PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE

Journal of Biological Chemistry, 2001

Archives of Biochemistry and Biophysics, 1999

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human ␣-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (k cat /K m) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s ؊1 mM ؊1 , respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s ؊1 mM ؊1 , respectively. Immunotitration studies with human liver extracts showed that the antibodies against human ␣-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the ␣-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetrae-noic acid, an important intermediate in the 5-lipoxygenase pathway.

Archives of Biochemistry and Biophysics, 1989

Rat liver microsomal glutathione transferase displays glutathione peroxidase activity with linoleic acid hydroperoxide, linoleic acid ethyl ester hydroperoxide, and dilinoleoyl phosphatidylcholine hydroperoxide, with rates of 0.2,0.3, and 0.3 /*mol/min/mg, respectively. The activities are increased between three-and fourfold when the enzyme is activated with N-ethylmaleimide. Microsomal glutathione transferase can also conjugate 4-hydroxynon-2-enal with a specific activity of 0.5 pmol/min/mg. These findings show that the enzyme can remove harmful products of lipid peroxidation and thereby possibly protect intracellular membranes against oxidative stress. A set of glutathione transferase inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue, and bromosulfophtalein) which abolish the glutathione-dependent protection against lipid peroxidation in liver microsomes have been characterized. These inhibitors were found to be effective in the micromolar range and could prove valuable in studying the factor responsible for glutathione-dependent protection against lipid peroxidation. 0 is89 Academic press, h. Glutathione transferases are a group of proteins (see (1) for review) involved in the conjugation of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds (2). Interest has also focused on the ability of these proteins to protect against harmful products formed during oxidative stress, such as hydroperoxides from lipids and DNA (3), as well as 4-hydroxyalk-2-enals (4). The liver of mammalian species contains numerous cytosolic forms and one membrane-bound form of glutathione transferase. The membrane-bound microsomal glutathione transferase (see (5, 6) for reviews) bears no obvious structural resemblance (amino acid sequence, molecular

Biochemical and Biophysical Research Communications, 1993

Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order : GST A1-1 GST T1-1 GST M1-1 GST A2-2 GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11octadecadienoyl)--3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol\min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-

Biochimica et Biophysica Acta (BBA) - General Subjects, 1985

Archives of Biochemistry and Biophysics, 1992

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 m-, g-, and a-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 k-and the a-class GSTs of human lung exhibited heterogeneity. The two p-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 p-class GSTs. The a-class GST isozymes of lung resolved into three peaks during isoelectric focusing corresponding to plvalues of 9.2,8.95, and 8.8. All three a-class GSTs isozymes had blocked N-termini and were immunologically similar to human liver a-class GSTs. Peptide fingerprints generated by SV-8 protease digestion and CNBr cleavage indicated minor structural differences between the liver and the lung a-class GSTs. The three a-class GSTs of lung expressed glutathione peroxidase activities toward the hydroperoxides of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with K, values in the range of 22 to 87 MM and V,,, values in the range of 67-120 mol/mol/ min, indicating the involvement of the a-class GSTs in the protection mechanisms against peroxidation. All ' This investigation was supported in part by USPHS Grant CA27967 awarded to Y.C.A. by the National Cancer Institute. Support by the Department of Internal Medicine to S.A. is acknowledged.

Journal of Biological Chemistry, 2002

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (1999) J. Biol. Chem. 274, 2866 -2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1990

An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO 3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of I0 or 500 /Lg/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ most sensitive to oxidative stress.

Journal of lipid research, 1996

Two enzymatic mechanisms have been proposed for the metabolism of hydroperoxy-phospholipids: i) the combined action of phospholipase A2 and glutathione peroxidase, and/or ii) direct enzymatic reduction. The latter reaction may be catalyzed by selenium-dependent phospholipid hydroperoxide glutathione peroxidase and/or by glutathione S-transferase alpha. To study the pathway of this reaction, we used human hepatoma HepG2 cells into which was incorporated labeled, hydroperoxy-phospholipids. The major product of incorporated l-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine was the corresponding hydroxy-phospholipid with no hydroxy- or hydroperoxy-fatty acids. The contributions to reduction of hydroperoxy-phospholipids in HepG2 cells from glutathione S-transferase Al and phospholipid hydroperoxide glutathione peroxidase were calculated to be 0.5% and 99.5%, respectively. Increasing selenium in the cell culture medium led to increases in selenium-depe...

Biochimica et Biophysica Acta (BBA) - General Subjects, 1986

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the a, fl, ~, 8 and e set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosois.