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S S Singhal

Publications and source records attributed to S S Singhal.

At least 37 records · Page 2Linked to original sources
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

Adenosine Triphosphatases↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity.

Purified dinitrophenyl S-glutathione (DNP-SG) ATPase was reconstituted into artificial liposomes prepared from soybean asolectin. Electron micrography confirmed the formation of unilamellar vesicles with an average radius of 0.25 micron. Intravesicular volume estimated by incorporation of radiolabled inulin into the vesicles was found to be 19.7 +/- 1.3 microL/mL reconstitution solution. Accumulation of the glutathione-conjugate of CDNB, DNP-SG, and of doxorubicin (DOX) in the proteoliposomes was increased in the presence of ATP as compared to equimolar ADP or adenosine 5'-[beta,gamma-methylene]triphosphate tetralithium. ATP-dependent transmembrane movement of DOX and DNP-SG into DNP-SG ATPase-reconstituted vesicles was saturable with respect to time, sensitive to the osmolarity of the assay medium, and temperature dependent. The energy of activation was found to be 12 and 15 kcal/mol for DNP-SG and DOX, respectively. Optimal temperature for transport was 37 degrees C. Saturable transport was demonstrated for DNP-SG (Vmax of 433 +/- 20 nmol/min/mg of protein, KmATP = 2.4 +/- 0. 3 mM and KmDNP-SG = 36 +/- 5 microM) as well as DOX (Vmax = 194 +/- 19 nmol/min/mg of protein, KmATP = 2.5 +/- 0.6 mM and KmDOX = 2.4 +/- 0.7 microM). The kinetic data for both DNP-SG and DOX transport were consistent with a random bi-bi sequential reaction mechanism. DOX was found to be a competitive inhibitor of DNP-SG transport with Kis of 1.2 +/- 0.2 microM and DNP-SG was found to be a competitive inhibitor of DOX transport with Kis of 13.3 +/- 2.6 microM.

Adenosine Triphosphatases↗
PubMed1998

Glutathione S-transferases of rabbit lung macrophages.

The catalytic activities of glutathione S-transferases (GSTs), particularly the alpha-class isozymes, can provide protection against oxidative stress through GSH-mediated metabolism of reactive products of lipid peroxidation. Lipid peroxidation products from oxidative metabolism in alveolar macrophages play an important role in mediating and regulating inflammatory response and injury in the lung. The rabbit has been used as an important animal model for studies of the role of alveolar macrophages in pulmonary pathology. Although rabbit lung macrophages display GST activity, the isozyme-specific expression of GSTs and the catalytic properties of these isozymes has not previously been defined. In present studies, we have purified the GST isozymes of rabbit alveolar macrophages obtained by bronchoalveolar lavage and performed immunologic and kinetic characterization of the purified isozymes. Results of our studies indicate the presence of three alpha-class isozymes (pI 10.2, 9.3, and 6.0) and one micro-class isozyme (pI 7.2). N-terminal sequence analysis of the micro-class isozyme indicated that it was distinct from the two previously described micro-class isozymes of rabbit. Kinetic studies indicated that two cationic alpha-class GSTs (pI 10.2 and 9.3) contribute the large majority of selenium independent GSH-peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide (kcat/Km values of 83.4 and 31.9 s-1 . M-1 . 10(3), respectively). A third alpha-class GST (pI 6.0) was shown to have highest catalytic activity toward conjugation of the 4-hydroxynonenal (4HNE) with GSH (kcat/Km = 1900 s-1 . M-1 . 10(3)). Structural and immunologic characterization of this GST isozyme indicated that it belongs to a subclass of the alpha-classGSTs selectively expressed in mesodermal origin cells that are exposed to high levels of oxidative stress and are characterized by high specific activity toward both lipid hydroperoxides and 4-HNE.

Aldehydes↗
PubMed1998

The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall.

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Acrolein↗
PubMed1998

Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver.

