Glutathione S-transferase Isoenzymes Research Articles

Immunohistochemical demonstration of glycoprotein 170 and glutathione S-transferase isoenzymes in normal and neoplastic bronchial tissue : Wossner R, Toomes H, Fritz P, Linder A, Dierkesmann RE, Behrle E et al. Pathologie, Robert Bosch Krankenhaus, Auerbachstrasse 110, D-70376 Stuttgart. Onkologie 1994;17:28–34

Immunohistochemical demonstration of glycoprotein 170 and glutathione S-transferase isoenzymes in normal and neoplastic bronchial tissue : Wossner R, Toomes H, Fritz P, Linder A, Dierkesmann RE, Behrle E et al. Pathologie, Robert Bosch Krankenhaus, Auerbachstrasse 110, D-70376 Stuttgart. Onkologie 1994;17:28–34

Influences of dietary deoxycholic acid on progression of hepatocellular neoplasms and expression of glutathione S-transferases in rats.

Serial magnetic resonance imaging (MRI) was used to evaluate the influences of dietary deoxycholic acid (DCA) on the rate of progression of chemically induced hepatocellular neoplasms in rats. Male Fischer-344 rats with established persistent hepatocellular nodules generated by the Solt-Farber protocol were exposed to dietary DCA (0.3%) between 6 and 12 mo of age. Growth of nodules and carcinomas in vivo was measured by morphometric quantification of tumor images obtained every 6 wk. The final stages of neoplastic progression were determined by terminal histopathological examination and by expression and functional evaluation of glutathione S-transferase (GST) isoenzyme phenotypes. Dietary DCA increased the number of hepatocellular neoplasms per rat, accelerated the rate of growth of persistent nodules, and increased the histological progression of liver tumors. Expression of immunoreactive GST subunits Yf, Ya, and Yb1 was induced in early persistent nodules, a pattern that was maintained throughout the study in both basal diet and DCA-fed groups. However, 5% of early nodules and about 75% of advanced neoplasms were partially or completely deficient in GST Yb2 expression in both groups. DCA did not alter the cytosolic activity for the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) or trans-4-phenyl-3-buten-2-one (tPBO) in tumors or surrounding liver. However, in both groups, CDNB activity was increased in the tumors relative to the surrounding nonneoplastic tissue, whereas activity for tPBO, a substrate more specific for the Yb2 subunit, was reduced in the tumors. All advanced neoplasms were similarly more resistant than surrounding liver to DNA-binding metabolites of aflatoxin B1 or benzo[a]pyrene. These data demonstrate that DCA can increase the progression of established hepatocellular nodules to larger, more advanced neoplasms but does not preferentially select for a specific GST phenotype. Preferential loss of constitutively expressed GST Yb2 in both basal diet and DCA-fed groups may be an important aspect of progression from resistant nodules to advanced cancers in this model. These studies also demonstrate that serial MRI is a useful tool for measuring the rates of enlargement and patterns of growth in established hepatocellular neoplasms.

Stimulatory effects of sulfur and nitrogen oxides on carcinogen activation in human polymorphonuclear leukocytes.

The occurrence of inflammatory processes and of cancer in the human respiratory tract is intimately associated. One of the major factors in this is probably the recruitment of and stimulated activity of polymorphonuclear leukocytes (PML) in conjunction with the ability of these cells to convert various carcinogens to their ultimate active metabolites. In this study, we demonstrate that nitrite and sulfite, the major dissolution products of the environmental pollutants nitrogen dioxide and sulfur dioxide in water enhance the metabolic activation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-dihydrodiol), the proximal carcinogen of benzo[a]pyrene, to trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and tetraols, the corresponding hydrolysis products, in human PML prestimulated with 12-O-tetradecanoylphorbol-13-acetate. Nitrite was more efficient than sulfite in stimulating the formation of reactive intermediates of BP-7,8-dihydrodiol in PML that covalently bind to extracellular DNA and, in particular, to intracellular proteins. The mechanism by which sulfite stimulates the metabolism of BP-7,8-dihydrodiol most probably involves the intermediate formation of a sulfur trioxide radical anion (SO3.-) the subsequent formation of the corresponding sulfur peroxyl radical anion (.OOSO3-) in the presence of oxygen. The mechanism underlying the stimulatory action of nitrite is less clear but the major pathway seems to involve myeloperoxidase. These results offer an explanation for the increased incidence of lung cancer in cigarette smokers living in urban areas. The major glutathione transferase (GST) isoenzyme in human PML is GST P1-1, a Pi-class form. The GST activity of PML was found to be inversely correlated with the extent of binding of BP-7,8-dihydrodiol products to exogenous DNA. These results suggest that individuals exhibiting high GST-activity in the PML may be better protected against the type of carcinogenic dealt with in this study.

