A study of gender,strain and age differences in mouse liver glutathione-S-transferase

The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

Glutathione S-transferase activities in phytophagous insects: Induction and inhibition by plant phototoxins and phenols

The effects of two plant phototoxins (xanthotoxin and harmine) and three plant phenols (quercetin, ellagic acid, and juglone) on detoxification enzymes were studied in the polyphagous cabbage looper, Trichoplusia ni, and the oligophagous black swallowtail, Papilio polyxenes. In P. polyxenes, glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were 1840 and 1750 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut and fat body, respectively. Dietary xanthotoxin (0.1% fw) increased the activity 2.5 and 2.9-fold in the midgut and fat body, respectively. Xanthotoxin-conjugating GST activity was absent in both tissues. In T. ni, GST activity, 513 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut, was increased almost twofold by dietary xanthotoxin and harmine. Plant phenols effectively inhibited in vitro GST and Se-independent glutathione peroxidase (GPOX) activities in a dose-dependent manner in the two species. Both GST and GPOX of P. polyxenes were 2-fold less sensitive to phenol inhibitors than T. ni. GST inhibition differed according to the nature of the inhibitor in P. polyxenes. Quercetin is competitive with CDNB and is non-competitive with respect to GSH. In contrast, inhibition by ellagic acid is non-competitive with CDNB and competitive with GSH. Juglone showed competitive inhibition with both GSH and CDNB.     

Comparative study of the purification and characterization of the cytosolic glutathione S-transferases from two salmonid species: Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

In the present report, an efficient method for isolating multiple cytosolic forms of glutathione S-transferases from liver and kidney cytosolic samples of two salmonid species (brown trout and Atlantic salmon) is described, and some of the multiple properties of these enzymes are presented. Glutathione S-transferases were partially purified by low-pressure affinity chromatography on a column with glutathione coupled to agarose, which retained an average of 89.47% of the total activity. The GST activity was appropriated towards CDNB and ETHA as substrates. The application of an HPLC system associated to elestrospray ionization mass spectrometry allowed the identification of five GST cytosolic isoforms, corresponding to subunits with M(r) between 23,700 and 26,900 Da being the main form, with retention time of 17 min, a pi-class-related GST isoenzyme.     

Comparison of glutathione S-transferase and general esterase in two bugs, Eurygaster integriceps Puton (Heteroptera: Scutelleridae) and Brachynema germari Kolenati (Heteroptera: Pentatomidae)

Glutathione S -transferases (GSTs) and general esterases play important roles in the detoxification of many substances including allelochemicals from plants and chemical pesticides. In this study, to determine the feeding status and effects of agronomical practices on GST and general esterase, two bug species, Eurygaster integriceps and Brachynema germari , were selected with differences in these cases. GST and general esterase responses varied when switching from 1-chloro-2,4-dinitrobenzene (CDNB) and α -naphtyl to 1,2-dichloro-4-nitro-benzene (DCNB) and β -naphtyl in E. integriceps and B. germari , respectively. The activities of GST and esterase by using CDNB and α -naphtyl were higher than that of DCNB and β -naphtyl for both insects. It was shown that the optimal pH for GST and general esterase activity varied between pH 5.5 and 6. While the Michaelis constant (K_m) value related to E. integriceps toward CDNB and α -naphtyl was lower than K_m for B. germari , K_m values calculated for E. integriceps toward DCNB and β -naphtyl corresponding to those related to A. hejeri were similar to CDNB and α -naphtyl values. The maximal reaction velocity (V_max) values related to E. integriceps for both substrates (CDNB and α -naphtyl) were always higher than those from B. germari and showed significant differences. At the basement of the native-PAGE electrophoresis, five bands were observed in for GST and three were visualized for B. germari , with a large, darker band for E. integriceps in the case of esterases. Studies on the detoxification enzymes of herbivores should be undertaken to determine accurately the effect of the host plants on the organisms eating them, particularly in terms of biochemical and ecological advantages.     

The use of acetylcholinesterase activity in Ruditapes decussatus and Mytilus galloprovincialis in the biomonitoring of Bizerta lagoon

Glutathione S-transferases (GST) form an important family of biotransformation enzymes catalyzing the conjugation of glutathione to a great variety of xenobiotic compounds. The objective of this study was to compare the different characteristics of GST from freshly isolated rainbow trout hepatocytes with those corresponding to the total liver of the same fish, in order to establish the similarities. GST was purified by affinity chromatography and enzymatic activity was determined towards two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA). The different isoenzymes were determined by HPLC associated with SDS-PAGE. Slight differences between the samples were obtained when the results corresponding to the enzyme activity were compared. HPLC results showed that all GST isoforms present in the total liver samples were represented in the isolated cells too, corresponding to isoforms with molecular masses of approximately 25.5 and 23.0 kDa.     