Curcumin, an antioxidant isolated from turmeric (curcuma longa), has been shown to attenuate chemical carcinogenesis in rodents. Previous studies have shown that curcumin causes an increase in glutathione S-transferase (GST) activity in rodent liver which may contribute to its anti-cancer and anti-inflammatory activities. Since the effects of curcumin on specific GST isozymes and other glutathione (GSH)-linked enzymes are incompletely defined, we have examined in the present studies the effect of curcumin on hepatic non-protein sulfhydryls and GSH-linked enzymes in male Sprague-Dawley rats. When rats were fed curcumin at doses from 1 to 500 mg kg-1 body weight daily for 14 days, the induction of hepatic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was found to be biphasic, with maximal induction of about 1.5 fold at the 25 to 50 mg kg-1 body weight dosage. At higher doses, a decrease was observed in the activity and in the rats treated with 500 mg kg-1 curcumin this activity was below the levels observed in controls. In contrast, GST activity towards 4-hydroxynonenal (4-HNE) increased in a saturable, dose dependent manner. Western-blot analyses of liver cytosols revealed that curcumin caused a dose dependent induction of rGST 8-8, an isozyme which is known to display the highest activity towards 4-HNE, a highly toxic product of lipid peroxidation. Glutathione peroxidase (GPx) activity towards cumene hydroperoxide in liver homogenate was also found to be increased in a saturable manner with respect to curcumin dose. Our results suggest that induction of enzymes involved in the detoxification of the electrophilic products of lipid peroxidation may contribute to the anti-inflammatory and anti-cancer activities of curcumin.

Animals↗
PubMed1997

Rat GST 8-8 is expressed predominantly in myeloid origin cells infiltrating the gravid uterus.

Previous studies from our laboratory have shown a relatively high expression of rGST8-8 in uterine tissues. This GST isozyme displays relatively high glutathione-peroxidase activity towards lipid-hydroperoxides and towards toxic 4-hydroxyalkenals generated from lipid peroxidation. Since the uterus is a unique organ, subject to oxidative stress due to infiltration by immune effector cells during gestation and because this infiltration is readily identifiable histologically, the studies reported herein were performed to localize the cell specific expression of rGST8-8 to determine whether immune effector cells infiltrating the pregnant rat uterus specifically expressed rGST8-8. A 75 bp end-radiolabeled cRNA probe was prepared from the full length mGSTA4-4 cDNA from the region which is highly homologous with rGST8-8. This cRNA probe was used for in situ hybridization studies to localize rGST8-8 in specific cell types of gravid rat uterus. Results of these studies indicate that this GST isozyme is selectively expressed in myeloid origin cells such as monocytes/macrophages, and neutrophils infiltrating the uterine endometrium and in vascular walls. Selective expression of rGST8-8 in the myeloid origin cells, which are known to generate higher levels of reactive oxygen species, suggests that this GST isozyme plays an important role in the protection mechanisms against lipid peroxidation.

Animals↗
PubMed1996

Purification and characterization of a 4-hydroxynonenal metabolizing glutathione S-transferase isozyme from bovine pulmonary microvessel endothelial cells.

Previous studies have suggested that a group of structurally and immunologically related mammalian glutathione S-transferases (GSTs) which utilize 4-hydroxynonenal (4-HNE) as the preferred substrate and show glutathione peroxidase activity towards phospholipid hydroperoxides may be important for the defense of cells against lipid peroxidation. In present studies we have purified and characterized GST isozymes of bovine pulmonary microvessel endothelial (BPMVE) cells. The results of these studies indicate that BPMVE cells express relatively high amounts of a GST isozyme which utilizes 4-HNE as the preferred substrate. This GST isozyme purified to homogeneity from BPMVE cells showed remarkably high specific activity towards 4-HNE (48.3 units/mg protein) and had similar immunological, kinetic, and structural characteristics as reported for mouse enzyme mGSTA4-4 and other mammalian GSTs of this group. Since the endothelial cells are exposed to constant oxidative stress, we suggest that this GST isozyme may be important for the defense of these cells against lipid peroxidation.