Induction of specific cytochrome P450s involved in aflatoxin B1 metabolism in hepatitis B virus transgenic mice.

The relative roles of hepatitis B virus (HBV) and aflatoxin and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen-metabolizing enzymes. We tested this hypothesis in an HBV-transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278-1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a-5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an alpha-class glutathione S-transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a-5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV-transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV-transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver-damaging agents.

Heterogeneous expression and polymorphic genotype of glutathione S-transferases in human lung.

Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics by conjugation with glutathione. One of the mu class genes of this superfamily of enzymes, GSTM1, is polymorphic because of a partial gene deletion. This results in a failure to express GSTM1 in approximately 50% of individuals. Several studies have linked GSTM1 null status to an increased risk of lung carcinoma. This study investigated the expression and distribution of GST isoenzymes in human lung, and developed a polymerase chain reaction (PCR) assay which would allow genotyping of archival, paraffin embedded lung tissue. Distribution was examined using a panel of polyclonal anti-GST antibodies for immunohistochemistry in normal tissue of 21 tumour-bearing lungs. DNA for PCR was extracted from paraffin blocks and a control group of 350 blood lysates. As a positive control each assay amplified part of GSTM4, a mu class gene which is not polymorphic but which shows strong sequence homology to GSTM1. The presence of GST in bronchoalveolar lavage fluid was sought by Western analysis. Proximal airways contained pi class GST, alpha class GST, and mu class GST with expression concentrated in the brush border. In distal airspaces no alpha GST was expressed but pi GST and mu GST were present in alveolar cells and also alveolar macrophages. Pi class GST was present in bronchoalveolar lavage fluid. The PCR assay enabled genotypic determination using DNA extracted from archival material. Of the control group 56% were null at the GSTM1 locus. The distribution of GST isoenzymes in the lung is heterogeneous with an apparent decrease in GST in distal lung. Since GSTM1 status has already been associated with susceptibility to disease, the PCR assay developed will allow further studies of the relation between genotype and structural disorders in the lung using archival pathological material.

Active-site tyrosyl residues are targets in the irreversible inhibition of a class Mu glutathione transferase by 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone.

The mode of inactivation of glutathione S-transferase isoenzyme 3-3 from rat by the active site-directed inhibitor 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GSTCBQ) has been investigated by a combination of site-specific mutagenesis and mass spectrometric analysis of the sites of reaction of the reagent with the enzyme. This very reactive reagent is shown to target 3 residues in or near the active site, including the hydroxyl groups of Tyr-6 and Tyr-115 and the sulfhydryl group of Cys-114. Although the covalent attachment of one 2-(S-glutathionyl)dichloro-1,4-benzoquinonyl group/active site is sufficient to inactivate the enzyme ( < 5% residual activity), the 1 mol of reagent appears to be distributed among all three target sites. Mutant enzymes in which the reactive functional groups of these 3 residues have been individually removed remain susceptible to GSTCBQ. Evidence from amino acid sequencing and peptide maps visualized by matrix-assisted laser desorption/ionization mass spectrometry suggests that both Tyr-6 and Tyr-115 are primary targets of the reagent in the native enzyme. Docking of a model of GSTCBQ in a model of the active site derived from the crystal structure of the enzyme indicates that the trichlorobenzoquinonyl group can be positioned so that both tyrosine hydroxyl groups can act as nucleophiles to add to the reagent or alternatively act as electrophiles to assist in the nucleophilic addition of the other. The reaction of GSTCBQ with Cys-114 appears to require a conformation different from that in the crystal structure.

Glutathione transferases in the urine: sensitive methods for detection of kidney damage induced by nephrotoxic agents in humans.

