to the bioconversion of nitroglycerin to NO., but instead act as a degradative pathway for nitroglycerin, and (2) the release of NO. from nitroglycerin is not dependent on the formation of NO2-.

Animals↗
PubMed1990

Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes.

A large number of human tumor cell lines of various origins have been investigated with respect to expression of glutathione-linked enzymes in the cytosol fraction. The amounts of the different enzymes were estimated by use of activity measurements and by silver staining or immunoblot analysis after electrophoresis of cytosol fractions purified by affinity chromatography on S-hexylglutathione Sepharose. Class Pi glutathione transferase was the most abundant enzyme in most tumor cells; the cell lines HepG2 and Raji were exceptions in not expressing significant amounts of this enzyme. HepG2 cells derive from hepatocytes, which normally do not express the class Pi enzyme, whereas Raji cells originate from B-lymphocytes, which normally do express a class Pi glutathione transferase. The highest level of the class Pi transferase, in terms of protein reacting with antibodies as well as enzyme activity, was noted in the colon carcinoma cell line LS174T. Hu549Pat cells, EBV-transformed B-lymphocytes, also expressed high levels of a protein reacting with antibodies specific for class Pi glutathione transferases, but did not display any significant activity with ethacrynic acid, a substrate characteristic for this class. Class Alpha and class Mu glutathione transferases, in cell lines expressing these isoenzymes, were present in significantly lower concentrations than the class Pi enzyme. Most of the tumor cells contained a class Alpha transferase composed of 27.5 kd subunits, which has the physicochemical and immunological properties of the most basic glutathione transferase found in human skin. In several cell lines, a protein was detected with an apparent subunit Mr value of 30 kd that was tentatively identified as an additional class Alpha glutathione transferase not previously described. In addition, other glutathione-linked enzyme activities, namely glutathione peroxidase, glutathione reductase and glyoxalase I, were assayed with specific substrates in the cytosolic fraction of the tumor cells; glyoxalase I could also be estimated semiquantitatively by silver staining of SDS-PAGE cells after affinity chromatography. Like the glutathione transferases, these enzymes displayed distinctly different levels of expression in the various cell lines. Thus, virtually every cell line was found to have a unique pattern of glutathione-linked enzymes, suggesting that the resistance phenotypes of the cells differ accordingly.

Cell Line↗
PubMed1982

Regulation of epididymal glutathione S-transferases: effects of orchidectomy and androgen replacement.

Glutathione S-transferases, a family of enzymes that catalyze the conjugation of glutathione to a variety of substrates, are present in rat epididymis. In order to study the hormonal regulation of these enzymes in this tissue, adult rats were orchidectomized and implanted with empty or androgen-filled polydimethylsiloxane capsules. Orchidectomy alone significantly decreased caput-corpus epididymal glutathione S-transferase activity toward 2 substrates, 1-chloro-2,4-dinitrobenzene and trans-4-phenylbut-3-en-2-one, but had no effect on transferase activity toward the third substrate, 1,2-dichloro-4-nitrobenzene. In contrast to these results, orchidectomy did not alter glutathione S-transferase activity towards these substrates in the cauda epididymidis. Androgen replacement with testosterone prevented the orchidectomy-induced decrease in caput-corpus glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and trans-4-phenylbut-3-en-2-one and had no effect on transferase activity toward 1,2-dichloro-4-nitrobenzene. The effects of 5 alpha-reduced metabolites of testosterone were also studied. Both dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol maintained caput-corpus glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, although a lower dose of dihydrotestosterone was sufficient; these 1 androgens were unable to maintain activity toward trans-4-phenylbut-3-en-2-one and caused a suprastimulation of activity toward 1,2-dichloro-4-nitrobenzene above control values. The third 5 alpha-reduced androgen studied, 5 alpha-androstan-3 beta, 17 beta-diol had no effect on the transferase activity toward any of the 3 substrates. These results demonstrate that the epididymal glutathione S-transferases are under separate control and are differentially regulated by testosterone and its 5 alpha-reduced metabolites.

Androgens↗
PubMed2002

Farnesyl protein transferase inhibition interferes with activation of MAP kinase family members in human peripheral blood monocytes.

BACKGROUND: Farnesyl protein transferase inhibitors have emerged as promising novel agents for combating cancerous disease. Nevertheless, the importance for farnesyl protein transferase enzymatic activity for cellular physiology of untransformed cells remains poorly investigated. MATERIALS AND METHODS: Peripheral blood monocytes, isolated from the blood of eight healthy volunteers, were treated with a farnesyl protein transferase inhibitor (FTI 744,832) or vehicle control for 16 hr. Subsequently cells were challenged with different concentrations of lipopolysaccharide (LPS), colony stimulating factor-1 (CSF-1), or phorbol esters for 10 min, after which the activation state of p42/p44 MAP kinase, p38 MAP kinase, and Jun-N-terminal kinase was investigated using Western blotting and phosphospecific antibodies. RESULTS: We observed that farnesyl protein transferase inhibition abrogated activation of p38 MAP kinase by LPS, CSF-1, and phorbol esters. Also the activation of Jun-N-terminal kinase by LPS was not seen after farnesyl protein transferase inhibition. Finally, stimulation of p42/p44 MAP kinase with CSF-1 was strongly reduced by farnesyl protein transferase inhibition, whereas activation of p42/p44 MAP kinase by phorbol ester was only slightly effected. CONCLUSIONS: Farnesyl protein transferase enzymatic activity is required for proper activation of all major members of the MAP kinase family. The observation that activation the p38 MAP kinase and Jun-N-terminal kinase is sensitive to farnesyl protein transferase inhibition raises the possibility that, in addition to cancerous disease, farnesyl protein transferase inhibitors may be useful compounds in combating inflammatory disease.