Glutathione conjugation in male reproductive system: studies on glutathione-S-transferase of bull and boar epididymis

Male reproductive organs are extremely sensitive to the negative influence of toxic environmental factors as well as drugs, and until now not many attempts have been made at studying the detoxication enzymes and the relationship between the activity of those enzymes and spermatozoa fertility. In the present work we studied cytosolic glutathione-S-transferases (GST, EC 2.5.1.18) from different parts (head, corpus and tail) of bull and boar epididymis. We isolated two molecular forms of GST from each part of epididymis, characterized their biochemical properties and examined the mechanism of the catalyzed reaction. On the basis of their substrate specificity and isoelectric point, the isoforms were found to belong to the near neutral GST class mi. All examined GST forms exhibited higher affinity towards GSH than towards 1-chloro-2,4-dinitrobenzene (CDNB) and bull epididymis GST forms showed biphasic Lineweaver-Burk double reciprocal curves in the presence of GSH as a variable substrate. Boar epididymis anionic GST had the -SH groups both in the GSH and the CDNB binding place, whereas the cationic GST form--arginine residues in the CDNB binding place. Bull epididymis GST forms contained neither thiol nor arginine residues essential for catalytic activity.     

Plants for waste water treatment--effects of heavy metals on the detoxification system of Typha latifolia

Upon treatment with Cd and As cattail (Typha latifolia) showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes.Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations. Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Some Biochemical Evidence on the Selective Insecticide Toxicity between the Two Aphids,Aphis citricola and Myzus malisuctus (Homoptera: Phididae), and Their Predator,Harmonia axyridis (Coleoptera: Coccinellidae)

This experiment was carried out to compare the differences in biochemical enzyme activity on the selective insecticide toxicity between the two species of aphid, Aphis citricola van der Goot and Myzus malisuctus Matsumura, and their predator, Harmonia axyridis Pallas. Esterase activities between the two species of aphids and between the two stages of H. axyridis were significant different. Glutathione S-traasferase (GST) activity toward 1-chloro-2, 4-dinitrobenzene (CDNB) was much higher than 1, 2-dichloro-4-nirobenzene (DCNB) in all species tested. No DCNB conjugation was detected in A. citricola and M. malisuctus. The predator, H. axyridis, had much higher GST activity than the preys, A. citricola and M. malisuctus. GST activity toward CDNB in H. axyridis adult was highest, even 6.2-fold higher activity than H. axyridis larva. M. malisuctus had much higher GST activity than A. citricola. The degree of acetylcholinesterase (AChE) inhibition by phosphamidon among all three species tested was significantly varied. The concentration of phosphamidon required for 50% AChE inhibition was lowest in H. axyridis larva, while highest in M. malisuctus. There fore, elevated GST activity and target-site insensitivity may be largely associated with the differential susceptibility between larva and adult of H. axyridis. However, differential susceptibility between A. citricola and M. malisuctus may be due to other various biochemical mechanisms responsible for the multiple selective toxicity, including elevated GST activity and target-site insensitivity.     

Modulation of substrate-specific glutathione S-transferase activity in Daphnia magna with concomitant effects on toxicity tolerance

Glutathione S-transferase (GST) activity was measured in Daphnia magna and Ceriodaphnia reticulata using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as conjugation substrates. Levels of GST activity were comparable between species with CDNB; however, D. magna had nearly twice the GST activity with EA as compared to C. reticulata. GST activity with CDNB was elevated from exposure of daphnids to either CDNB or sodium pentachlorophenate (PCP), but not from exposure to EA. GST activity with EA could not be modulated from exposure to CDNB or EA. GST activity towards CDNB and EA was biochemically separated into different protein fractions suggesting the existence of two distinct isozymes. Preexposure of daphnids to CDNB or PCP increased the organisms' tolerance to the toxic effects of PCP, but not CDNB.     