Aldehydes↗
PubMed1996

Modulation of doxorubicin cytotoxicity by ethacrynic acid.

Energy-dependent membrane efflux pumps have been implicated in mediating resistance to doxorubicin (DOX). Membrane-transport mechanisms distinct from P-glycoprotein, capable of transporting DOX and glutathione conjugates have been reported in human cells. Since glutathione-conjugate-forming compounds may be candidates for modulating the cytotoxicity of certain anti-neoplastic agents transported by such transport mechanism, the present studies were performed (i) to determine whether ethacrynic acid, a glutathione-conjugate-forming diuretic, can increase DOX cytotoxicity, and (ii) to study the kinetics of DOX transport and its inhibition by the glutathione conjugate of ethacrynic acid (EA-SG) in the H69 human small-cell-lung-cancer cell line and 2 derived DOX-resistant sublines. Our results indicate that more than one DOX transport mechanism may exist in these cell lines, and that glutathione conjugates may be useful for modulating the cytotoxic effects of DOX.

ATP-Binding Cassette Transporters↗
PubMed1996

Transfection of a 4-hydroxynonenal metabolizing glutathione S-transferase isozyme, mouse GSTA4-4, confers doxorubicin resistance to Chinese hamster ovary cells.

It has previously been suggested that adriamycin (doxorubicin, DOX), which is used to treat various types of malignancies, exerts its cytotoxic effects through interactions with DNA and by accelerating membrane lipid peroxidation. Our previous studies have indicated that a mouse glutathione S-transferases isozyme, mGSTA4-4, which shows high activity toward 4-hydroxyalkenals and the hydroperoxides formed during lipid peroxidation, may play an important role in defense mechanism against lipid peroxidation. In order to test the hypothesis that the enhanced protection against lipid peroxidation confers DOX-resistance to cells, we have compared the cytotoxicity of DOX to Chinese hamster ovary cells transfected with mGSTA4-4 with that of controls cells transfected with the vector alone. The results of these studies show that mGSTA4-4-transfected cells have a twofold higher resistance to DOX as compared to the controls. Upon exposure to DOX, the transfected cells showed about 40% less membrane lipid peroxidation as compared to the controls, indicating a positive relationship between DOX-cytotoxicity and lipid peroxidation. These results suggest that mGSTA4-4 provides protection to cells against lipid peroxidation and that DOX cytotoxicity, at least in part, is mediated through oxidative stress resulting in peroxidation of membrane lipids which would explain the results of previous studies showing that DOX can kill cells without being internalized (T.R. Tritton and G. Yee (1982) Science 217, 248-250).

Aldehydes↗
PubMed1996

A glutathione S-transferases isozyme (bGST 5.8) involved in the metabolism of 4-hydroxy-2-trans-nonenal is localized in bovine lens epithelium.

Previous studies have suggested that a group of GST isozymes (bGST 5.8) with substrate preference for 4-hydroxy-2-trans-nonenal (4-HNE) were present in bovine retina, cornea, iris-ciliary body and sclera, but not in lens. The present studies demonstrate that bGST 5.8 is present in bovine lens epithelium and absent in the cortex and nucleus. Immunochemical studies demonstrated that the enzyme is selectively expressed in epithelium where it can be induced by about 2.5-fold when the lenses are cultured in Medium-199 for 24 hr in the presence of 10 microM BHT. bGST 5.8 was purified to homogeneity from the epithelium using immunoaffinity chromatography. Upon SDS-PAGE, the enzyme showed a single band corresponding to an M(r) value of 25 kDa and its CNBr-peptide maps in SDS-gels were identical to those of the isozymes of this group of GSTs reported previously. The enzyme exhibited high activity towards 4-HNE, and showed glutathione peroxidase activity towards phospholipid hydroperoxides. The Km values of the enzyme for 4-HNE (57 microM from control and 52 microM from BHT-treated) were in the same range as those reported for GSTs 5.8 of human ocular tissues. However, the Kcat value of the lens epithelium enzyme for 4-HNE (15.4 mol mol-1 sec-1 from control, and 20.2 mol mol-1 sec-1 from BHT treated) were considerably less than those reported for the human ocular GST 5.8. Results of these studies suggested that a GST isozyme involved in the detoxification of the electrophilic products of lipid peroxidation was localized in the epithelium of bovine lens.