With the aid of immunohistochemical methods the localization of the various isoenzymes of glutathione S-transferase was investigated. The alpha isoenzyme was present solely in the proximal tubular cells of the human kidney, while the pi form was restricted to the distal convoluted tubules, the thin loop of Henle, and the collecting ducts. Damage to the epithelial cell membranes results in the increased excretion of these enzymes with the urine. The alpha and pi isoenzymes have been isolated in a highly purified form and used for the production of polyclonal antisera. Subsequently, radioimmunological and ELISA techniques were developed for quantitation of these proteins in the urine; the methods exhibited a high specificity and were sufficiently sensitive to determine nanogram quantities or less. Disease affecting tubular function, cyclosporine A treatment, administration of nephrotoxic antibiotics, and exposure to cadmium all resulted in characteristic changes in the pattern of the glutathione transferase isoenzymes present in urine. Such effects were seen also in patients who had previously been exposed to nephrotoxic agents, but in whom conventional tests for kidney function were apparently normal. Thus, it appears that radioimmunologic or immunochemical quantitation of alpha and pi forms of the enzyme can be used as sensitive and relatively simple markers for the early detection of toxic effects with respect to the renal tubuli.

Immunochemical evidence for the occurrence of Mu class glutathione S-transferase in human fetal livers.

Immunoblot analysis showed that alpha-class glutathione S-transferase (GST), which is one of the major forms in adult human liver, was expressed in human fetal liver. Mu-class GST was also expressed in fetal liver. The majority of mu-class GST expressed in adult liver consisted of a subunit with a molecular weight of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two subunits of 27 and 26 kDa were detected in fetal liver as proteins immunochemically related to mu-class GST. On reverse-phase HPLC, these two subunits cross-reactive with antibodies to rat GST 3-3 in fetal liver were indistinguishable from each other in their retention time; though, they could be separated by chromatofocusing analysis. The molecular weights of GSTs immunochemically related to rat GST 3-3, eluted at pH 7.1, 6.4, and 5.7, were 27, 27 and 26, and 26 kDa, respectively. In addition, the N-terminal amino acid sequence of these subunits suggested that GSTs related to rat GST 3-3 expressed in fetal liver may be homodimeric and heterodimeric proteins. As expected, pi-class GST was found to be a major form of GST in fetal liver but not in adult liver. In contrast, the GST immunochemically related to rat GST Yrs-Yrs, which is classified as theta-class GST, was detected in adult liver but not in fetal liver. These results indicate that several isoenzymes of GST are expressed in human fetal liver, but they are not the same as those in adult liver.

Purification and characterization of glutathione S-transferase isoenzymes from grey mullet ( Mugil cephalus). Relationship to xenoblotic exposure

Purification and characterization of glutathione S-transferase isoenzymes from grey mullet ( Mugil cephalus). Relationship to xenoblotic exposure

Use of P53-deficient mice for in vivo chemical testing: Preliminary data

Glutathione-S-transferase (GST) activity has been examined in liver cytosol fractions from guinea pigs, mice, control fed rats or rats with pre-neoplastic nodular liver lesions. The levels of activity in unfractionated cytosols have been assayed using the model substrates 1-chloro-2,4-dinitrobenzene (CDNB), 3, 4-dichloronitrobenzene (DCNB) and monobromobimane (mBrB) with reduced glutathione (GSH). The order of activities in the various liver fractions using CDNB as substrate were: mouse > preneoplastic nodular rat > guinea pig >Y control rat and paralleled the capacities of the cytosols to catalyse the formation of aflatoxin B1-GSH from microsomally-activated aflatoxin B1 (AFB1) and GSH. Quantitative differences between the activities of the cytosols using the three model substrates were observed. In the mouse fractionation of GST activity by isoelectric focusing (I.E.F.) on preparative granular gels showed that the most basic component (isoelectric point pH 9.4) with the highest conjugating activity with respect to microsomally-activated AFB1 did not correspond with the peak of most activity for conjugating CDNB. In the pre-neoplastic nodular rat liver the CDNB conjugating activities of all fractions separated on granular I.E.F. gels, were higher than the corresponding fractions isolated from control rat liver, with particular enhancement of the peak containing the 3:3 isoenzyme. In contrast to control rat liver the 7:7 isoenzyme was detected in pre-neoplastic nodular liver preparations. These isoenzymes (3:3 and 7:7) did not contribute significantly to the enhanced level of AFB1-GSH formation catalysed by cytosol fractions prepared from pre-neoplastic nodular rat liver. The microsomally-activated AFB1-conjugating activity of unfractionated rat liver cytosols was increased to a relatively greater extent than CDNB conjugating activity during the induction of pre-neoplastic nodular liver lesions, and the elevated level of the activated AFB1-conjugating activity was found to be associated with the most basic fraction (isoelectric point pH 9.0). Analytical isoelectric focusing gels using mBrB as substrate demonstrated the presence of a basic GST isoenzyme in the pre-neoplastic nodular rat liver, not detected in preparations from the livers of control rats. The low level of activated AFB1-conjugating activity present in unfractionated guinea-pig cytosol was found to correspond with the fraction containing the peak of CDNB conjugating activity on preparative isoelectric focusing (isoelectric point pH 7.5).The lack of correlation between the conjugation of model substrates and the conjugation of xenobiotics could be of importance in studies on drug resistance.

Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters

The effects of microsomal enzyme inducers on glutathione S-transferase (GST) isoenzymes were studied in livers of rats and hamsters using three hypolipidemic drugs of the peroxisome proliferator type and the two model substances phenobarbital (PB) and 3-methylcholanthrene (MC). The effects were investigated by immunoblot analysis of the various GST subunits using polyclonal antibodies directed to rat subunits 1–4. In untreated animals the subunit composition was different, with hamsters having a much higher content of class mu isoenzymes. Administration of all three hypolipidemic drugs reduced the protein concentration of both alpha and mu class GSTs in rats but reduced only class mu subunits in hamsters. This reduction was in good agreement with the decreased activity observed with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB) in both species. As expected, PB and MC increased GST activity together with the concentration of subunits 1 and 3 in rats. In hamsters, PB significantly increased subunit 1 and slightly reduced subunits 3 and 4, although this decrease was not significant. Total GST, measured with CDNB, was reduced by 17%. In contrast, MC slightly decreased subunit 1 and markedly raised subunits 3 and 4, resulting in a net increase in total GST activity. All drugs increased relative liver weight, microsomal protein concentration and total P450 in both species; in contrast, total cytosolic proteins were raised by all drugs in rats but not in hamsters, except for MC. The results obtained in these two species show that GST activity is not always increased by microsomal enzyme inducers. The response may depend in part on isoenzyme profile, and varies with the subunit considered.

Reduced expression of glutathione S-transferase Yb2 during progression of chemically induced hepatocellular carcinomas in fischer 344 rats

We followed the expression of several glutathione S-transferase subunits in altered foci, liver neoplasms and metastases produced in male Fischer 344 rats by a modified Solt-Farber protocol, to determine whether components of the resistant phenotype are lost during neoplastic progression. At 6 mo after initiation, altered foci and persistent nodules displayed increased immunohistochemical expression of glutathione S-transferase subunits Yf (pi-class), Ya (alpha-class) and Yb1 (mu-class) in comparison with normal or surrounding liver tissue. However, although most altered foci exhibited little change in glutathione S-transferase Yb2 (mu-class) subunit expression, 5% of Yf-positive foci and nodules were partially or completely deficient in Yb2 expression. At 12 and 18 mo after initiation, most grossly visible hepatocellular tumors retained induced expression of glutathione S-transferase subunits Yf, Ya and Yb1, but 63% of the carcinomas, 88% of the primary metastatic carcinomas and 94% of the pulmonary metastases were deficient in Yb2 expression. These differences in glutathione S-transferase subunit expression were confirmed by quantitative analysis by reverse-phase HPLC of S-hexylglutathione affinity-purified glutathione S-transferases from advanced tumors. Cytosolic glutathione S-transferase activity for trans-4-phenyl-3-buten-2-one in advanced tumors ranged from 42% to 66% of the activity in matched surrounding liver, whereas glutathione S-transferase activities for 1-chloro-2,4-dinitrobenzene were increased by 140% to 161%. These studies demonstrate that progression of hepatocellular carcinomas in the resistant hepatocyte model of carcinogenesis in which several glutathione S-transferase subunits are induced is associated with the loss of a major constitutive mu-class hepatic glutathione S-transferase. Although the mechanism and role of the reduction or loss of glutathione S-transferase Yb2 during malignant progression are unknown, we propose that loss of glutathione S-transferase Yb2 in some preneoplastic populations of hepatocytes might be conducive to further DNA damage by presently unknown environmental or endogenous compounds that are normally detoxified preferentially by glutathione S-transferase isoenzymes containing this subunit.

