Glutathione S-transferase Composition Research Articles

Human glutathione S-transferases

1. 1. Multiple forms of glutathione S-transferase (GST) isoenzymes present in human tissues are dimers of subunits belonging to three distinct gene families namely α, μ and π. Only the subunits within each class hybridize to give active dimers. 2. 2. These subunits are differentially expressed in a tissue-specific manner and the composition of glutathione S-transferases in various tissues differs significantly. 3. 3. Minor GST subunits not belonging to these three classes are also present in some tissues. 4. 4. An ortholog of rat GST 8-8 and mouse mGSTA4-4 is selectively expressed in some human tissues including bladder, brain, heart, liver, and pancreas. This isoenzyme designated as GST 5.8 expresses several fold higher activity towards 4-hydroxy-2,3-trans-nonenal as compared to the routinely used substrate 1-chloro-2,4-dinitrobenzene.

Glutathione and glutathione S-transferases in Barrett's epithelium

Glutathione content, enzyme activity and isoenzyme composition of glutathione S-transferases were assayed in normal and Barrett's esophageal epithelium of ten patients with Barrett's esophagus. In addition, gastric and duodenal specimens from the same patients were also investigated. Glutathione content, glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were all significantly lower in Barrett's epithelium as compared to normal esophageal mucosa. In contrast, glutathione S-transferase class alpha enzymes are markedly expressed in Barrett's epithelium, whereas only low amounts are present in normal esophageal epithelium. Glutathione and glutathione S-transferase composition in Barrett's epithelium show striking similarities with gastric epithelium, whereas duodenal epithelium is provided with considerable higher amounts of glutathione and glutathione S-transferases, except for levels of glutathione S-transferase class pi, which are lower. A significant negative correlation exists between glutathione S-transferase enzyme activity in the mucosa along the gastrointestinal tract, and the tumour incidence. Since glutathione and glutathione S-transferase are correlated with protection against cellular or cytogenetic damage, the low content of glutathione and glutathione S-transferases in the Barrett's esophagus may be a factor of relevance for the increased tumour risk in this tissue.

Purification and Characterization of Class μ Glutathione S-Transferase Isozymes from Rabbit Hepatic Tissue

Class μ glutathione S-transferases (GSTs) are important in the detoxication of epoxides generated by oxidative metabolism. Phenobarbital, 3-methylcholanthrene, and pyridine have failed to enhance the expression of class μ GST isozymes in rabbit hepatic tissue (T. Primiano, S. G. Kim, and R. F. Novak, Toxicol. Appi. Pharmacol., 113, 64-73, 1992). Two class μ GST isozymes have been isolated from rabbit hepatic cytosol and purified to homogeneity using S-hexylglutathioneagarose, CM-Sepharose, and PBE94 chromatofocusing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses showed that both isozymes possessed M r values of ≍25,500 and cross-reacted with class μ-specific GST IgG. Gel filtration analysis revealed that these isozymes were dimers with molecular weights of approximately 45 kDa. The class μ GST isozymes had pls of 7.8 and 7.2 as determined by nonequilibrium pH gel electrophoresis. The class μ GST 7.8 and 7.2 isozymes exhibited different metabolic activities toward the substrates 1-chloro-2,4-dinitrobenzene, bromosulfophthalein, 1,2-epoxy-3-( p-nitrophenoxy)propane, trans-4-phenyl-3-buten-2-one, p-nitrobenzyl chloride, and 3,4-dichloronitrobenzene. Metabolic activity of the two GSTs toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by Cibacron blue, triethyltin bromide, S-hexylglutathione, bromosulfophthalein, and indomethacin. The amino acid composition of GST μ 7.8 and 7.2 was determined and found to be very similar to those of purified rat class μ GST isozymes. N-terminal analysis of the first 21 residues of the p I 7.8 class μ GST isozyme revealed that it had 71 and 81% sequence identity with the Yb1 and Yb2 subunits, respectively. Similarly, N-terminal analysis of the first 21 residues of the p I 7.2 class μ GST isozyme revealed a 75% sequence identity with either the rat Yb1 or Yb2 subunit. Examination of class μ GST expression in rabbit hepatic cytosol following treatment with a series of known inducers including phenobarbital, 3-methyl-cholanthrene, isosafrole, pyrazine, trans-stilbene oxide, butylated hydroxyanisole, and tert-butyihydroquinone was accomplished. The data show that these agents not only failed to enhance class μ GST expression, but that 3-methylcholanthrene and isosafrole caused suppression of class μ GSTs. These results provide evidence for the existence of two closely related class μ GST isozymes in rabbit hepatic tissue and suggest that the molecular mechanisms regulating GST expression differ between rat and rabbit in response to these xenobiotics.

Multiplicity of cytosolic glutathione S-transferases in several animal species.

Glutathione S-transferases (GSTs) are one of the important enzymes in terms of not only drug metabolism but also physiological functions. The marked sex difference in GST activity has been found in rat liver cytosol, and such differences are suggested to be primarily due to the differences in the subunit composition of GSTs in both sexes. GSTs are widely distributed in mammalian species, and can be grouped into three distinct species-independent classes named alpha, mu and pi. The pi class GST 7-7 as a new mark er for preneoplasia is almost undetectable in normal rat liver. Normal dog liver cytosol has, however, possessed abundantly GST isozymes which are immunologically identical to GST 7-7. The present review also describes multiple forms of several animal species.

Time-dependent activity and expression of glutathione S-transferases in the human colon adenocarcinoma cell line Caco-2.

The human colon carcinoma cell line Caco-2 was examined for glutathione S-transferase (GST) composition and activity. Freshly seeded cells and cells until 4 days after confluency contain only the placental (Pi) form of glutathione transferase. Cells in culture for longer periods start to express class-Alpha GST isoenzymes. Confluent cells in culture for 20 days or longer contain up to 90% class-Alpha GST. Class-Mu GSTs are not detectable. GST activity gradually increases from 564 +/- 28 to 5381 +/- 165 nmol/min per mg of protein at day 0 and 32 after confluency respectively. With regard to GST composition, Caco-2 cells in culture for longer periods most resemble small-intestinal cells, whereas short-time cultures have characteristics of colonic cells. This cell line is very well suited for the study of both the in vitro properties and the expression of class Alpha and Pi GSTs.

Sex and species differences in glutathione S-transferase activities.

Glutathione S-transferases (GSTs) are one of the important enzymes in terms of not only drug metabolism but also physiological functions. The marked sex difference in GST activity has been found in rat and mouse liver cytosol, and such differences in rat liver are suggested to be primarily due to the differences in the subunit composition of GSTs in both sexes. In addition, GST activities of rat liver cytosol are known to be largely influenced by treatment with inducers such as phenobarbital and 3-methylcholanthrene and various hormones. GSTs are widely distributed in mammalian species, and multiplicity of GST has been demonstrated so far. The present review also describes multiple forms of GST from the viewpoint of enzymology and immunology.

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WR r) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WR r cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WR r and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.