Aldehydes↗
PubMed1996

Rabbit aorta glutathione S-transferases and their role in bioactivation of trinitroglycerin.

The pharmacological action of glyceryl trinitrate (GTN), a widely used drug for the treatment of angina pectoris, is thought to be mediated through release of nitric oxide (NO) during its biotransformation. Since glutathione S-transferases (GST) can utilize GTN as substrate and GST inhibitors can attenuate GTN-induced relaxation of rabbit aorta in vitro it has been suggested that these enzymes are involved in the bioactivation of GTN in rabbit aorta. Because GSTs are multifunctional enzymes and a multitude of GST isozymes with varying substrate preferences are present in mammalian tissues, the role of specific GST isozymes in bioactivation of GTN in rabbit aorta needs to be established. Therefore, during the present studies we have purified and characterized GST isozymes from rabbit aorta and evaluated their possible roles in the biotransformation of GTN. The results of these studies showed that rabbit aorta contained three GST isozymes having pI values of 9.4, 7.7, and 5.4. Structural, immunological, and kinetic studies showed that GST 9.4, GST 7.7, and GST 5.4 belonged to the alpha-, pi-, and mu-classes, respectively. The relative abundance of these enzymes in rabbit aorta was alpha > pi > mu. The alpha- and mu-class GST isozymes had similar activities toward GTN (0.71 U/mg and 0.86 U/mg, respectively) while the pi-class GST showed much lower activity toward GTN. The catalytic efficiency k(cat)/Km of the mu- and alpha-class GSTs toward GTN were similar but these activities were differentially inhibited by ethacrynic acid, its GSH conjugate, bromosulfophthalein (BSP), and hematin. These results suggest that in rabbit aorta GSTs may be involved in bioactivation of GTN, and because of their higher abundance the alpha-class GSTs may be more important for the pharmacological effects of GTN than the mu-class GSTs. The results on kinetics of inhibition by various inhibitors suggest that hematin may be an effective inhibitor to delineate the role of specific GST isozymes in the bioactivation of GTN.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of glutathione S-transferases of rat uterus.

Glutathione S-transferases (GSTs) provide protection to cells against electrophilic xenobiotics as well as lipid hydroperoxides and 4-hydroxynonenal generated during lipid peroxidation. The catalytic properties of the alpha class GSTs are well suited for detoxification of electrophilic products of lipid peroxidation. An immunologically distinct subgroup of the alpha class GST isozymes having high activity towards 4-hydroxynonenal has been recently identified in several mammalian tissues [Zimniak et al. (1994) J. Biol. Chem. 269, 992-1000]. Since oxidative stress can exert deleterious effects during gestation, the present studies were performed to determine whether the rat homolog of this group of GSTs, rGST 8-8, is expressed in gravid rat uterus, where it may function as a defense mechanism against oxidative stress. GSTs were purified by GSH-affinity chromatography. Individual GST isozymes were separated by column isoelectric focusing and their immunologic identities were established using highly specific polyclonal antibodies in Western blot analysis. Their expression was quantified and kinetic properties were characterized. Rat uterus contained an alpha class GST (pI 9.8), a pi class GST (pI 8.1), two mu class GSTs (pI 6.7 and 6.2) and rGST 8-8. This result indicated that rGST subunits 1, 2, 3, 4, 7 and 8 are present in rat uterus. The relative abundance of rGST 8-8 in the gravid rat uterus was found to be about three-fold higher as compared with that previously seen in rat liver. Higher relative abundance of rGST8-8 in gravid rat uterus suggests that it may play a protective role against the deleterious effects of lipid peroxidation during gestation.

