Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol

1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gammaglutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated.2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1–2 days.3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1–3 days.4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 μM tocotrienol at 1–3 days in culture compared to the control.     

Distribution and some properties of the glutathione S-transferase and gamma-glutamyl transpeptidase activities of rainbow trout

1. Gills, kidney, intestinal caeca and liver of trout have glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene (200 500 nmol/min/mg protein), and reduced glutathione (0.5 2.0 mmol/kg tissue). 2. Only kidney and intestinal caeca have substantial gamma-glutamyl transpeptidase activity with gamma-glutamyl-rho-nitroanilide (2-9 nmol/min/mg protein). 3. Renal gamma-glutamyl transpeptidase is membrane-bound and has similar kinetic properties to its mammalian counterparts. 4. The data are consistent with the presence of a mercapturic acid pathway in trout.     

Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradation by gamma-glutamyl transpeptidase.

Inhibitors for glutathione S-transferase (GST) iso-enzymes from rat liver with high affinity for the glutathione-binding site (G-site) have been developed. In previous studies, a model was described for the G-site of GST (Adang, A. E. P., Brussee, J., van der Gen, A., and Mulder, G. J. (1990) Biochem. J. 269, 47-54) in terms of essential and nonessential interactions between groups in glutathione (GSH) and the G-site. Based on this model, compounds were designed that have high affinity for the G-site but cannot be conjugated. In the dipeptide gamma-L-glutamyl-D-aminoadipic acid (gamma-L-Glu-D-Aad), the L-cysteinylglycine moiety is replaced by D-aminoadipic acid. This dipeptide is an efficient competitive inhibitor (toward GSH) of mu class GST isoenzymes with Ki values of 34 microM for GST isoenzyme 3-3 and 8 microM for GST isoenzyme 4-4. Other GSH-dependent enzymes, such as gamma-glutamyl transpeptidase (gamma-GT), glutathione reductase, and glutathione peroxidase, were not inhibited by 1 mM of gamma-L-Glu-D-Aad. Inhibition is also highly stereospecific since gamma-L-Glu-L-Aad is only a poor inhibitor (Ki = 430 microM for GST 3-3). Gamma-L-Glutamyl-D-norleucine also had a much higher Ki value for GST 3-3. Thus, the presence of a delta-carboxylate group in D-Aad appears to be essential for a high affinity inhibitor. An additional hydrophobic group did not result in increased inhibitory potency. In a different approach, the gamma-L-glutamyl moiety in GSH was replaced by delta-L-aminoadipic acid; delta-L-Aad-L-Cys-Gly is an efficient cosubstrate analogue for GSTs with Km values comparable to GSH and Vmax values ranging from 0.24 to 57 mumol/min/mg for the different GSTs. The structures of the efficient inhibitor and the cosubstrate analogue were combined in delta-L-Aad-D-Aad, which had a Ki value of 68 microM with GST 3-3. In order to investigate their possible use in vivo studies, the degradation of gamma-L-Glu-D-Aad and delta-L-Aad-L-Cys-Gly by gamma-GT was investigated. The peptides showed no measurable hydrolysis rates under conditions where GSH was rapidly hydrolyzed. Thus, an efficient, mu class-specific GST inhibitor and a gamma-glutamyl-modified cosubstrate analogue of GSH were developed. Their gamma-GT stability offers the possibility to use these peptides in in vivo experiments.     

Modifications of gamma-glutamyl transpeptidase activity in duodenal mucosa of rats treated with different antiulcer drugs

The activity of gamma-glutamyl transpeptidase (gamma-GT) in duodenal mucosa both in healthy rats and in rats experimentally ulcerated with indomethacin increases significantly after oral administration of pirenzepine as well as ranitidine but not after oral administration of sucralphate. These increase in gamma-GT activity may contribute to the cytoprotective effects already described for pirenzepine and ranitidine.     

Inhibitory action of microwave radiation on gamma-glutamyl transpeptidase activity in liver of rats treated with hydrocortisone

The influence of microwave irradiation on the activity of gamma-glutamyl transpeptidase (GGT) induced by hydrocortisone (HC) in the liver of rats was investigated. Animals were subjected to microwave irradiation (frequency 53.57 GHz, power density 10 mW/cm2 and 1 mW/cm2) during and after hydrocortisone (HC) treatment (20 mg/kg for 60 days). The results indicate that microwave radiation may block an inducible effect of HC on GGT activity in the liver of rats. This effect depends on the power density of millimetre microwaves.     

Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate.

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. L-serine in borate buffer inhibits γ-glutamyl transpeptidase, the major catabolic enzyme for glutathione. Treatment of glutathione synthetase deficient fibroblasts with 40mM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione synthetase deficient fibroblasts, presumably secondary to feedback inhibition of γ-glutamylcysteine synthetase by the increased intracellular glutathione concentration. Cellular free amino acid content was generally unaffected by such exposure although increases were observed in serine and phosphoserine. This model system suggests that γ-glutamyl transpeptidase inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency.     

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Synthesis of S-alkyl L-homocysteine analogues of glutathione and their kinetic studies with gamma-glutamyl transpeptidase

A series of S-alkyl L-homocysteine analogues of glutathione was synthesized with varied oxidation state of the sulfur and tested for inhibition of rat kidney gamma-glutamyl transpeptidase (GGT). The strong selectivity of the enzyme with respect to the sulfur oxidation state reveals important information for the development of powerful competitive inhibitors.     

Interaction of gamma-glutamyl transpeptidase with glutathione involves specific arginine and lysine residues of the heavy subunit.

Gamma-glutamyl transpeptidase, an enzyme of importance in glutathione metabolism, consists of two subunits, one of which (the light subunit, Mr 22,000; residues 380-568; rat kidney) contains residue Thr-523, which selectively interacts with the substrate analog acivicin to form an adduct that is apparently analogous to the gamma-glutamyl enzyme intermediate formed in the normal reaction (Stole, E., Seddon, A. P., Wellner, D., and Meister, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1706-1709). The present studies indicate that specific arginine and lysine residues of the heavy subunit (Mr 51,000; residues 31-379) participate in catalysis by binding the substrates. Selective labeling studies of the enzyme with [14C]phenylglyoxal showed that Lys-99 and Arg-111 were modified. This appears to be the first instance in which phenylglyoxal was found to react with an enzyme lysine residue. Incorporation of [14C]phenylglyoxal into Lys-99 was decreased in the presence of acceptor site selective compounds. Incorporation into both Lys-99 and Arg-111 was decreased in the presence of glutathione. The findings suggest that Lys-99 and Arg-111 interact, respectively, with the omega- and alpha-carboxyl groups of glutathione. That these putative electrostatic binding sites are on the heavy subunit indicates that both subunits contribute to the active center. Two additional heavy subunit arginine residues become accessible to modification by phenylglyoxal when acivicin is bound, suggesting that interaction with acivicin is associated with a conformational change.     

Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.     

Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).     

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.     

Regulation of glutathione S-transferase subunits 3 and 4 in cultured rat hepatocytes

mRNA levels of glutathione S-transferase (GST) subunits 3 and 4 were measured with a specific cDNA probe in adult rat hepatocytes maintained either in conventional culture or in coculture with rat liver epithelial cells. Four media conditions were used, i.e. with or without fetal calf serum (FCS) and with nicotinamide or dimethylsulfoxide (DMSO). When FCS was present in the culture medium, GST subunit 3 and 4 mRNAs were expressed at a level close to that found in freshly isolated hepatocytes during the whole culture period both in conventional culture and in coculture. All other culture conditions resulted in an increase of GST 3 and 4 mRNA levels. After exposure to phenobarbital an increase in GST 3 and 4 mRNA levels was demonstrated in both culture systems. Comparison with previous findings on the expression of GST subunits 1, 2 and 7 in the same culture conditions indicates that the different classes of GST are regulated independently.     

Expression of multiple proteins structurally related to gamma-glutamyl transpeptidase in non-neoplastic adult rat hepatocytes in vivo and in culture.

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma-glutamyl transpeptidase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct-ligated rats. However, the induction of GGT activity in rat hepatocytes by two-thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross-react with 2 different anti-rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9- and 58.0-kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5-kDa protein represents 50% of the total GGT-immunoreactive protein, in contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5-kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79-kDa single-chain glycoprotein propeptide of GGT. It appears that in normal hepatocytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.