Alkyl and Aryl Transferases↗
PubMed1990

Glucocorticoid regulation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferases, and NAD(P)H:quinone oxidoreductase in cultured fetal rat hepatocytes.

The regulation of polycyclic aromatic hydrocarbon-inducible enzymes, cytochrome P450IA1, NAD(P)H:quinone oxidoreductase, and glutathione S-transferases, by glucocorticoids was investigated using primary fetal rat hepatocyte culture. Treatment of cells in culture with 1,2-benzanthracene (100 microM, 72 hr) resulted in 60-, 2-, and 6-fold increases in cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone reductase activities, respectively. The inductive effect of 1,2-benzanthracene on cytochrome P450IA1 and glutathione S-transferase (1-chloro-2,4-dinitrobenzene conjugation) activities was potentiated approximately 3- and 2- to 3-fold, respectively, when dexamethasone (0.01-1 microM) was included in the culture medium. In contrast, 1 microM dexamethasone was found not to potentiate the induction of NAD(P)H:quinone oxidoreductase activity by 1,2-benzanthracene. Treatment of cultured hepatocytes with dexamethasone alone, at concentrations of up to 100 microM, resulted in a 2- to 4-fold increase in glutathione S-transferase and NAD(P)H:quinone oxidoreductase activity. Both the induction of glutathione S-transferase activity by high concentrations of dexamethasone alone and the potentiation of 1,2-benzanthracene induction by lower concentrations of dexamethasone were observed for other steroids of the glucocorticoid class in conjunction with a variety of polycyclic aromatic hydrocarbons. Western immunoblot analyses indicated that low concentrations of dexamethasone (0.1-1 microM) potentiated 1,2-benzanthracene-dependent induction of cytochrome P450IA1, glutathione S-transferase Ya/Yc subunit and NAD(P)H:quinone oxidoreductase content. Additionally, increased glutathione S-transferase activity in response to concentrations of dexamethasone exceeding 1 microM was associated with concomitant increases in Ya/Yc and Yb subunit content. Potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase protein content by low concentrations of glucocorticoids and induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase by high concentrations of glucocorticoids alone indicates the importance of these endogenous compounds in the regulation of some hepatic enzymes involved in xenobiotic metabolism.

Animals↗
PubMed1990

Regulation of succinyl coenzyme A:acetoacetyl coenzyme A transferase in rat hepatoma cell lines.

The regulation of succinyl-CoA:acetoacetyl-CoA transferase (CoA transferase) has been studied in 8 rat hepatoma cell lines. Compared with normal rat hepatocytes, which have almost nondetectable activity of the enzyme, the hepatoma cell lines have a wide range of expression of CoA transferase activity, from as low as 45 nmol/min/mg to as high as 960 nmol/min/mg. Western blotting showed that the different levels of CoA transferase activity were due to differing amounts of the enzyme in the cells. This was further attributed to the varying amounts of the enzyme synthesized in the cells as monitored by L-[35S]methionine labeling followed by immunoprecipitation. To study further the differential expression of CoA transferase in the hepatoma cell lines, the relative quantity of functional CoA-transferase mRNA in the cells was measured by in vitro translation. The results showed that the levels of functional CoA transferase mRNA detected were consistent with the differences in the enzyme activity in the cells. Since CoA transferase is the key enzyme responsible for the utilization of ketone bodies as an alternative energy source, the expression of CoA transferase in hepatoma cells may play a role in energy production.

Animals↗
PubMed1986

Differences in stereoselectivity and catalytic efficiency of three human glutathione transferases in the conjugation of glutathione with 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The kinetics of the enzyme-catalyzed conjugation of glutathione with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha -oxy-7,8,9,10 -tetrahydrobenzo(a)pyrene [(+/-)-anti-BPDE] have been studied with the following human cytosolic glutathione transferases: the basic (alpha-epsilon) and near-neutral (mu) isoenzymes from liver, and the acidic (pi) isoenzyme from placenta. When the BPDE concentration was varied (using 5 mM glutathione) the apparent Vmax values for transferases alpha-epsilon, mu, and pi were 38, 570, and 825 nmol X mg-1 X min-1, respectively, with corresponding apparent Km values of 88, 27, and 54 microM. The apparent Km values for glutathione [using 80 microM (+/-)-anti-BPDE] were 0.4, 0.7, and 0.1 mM for transferase alpha-epsilon, mu, and pi, respectively. The glutathione conjugates formed with the two enantiomers of (+/-)-anti-BPDE were resolved by high performance liquid chromatography. The percentages of conjugates derived from the highly carcinogenic (+)-enantiomer were 59, 60, and greater than or equal to 90% for transferases alpha-epsilon, mu, and pi, respectively. The separate enantiomers of anti-BPDE were assayed in experiments with transferases mu and pi. Both enantiomers were substrates for transferase mu, but only the (+)-enantiomer gave measurable activity with transferase pi. A 3-fold increase in Vmax and Km values for transferase pi was obtained with (+)-anti-BPDE as compared with the racemic substrate and could be quantitatively accounted for by the finding that (-)-anti-BPDE serves as a competitive inhibitor for transferase pi.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells.

Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.

Animals↗
PubMed1985

Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites.

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.

Animals↗