Manju Saxena

Glutathione S-transferases of human lung: Characterization and evaluation of the protective role of the α-class isozymes against lipid peroxidation

Glutathione S-transferase (GST) isozymes of human lung have been purified, characterized, quantitated, and, based on their structural and immunological profiles, identified with their respective classes. The tau-, mu-, and alpha-class GSTs represented 94, 3, and 3% activities of total human lung GSTs toward CDNB, respectively, and 60, 10, and 30% of total GST protein, respectively. Both the mu- and the alpha-class GSTs of human lung exhibited heterogeneity. The two mu-class GSTs of human lung had pI values of 6.5 and 6.25 and were differentially expressed in humans. Significant differences were seen between the kinetic properties of these two isozymes and also between the lung and liver mu-class GSTs. The alpha-class GST isozymes of lung resolved into three peaks during isoelectric focusing corresponding to pI values of 9.2, 8.95, and 8.8. All three alpha-class GSTs isozymes had blocked N-termini and were immunologically similar to human liver alpha-class GSTs. Peptide fingerprints generated by SV-8 protease digestion and CNBr cleavage indicated minor structural differences between the liver and the lung alpha-class GSTs. The three alpha-class GSTs of lung expressed glutathione peroxidase activities toward the hydroperoxides of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with Km values in the range of 22 to 87 microM and Vmax values in the range of 67-120 mol/mol/min, indicating the involvement of the alpha-class GSTs in the protection mechanisms against peroxidation. All three classes of lung GSTs expressed activities toward leukotriene A4 methyl ester and epoxy stearic acid but the mu-class GSTs had relatively higher activities toward these substrates.

Glutathione S-transferases of mouse liver: sex-related differences in the expression of various isozymes

Sex-related differences in the expression of glutathione S-transferase (GST) isozymes of mouse liver have been described. There were no apparent qualitative differences in the isoelectric focusing profiles of the GST isozymes from male and female mouse liver. Both male and female mice have at least four GST isozymes in their liver with pI values of 9.8, 8.7, 6.4 and 5.7. Kinetic, immunological, and structural properties including the N-terminal region amino acid sequences of these isozymes have been determined and they have been classified into alpha, mu, and pi classes. The most cationic isozyme (pI 9.8) belongs to the alpha class and is comparatively more abundant in female liver. The isozyme having pI 8.7 belongs to the pi class and is more abundant in male liver. The mu class GST pI 6.4 as well as the isozyme having pI 5.7 which corresponded to the a class isozyme GST 8-8 of rat liver were more abundant (about 1.5-fold) in male mouse liver as compared to the female. Interestingly, present studies reveal sex-related differences in the heat stabilities of the alpha and pi class GSTs of mouse liver. The alpha class GST pI (9.8) isolated from female mouse liver was more thermostable as compared to the corresponding enzyme from male mouse liver. On the contrary, the pi class GST (pI 8.7) from male mouse was more thermostable as compared to the corresponding enzyme from the female mouse.

A subgroup of class α glutathione S-transferases Cloning of cDNA for mouse lung glutathione S-transferase GST 5.7

A full‐length cDNA clone encoding the previously purified mouse glutathione S‐transferase GST 5.7 [(1991), Biochem. J. 278, 793–799] has been isolated from a mouse lung cDNA library in λgt11. Sequencing of the clone revealed the presence of microheterogeneity in GST 5.7. Comparison of the deduced protein sequence with other glutathione S‐transferases, together with previous information available on GST 5.7, indicates that the enzyme belongs to a novel subgroup within the α class of glutathione S‐transferases. Members of the subgroup, which also include the rat GST 8‐8 and perhaps chicken GST CL3, show high sequence homology with each other, but only moderate similarity to other α class enzymes. They share a substrate specificity profile that resembles π‐class enzymes, and are active in the conjugation of lipid peroxidation products.

Glutathione S-transferases of human skin: qualitative and quantitative differences in men and women.

Glutathione S-transferase (GST) isozymes of male and female leg skin have been characterized. GST activities and protein have been quantified in a number of male and female skin samples and the results indicate that as compared to the male skin, female skin contains a higher amount of GST activity as well as protein. Both male and female leg skin contain three GST isozymes with pI values 9.9, 9.1 and 4.8. In accordance with previous findings the major isozyme, pI 4.8 belongs to the pi-class, whereas the two minor forms pI 9.1 and 9.9 belong to the alpha-class. Each of the three isozymes is more abundant in female skin. Surprisingly, the specific activities and Kcat values of the female skin GSTs, particularly of the pi-class isozyme were found to be significantly higher as compared to those of male skin isozyme. Studies into the kinetics of inhibition by hematin also indicated differences in male and female skin GSTs. Whereas we confirm the presence of an alpha-class GST, pI 9.9, in human skin with an apparently higher subunit M(r) value as compared to other human alpha-class GSTs, contrary to the previous report (Del Boccio et al. (1987) Biochem. J. 244, 21-25), the results of the present studies show that the N-terminus of this alpha-class GST is blocked.