Main drug- and carcinogen-metabolizing enzyme systems in human non-small cell lung cancer and peritumoral tissues : Toussaint C, Albin N, Massaad L, Grunenwald D, Parise O Jr, Morizet J et al. Institut Gustave-Roussy, Pavillon de recherche 2, 94805 Villejuif Cedex. Cancer Res 1993;53:4608–12

To better understand the importance of drug-metabolizing enzymes in carcinogenesis and anticancer drug sensitivity of human non-small cell lung cancer, we studied the main drug-metabolizing enzyme systems in both lung tumors and their corresponding nontumoral lung tissues in 12 patients. The following enzymes were assayed by Western blot analysis: cytochromes P-450 (1A1/A2, 2B1/B2, 2C8-10, 2E1, 3A4); epoxide hydrolase; and glutathione S-transferase isoenzymes (GST-alpha, -mu, and -pi). The activity of the following enzymes or cofactor were determined by spectrophotometric or fluorometric assays: glutathione S-transferase (GST); total glutathione; UDP-glucuronosyltransferase; beta-glucuronidase; sulfotransferase; and sulfatase. Results showed the presence of cytochrome P-450 1A1/1A2 in both tumoral and nontumoral tissues. P-450 1A1/1A2 levels were 3-fold lower in tumors compared to corresponding nontumoral tissues (P < 0.05). None of the other probed cytochromes P-450 were detected in either tumoral or nontumoral lung tissues. For the glutathione system, no significant difference between tumoral and nontumoral tissues was observed (GST activity, glutathione content, GST-alpha, -mu, and -pi). A positive linear correlation was observed between GST activity and GST-alpha or GST-pi. No significant difference was observed for the glucuronide and the sulfate pathways and their corresponding hydrolytic enzymes. Epoxide hydrolase was significantly decreased in tumors compared to nontumoral lung tissues (P < 0.05). In conclusion, these results showed differences between non-small cell lung tumors and nontumoral tissues for cytochrome P-450 1A1/1A2 and epoxide hydrolase. These differences between tumors and peritumoral tissues with regard to these drug-metabolizing enzymes could reflect differences occurring after malignant transformation and may play a role in drug sensitivity to anticancer drugs.

Reduced expression of glutathione S-transferase Yb2 during progression of chemically induced hepatocellular carcinomas in Fischer 344 rats.

We followed the expression of several glutathione S-transferase subunits in altered foci, liver neoplasms and metastases produced in male Fischer 344 rats by a modified Solt-Farber protocol, to determine whether components of the resistant phenotype are lost during neoplastic progression. At 6 mo after initiation, altered foci and persistent nodules displayed increased immunohistochemical expression of glutathione S-transferase subunits Yf (pi-class), Ya (alpha-class) and Yb1 (mu-class) in comparison with normal or surrounding liver tissue. However, although most altered foci exhibited little change in glutathione S-transferase Yb2 (mu-class) subunit expression, 5% of Yf-positive foci and nodules were partially or completely deficient in Yb2 expression. At 12 and 18 mo after initiation, most grossly visible hepatocellular tumors retained induced expression of glutathione S-transferase subunits Yf, Ya and Yb1, but 63% of the carcinomas, 88% of the primary metastatic carcinomas and 94% of the pulmonary metastases were deficient in Yb2 expression. These differences in glutathione S-transferase subunit expression were confirmed by quantitative analysis by reverse-phase HPLC of S-hexylglutathione affinity-purified glutathione S-transferases from advanced tumors. Cytosolic glutathione S-transferase activity for trans-4-phenyl-3-buten-2-one in advanced tumors ranged from 42% to 66% of the activity in matched surrounding liver, whereas glutathione S-transferase activities for 1-chloro-2,4-dinitrobenzene were increased by 140% to 161%. These studies demonstrate that progression of hepatocellular carcinomas in the resistant hepatocyte model of carcinogenesis in which several glutathione S-transferase subunits are induced is associated with the loss of a major constitutive mu-class hepatic glutathione S-transferase. Although the mechanism and role of the reduction or loss of glutathione S-transferase Yb2 during malignant progression are unknown, we propose that loss of glutathione S-transferase Yb2 in some preneoplastic populations of hepatocytes might be conducive to further DNA damage by presently unknown environmental or endogenous compounds that are normally detoxified preferentially by glutathione S-transferase isoenzymes containing this subunit.