Amino Acid Sequence↗
PubMed1996

Curcumin protects against 4-hydroxy-2-trans-nonenal-induced cataract formation in rat lenses.

Age-related cataractogenesis is a significant health problem worldwide. Oxidative stress has been suggested to be a common underlying mechanism of cataractogenesis, and augmentation of the antioxidant defenses of the ocular lens has been shown to prevent or delay cataractogenesis. The present studies were designed to test the efficacy of curcumin, an antioxidant present in the commonly used spice turmeric, in preventing cataractogenesis in an in vitro rat model. Rats were maintained on an AIN-76 diet (ICN Pharmaceuticals Inc, Cleveland) for 2 wk, after which they were given a daily dose of corn oil alone or 75 mg curcumin/kg in corn oil for 14 d. Their lenses were removed and cultured for 72 h in vitro in the presence or absence of 100 mumol 4-hydroxy-2-nonenal (4-HNE)/L, a highly electrophilic product of lipid peroxidation. The results of these studies showed that 4-HNE caused opacifications of cultured lenses as indicated by the measurements of transmitted light intensity using digital image analysis. However, the lenses from curcumin-treated rats were much more resistant to 4-HNE-induced opacification than were lenses from control animals. Curcumin treatment caused a significant induction of the glutathione S-transferase (GST) isozyme rGST8-8 in rat lens epithelium. Because rGST8-8 utilizes 4-HNE as a preferred substrate, we suggest that the protective effect of curcumin may be mediated through the induction of this GST isozyme. These studies suggest that curcumin may be an effective protective agent against cataractogenesis induced by lipid peroxidation.

Aldehydes↗
PubMed1995

Iron-induced lipid peroxidation in rat liver is accompanied by preferential induction of glutathione S-transferase 8-8 isozyme.

Since previous studies from this laboratory have suggested that glutathione S-transferase (GST) 8-8 of rat belongs to a distinct subgroup of GST isozymes which may be involved in the detoxification of the products of lipid peroxidation (Zimniak et al., J. Biol. Chem. 269, 992-1000, 1994), during the present studies we examined the effect of iron-induced lipid peroxidation on the expression of GST 8-8 in rat liver. Rats treated with 100 mg/kg body wt iron showed a significant increase in lipid peroxidation in liver. This was accompanied by a concomitant increase in the expression of GST 8-8 in liver as observed in isoelectrophoretic analysis of rat liver GSTs, and an increase in GST activity toward 4-HNE, a toxic product of lipid peroxidation toward which GST 8-8 displays high specific activity. Western blot studies using polyclonal antibodies specifically recognizing GST 8-8 also indicated that, among the GST isozymes of rat liver, GST 8-8 was preferentially induced upon iron treatment. These findings were further confirmed by purifying and quantitating GST 8-8 protein from the controls and iron-treated rats. Significant differences in the specific activities of GST 8-8 purified from the controls and iron-treated rats were observed, indicating that more than one GST isozyme related to GST 8-8 may be present in rat liver. This observation is consistent with the observed heterogeneity in mouse mGSTA4-4 which is an ortholog of rat GST 8-8. Iron treatment also caused significant increase in GSH levels probably because of de novo synthesis as indicated by an increase in gamma-glutamyl cysteine synthetase activity. The results of these studies suggest that GST 8-8, and possibly other related GST isozymes, may play an important role in defense mechanisms against lipid peroxidation.

Aldehydes↗
PubMed1995

Glutathione S-transferase 8-8 is localized in smooth muscle cells of rat aorta and is induced in an experimental model of atherosclerosis.