Gender related differences in the expression and characteristics of glutathione S-transferases of human colon

In the present study, the expression of glutathione S-transferase (GST) isozymes was compared in human male and female colon tissues. GST isozymes were purified and quantified in five male and five female colon tissue samples. Noticeable differences were observed in the isoelectric focusing profiles (IEF) of the GSTs, from male and female colon tissues. Both male and female colon tissues had three common GST peaks with pI values of 9.2, 6.7 and 4.8. An additional GST peak with a pI value of 6.2 found in all females was not found in males. Based on kinetic, immunological and structural properties, these isozymes were classified into alpha (pI 9.2), mu (pI 6.7 and 6.2) and pi (pI 4.8) classes. Activity of the alpha-class GST in male colon was approx. 2-fold higher than the corresponding isozyme in female colon. The pi-class GST 4.8 was the most predominant GST in both the sexes and its activity with CDNB as substrate was more abundant (about 1.6-fold) in female colon as compared to that in male colon. Significant differences were seen in substrate specificities between male and female colon GST 4.8. Sex related differences were also observed in the inhibition kinetics of GST pi from male and female colon in the presence of hematin. In addition, GST pi isolated from female colon was more thermostable as compared to the corresponding male isozyme. The thermostability of purified GST pi isozyme from males or females was not affected by incubation of the enzyme with either estrogen, testosterone or progesterone.

Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes.

Dinitrophenyl S-glutathione (Dnp-SG) ATPase has been purified from human muscle to apparent homogeneity using Dnp-SG affinity chromatography and immunoaffinity chromatography using antibodies raised against human erythrocyte Dnp-SG ATPase. The enzyme purified from human muscle showed a subunit M(r) value of about 38 kDa in denaturing gels. The M(r) value of the native enzyme as determined by Sephadex G-200 gel filtration was found to be about 80 kDa, which indicates that it is a dimer. The N-terminus of the enzyme was blocked. Its immunological and kinetic properties were similar to Dnp-SG ATPase of human erythrocytes. Besides catalyzing the ATP hydrolysis in the presence of Dnp-SG, the muscle enzyme also catalyzed ATP hydrolysis in the presence of various leukotrienes, namely LTC4.LTD4, LTE4, and N-acetyl LTE4. The specific activity of the enzyme toward LTC4 was relatively higher than other GSH-xenobiotic conjugates. The muscle enzyme exhibits a low Km value for all leukotrienes as compared to Dnp-SG, indicating high affinity of the enzyme for leukotrienes as activators. The enzyme also catalyzed ATP hydrolysis in the presence of GSH conjugates of endogenously generated fatty acid epoxides. Our results might suggest that Dnp-SG ATPase is involved in the transport of GSH conjugates, leukotrienes, and other organic anions in muscle, erythrocytes, liver, and probably other tissues.

IDENTIFICATION OF AN ANION-TRANSPORT ATPASE THAT CATALYZES GLUTATHIONE CONJUGATE-DEPENDENT ATP HYDROLYSIS IN CANALICULAR PLASMA-MEMBRANES FROM NORMAL RATS AND RATS WITH CONJUGATED HYPERBILIRUBINEMIA (GY MUTANT)

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.

Comparative studies on the effect of butylated hydroxyanisole on glutathione and glutathione S-transferases in the tissues of male and female CD-1 mice.

1. Male CD-1 mice had about 1.6-fold higher glutathione (GSH), 2-fold higher glutathione S-transferase (GST) activity and 2.8-fold higher GST protein in their livers as compared to the female mice. 2. When mice were fed a diet containing 0.75% BHA for 2 weeks, a 1.8-fold increase was observed in GSH levels of female mice liver as opposed to only 1.2-fold increase in male mice. BHA caused 10-fold increase in GST activity and protein in livers of female mice as compared to only about 3-4-fold increase in livers of males. Differential induction of GSH and GST in males and females was also observed in other tissue besides liver but was not as remarkable. 3. Sex-related differences were also observed in the induction of the alpha- and mu- and pi-classes of GSTs by BHA; most noticeable being GST pi, which was induced to about 10-fold in female liver as opposed to only 3.4-fold in male liver.

A specific, sensitive, and rapid method for the determination of glutathione and its application in ocular tissues.

A method for specific determination of glutathione (GSH) is described. This method utilizes the enzymatic conjugation of GSH to 1-chloro-2,4-dinitrobenzene through reaction catalyzed by glutathione S-transferase. The recovery of GSH as determined by this method is comparable to that in currently used methods. The method is specific for GSH determination. Other sulfhydryl (-SH) compounds including the protein -SH or beta-mercaptoethanol, which are often included in tissue homogenates, do not interfere with GSH determination. Acid extraction of the tissue is not required in this method and comparatively smaller amounts of tissue samples (as little as 20 microliters of a 10% w/v tissue homogenate) are needed for the analyses. The method when applied for GSH determination in ocular tissues yielded results in agreement with the reported values in literature. Evidence for the sensitivity, accuracy, and convenience of the method is provided by analysing the sample containing GSH in the range of 1-200 nmol by this method.

Characterization of two novel subunits of the α-class glutathione S-transferases of human liver

Abstract More than 85% of the complete amino-acid sequence of the α-class glutathione S- transferase ω (GST ω) of human liver, described for the first time in this communication, show that GST ω is a heterodimer of two closely related novel α-class GST subunits. The sequences of these subunits, ω1 and ω2, have over 97% homology between them and are also highly homologous to the two α-class subunits characterized previously. Characterization of these two novel α-class subunits described in this report would explain the molecular basis for high degree of heterogeneity observed among the α-class human GSTs.