Study of chromatographic parameters for glutathione S transferases on an high-performance liquid chromatography affinity stationary phase

The chromatographic parameters were examined for recombinant glutathione S-transferases (GSTs) on a new HPLC affinity packing containing the immobilized ligand S-octylglutathione. The k′ values of both rA1-1 and rP1-1 were determined under isocratic conditions with increasing concentrations of the mobile phase ligand S-butyl glutathione. Plots of 1/ k′ vs. S-butylglutathione concentration were non-linear which is consistent with a bivalent model for the association of these dimeric enzymes and the stationary phase. Low flow-rates were found to be decisive in obtaining good resolution of the isoenzymes, and at 0.10 ml/min it was possible to obtain baseline resolution of rP1-1, rA1-1 and rM1a-1a using shallow, linear gradients of GST competitive inhibitors. Association constants were determined from solution phase kinetics assuming a rapid equilibrium random Bi Bi mechanism. Solution phase association constants provide an approximate guide for the selection of ligands useful in this affinity phase HPLC separation of GST isoenzymes. A good fit ( r 2= 0.998) for the rA1-1 binding data was obtained using the solution phase binding constant but this was not the case for rP1-1. A comparison of the selectivities for the separation of rP1-1, rA1-1 and rM1a-1a was made using the GST competitive inhibitors S-hexylglutathione, S-butylglutathione and γ-glutamyl-(S-hexyl)cysteinyl-phenylglycine as mobile phase modifiers. The association constants determined in solution did not always predict the elution order of the recombinant GSTs (rGSTs) using the mobile phase inhibitors. Yields of active rGSTs from the column were 90%, 88% and 61% for rP1-1, rA1-1 and rM1a-1a, respectively. This technique was used in the fractionation of GSTs in placental and liver cytosols.

Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 × 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0–200 m M sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.

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.

Multiple glutathione S-transferases in Galleria mellonella; their detection with Fluorigenic substrates

Multiple glutathione S-transferase (GST) isoenzymes from Galleria mellonella have been purified by use of a combination of BSP affinity chromatography and isoelectric focusing to give catalytically active proteins isoelectric at pI 5.2, 6.9, 7.7 and 8.2. All isoforms were active with 1-chloro-2,4-dinitrobenzene (CDNB), 3-4-dichloro-nitrobenzene (DCNB) and were capable of cleaving β-methyl-umbelliferyl acetate (MUA). The pI 8.2 isoenzyme had by far the greatest activity with respect to CDNB. All four isoforms had activities of approximately the same magnitude with the other two substrates. All isoforms had detectable activity with monobromobimane and this substrate, or β-methyl umbelliferyl acetate could be used to localize the isoenzymes in electrophoresis gels by virtue of the fluorescent products of the reactions.

Role of glutathione S-transferase isoenzymes in cisplatin-induced nephrotoxicity in the rat

cis-Diamminedichloroplatinum(II) (cisplatin) is an effective antitumor agent but causes dose-dependent nephrotoxicity. We examined the changes of glutathione S-transferase (GST) isoenzymes in the rat kidney after cisplatin administration. Renal GST-α activity was decreased to 33.4% of the control level and GST-μ activity was increased 1.9-fold after cisplatin administration. These results were confirmed by affinity chromatography of rat renal GST isoenzymes. Our results showed that changes of GST isoenzymes were associated with cisplatin-induced nephrotoxicity. We examined whether GST isoenzymes leaked into the urine by proximal tubular damage could provide a useful marker of cisplatin-induced nephrotoxicity. The total GST and GST-μ activities in urine correlated well with the changes of BUN, which closely parallels the course of nephrotoxicity after cisplatin administration. Our results indicated that renal GST-μ activity was decreased by cisplatin, and although GST-μ activity increased as a compensation mechanism, nephrotoxicity still appeared because of differences in substrate specificity between these two isoenzymes.

Haem peptide/protein interaction part 6: The kinetic mechanisms of the interactions with, and inhibition of enzymic activity of the human erythrocyte glutathione S-transferase isoenzyme rho ( p), by haem octa-, nona-, and undecapeptides MP-8/-9/-11

The binding of the cytochrome- c derived haem peptides microperoxidase-8, -9, and -11 (MP-8, -9, and -11) to the human erythrocyte glutathione S-transferase rho (GST- p) enzyme is demonstrated. Inhibition by the haem peptides of the enzymic conjugation of glutathione (GSH) with the electrophilic cosubstrate 1-chloro-2, 4-dinitrobenzene (CDNB) is mixed-type with respect to CDNB, and K i, the inhibition constant, increases with increasing length of the peptide chain. The results obtained here for the GST- p are compared to those published recently for the previously-supposed identical isoenzyme human placental GST-π.