Allylamine (AA) is an electrophilic amine with a long history of experimental usage because of its extremely potent and relatively specific cardiovascular toxicity; it has been utilized in a variety of experimental models attempting to mimic human atherosclerotic lesions, myocardial infarction, and vascular injury. Even though the exact mechanisms by which AA causes vascular lesions remain unresolved, recent studies on the acute effects of AA exposure in rats strongly suggest that deamination to the aldehyde acrolein, oxidative stress, and the resultant increase in lipid peroxidation, generation of .OH radicals, and acute depletion of glutathione (GSH) may be some of the causative factors in AA-induced vascular lesions. Since glutathione S-transferase 8-8 (GST8-8) of rat belongs to a distinct subgroup of GST isozymes involved in the detoxification of products of lipid peroxidation, we designed studies to examine the effects of AA exposure on this GST isoform in rat aorta using Western blotting and immunohistochemical techniques. The results of these studies demonstrate that GST8-8 is expressed in rat aorta and is dramatically induced upon AA exposure. By immunohistochemistry, GST8-8 was localized in the smooth muscle cells of the vascular media which is believed to be the site of metabolism of AA. A significant increase in gamma-glutamylcysteine synthetase activity and GST activity toward 4-hydroxynonenal and acrolein, which are preferred substrates of GST8-8, was seen as early as 3 days following AA treatment. Alterations in GSH and other GSH-related enzymes at 3 and 10 days support the concept that--upon AA exposure--aortic defense mechanisms respond early and induction of GSH biosynthesis and rat GST8-8 occur to alleviate the toxic effects of acrolein, a major, genotoxic product of AA metabolism. The presence of GST8-8 in the vasculature, which is constantly exposed to products of lipid peroxidation, and its induction by AA, suggest that GST8-8 plays a key role in protecting blood vessels against oxidative stress and hence, may be involved in the atherogenic process.

Allylamine↗
PubMed1995

Novel human ocular glutathione S-transferases with high activity toward 4-hydroxynonenal.

PURPOSE: To study the distribution and expression of glutathione S-transferase isozymes involved in detoxification of endogenously generated toxic products of lipid peroxidation, namely, 4-hydroxynonenal (4-HNE) in human lens, retina, cornea, iris, ciliary body and to study their kinetic and structural properties. METHODS: The authors have previously cloned and sequenced cDNA of mouse mGSTA4-4, which shows high activity towards 4-HNE. They have expressed it in Escherichia coli and have raised antibodies against the recombinant mGSTA4-4. In the present study, these antibodies were used in Western blot analysis and immunoaffinity chromatography to study the expression and to purify the human ortholog(s) of mGSTA4-4 from ocular tissues. RESULTS: Western blot analyses of human ocular tissues indicated that a glutathione S-transferases (GST) isozyme immunologically similar to mGSTA4-4 was expressed in cornea, retina, and iris and ciliary body, but not in lens. This isozyme designated as hGST 5.8 was purified to homogeneity from human retina, cornea, and iris and ciliary body by immunoabsorption on immobilized antibodies against mGSTA4-4. The human ortholog of mGSTA4-4, designated as hGST 5.8 purified from all these tissues and pI value of 5.8, subunit Mr value of 25 k and blocked N-terminal. Amino acid sequences of CNBr fragments of hGST 5.8 isozymes of human ocular tissues showed a high degree of primary structure homologies with the corresponding regions of mGSTA4-4. There were noticeable differences in the amino acid sequences of hGST 5.8 of cornea, retina, and iris and ciliary body, suggesting the presence of several closely related hGST 5.8 subunits in the ocular tissues. This heterogeneity was due to tissue-specific expression rather than simple allelic polymorphism. The hGST 5.8 had about sixfold to eightfold higher activity toward 4-hydroxynonenal than 1-chloro-2,4-dinitrobenzene, or CDNB. The catalytic efficiency (Kcat/Km) of ocular hGST 5.8 for 4-HNE was about 100-fold higher than those for the alpha, mu, or pi classes of GST. In addition, hGST 5.8 expressed glutathione peroxidase activity toward phospholipid hydroperoxides and GSH-conjugating activity toward 9,10-epoxy stearic acid. CONCLUSIONS: The results indicate that hGST 5.8 isozyme(s) distinct from the alpha, mu, and pi classes of GSTs, are differentially expressed in human ocular tissues and may play an important role in protective mechanisms against endogenous toxicants generated during lipid peroxidation.

Aldehydes↗