Combined effects of cadmium and nickel on testicular xenobiotic metabolizing enzymes in rats

When male rats were given a single dose of cadmium (Cd) (3.58 mg CdCl₂·H₂O/kg, ip) 72 hr prior to sacrifice, the testicular 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP), and cumene hydroperoxide (CHPx) decreased significantly as compared to controls. Cd also inhibited reduced glutathione (GSH) level while increasing the lipid peroxidation (LP) level significantly. When the animals were given a single dose of nickel (Ni) (59.5 mg NiCl₂·6H₂O/kg, ip) 16 hr prior to sacrifice, significant decreases were observed in EROD and GST activities toward CDNB, EAA, EPNP, and CHPx, and GSH level. No significant alterations were noted in DCNB GST activity and LP level by Ni. For the combined treatment, rats received the single dose of Ni 56 hr after the single dose of Cd and were killed 16 hr later. In these animals, lesser depressions were observed on EROD activity and LP level than those of Cd alone. The combination of metals significantly inhibited GST activities and GSH level but not to a greater degree than noted by Cd or Ni alone. Plasma testosterone levels of Cd-, Ni-, and combination-treated rats decreased significantly compared to controls. The strongest depression was achieved by Cd alone. Cd, both alone and in combination with Ni, increased the tissue Ni uptake significantly. Ni, however, did not produce such an effect on the tissue uptake of Cd in either case. Cd treatment caused interstitial edema and coagulation necrosis in seminiferous tubules and also caused fibrinoidal necrosis in vascular endothelium. Ni treatment did not produce any pathological testicular alterations compared to controls. Combined treatment produced fewer pathological alterations (i.e., only interstitial edema) than that of Cd treatment. These results reveal that the combination of Cd and Ni does not have a synergistic effect on testicular xenobiotic metabolizing enzymes, and in contrast, Ni has an ameliorating effect on pathological disturbances caused by Cd alone in the rat testis.     

Antioxidant and detoxifying fish enzymes as biomarkers of river pollution

The activity of several antioxidant and detoxifying enzymes, superoxide dismutase SOD , GSH peroxidase GSHPx , GSSG reductase GSR and GSH S transferase GST , the contents of thiobarbituric acid reactive substances TBARS , and the SOD and GST isoenzyme patterns were studied in the livers of chubs Leuciscus cephalus from reference river areas and polluted urban sites. Livers of polluted fish contained higher concentrations of transition metals, especially copper and iron. Total GSHPx activity was 1.8 fold higher in the polluted fish than in reference animals, while the SOD and GSR activities and the TBARS content were not significantly changed. Three new SOD isoforms pI 4.45, 5.1, 5.2 and a higher intensity of the band pI 4.2 were observed after isoelectrofocusing of polluted fish extracts. Total GST activity was higher in fish from polluted areas. The GST isoenzyme pattern was studied using subunit specific substrates DCNB, EPNP, EA, NPB, NBC and by Western blot using antibodies specific to rat GST subunits 1, 8 Alpha class , 3 Mu class and 7 Pi class . Reference and polluted fish lacked cross reactivity towards Alpha class GSTs. Reference fish displayed weaker cross reactivity towards CST 7 and 2.3 fold lower activity with EA, while higher cross reaction with GST 3 was observed in polluted fish.     

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.     

Antioxidant and detoxifying fish enzymes as biomarkers of river pollution

The activity of several antioxidant and detoxifying enzymes, superoxide dismutase SOD, GSH peroxidase GSHPx, GSSG reductase GSR and GSH S transferase GST, the contents of thiobarbituric acid reactive substances TBARS, and the SOD and GST isoenzyme patterns were studied in the livers of chubs Leuciscus cephalus from reference river areas and polluted urban sites. Livers of polluted fish contained higher concentrations of transition metals, especially copper and iron. Total GSHPx activity was 1.8 fold higher in the polluted fish than in reference animals, while the SOD and GSR activities and the TBARS content were not significantly changed. Three new SOD isoforms pI 4.45, 5.1, 5.2 and a higher intensity of the band pI 4.2 were observed after isoelectrofocusing of polluted fish extracts. Total GST activity was higher in fish from polluted areas. The GST isoenzyme pattern was studied using subunit specific substrates DCNB, EPNP, EA, NPB, NBC and by Western blot using antibodies specific to rat GST subunits 1, 8 Alpha class, 3 Mu class and 7 Pi class. Reference and polluted fish lacked cross reactivity towards Alpha class GSTs. Reference fish displayed weaker cross reactivity towards CST 7 and 2.3 fold lower activity with EA, while higher cross reaction with GST 3 was observed in polluted fish.     

Optical biosensor consisting of glutathione-S-transferase for detection of captan

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 μg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min.     

Single chain antibody displays glutathione S-transferase activity

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.