Key points are not available for this paper at this time.
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 (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (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. 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 (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (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. Glutathione peroxidases (GPx) 1The abbreviations used are: GPx, glutathione peroxidase; GSH, reduced glutathione; cGPx, cellular glutathione peroxidase; PHGPx, phospholipid hydroperoxide glutathione peroxidase; eGPx, extracellular glutathione peroxidase; GIGPx, gastrointestinal GPx; SeP, selenoprotein P; t-Bu-OOH, tertiary butyl hydroperoxide; PC-OOH, phosphatidylcholine hydroperoxide; HPLC, high performance liquid chromatography; TRX, thioredoxin. containing selenocysteine at the catalytic center play an important role in the detoxification of various hydroperoxides (1Ursini F. Maiorino M. Brigelius-Flohe R. Aumann K.D. Roveri A. Schomburg D. Flohe L. Methods Enzymol. 1995; 252: 38-53Google Scholar, 2Brigelius-Flohe R. Free Radical Biol. Med. 1999; 27: 951-965Google Scholar). Four types of GPx (cellular GPx (cGPx), gastrointestinal GPx (GIGPx), extracellular GPx (eGPx), and phospholipid hydroperoxide GPx (PHGPx)) have been identified, and each enzyme is antigenetically, structurally, and enzymatically different. cGPx is a ubiquitously distributed enzyme that was the first of the GPx family to be discovered (3Flohe L. Gunzler W.A. Schock H.H. FEBS Lett. 1973; 32: 132-134Google Scholar). GIGPx is also an intracellular enzyme but is expressed only at the epithelium of the gastrointestinal tract as a first line of defense against ingested lipid hydroperoxides (4Chu F.F. Doroshow J.H. Esworthy R.S. J. Biol. Chem. 1993; 268: 2571-2576Google Scholar). eGPx is mainly expressed by the kidneys from where it is released into the blood circulation (5Takahashi K. Akasaka M. Yamamoto Y. Kobayashi C. Mizoguchi J. Koyama J. J. Biochem. (Tokyo). 1990; 108: 145-148Google Scholar, 6Avissar N. Eisenmann C. Breen J.G. Horowitz S. Miller R.K. Cohen H.J. Am. J. Physiol. 1994; 267: E68-E76Google Scholar). Of the GPx family, eGPx is the only enzyme distributed in the extracellular fluids such as plasma (7Takahashi K. Avissar N. Whitin J. Cohen H. Arch. Biochem. Biophys. 1987; 256: 677-686Google Scholar). PHGPx can reduce a variety of hydroperoxides including hydroperoxides integrated in membranes, hydroperoxy lipids in low density lipoprotein (8Sattler W. Maiorino M. Stocker R. Arch. Biochem. Biophys. 1994; 309: 214-221Google Scholar), or thymine (9Bao Y. Jemth P. Mannervik B. Williamson G. FEBS Lett. 1997; 410: 210-212Google Scholar). Although cGPx, GIGPx, and eGPx are homotetramers, PHGPx is a monomer with a molecular size smaller than the subunits of the other GPxs (10Ursini F. Maiorino M. Valente M. Ferri L. Gregolin C. Biochim. Biophys. Acta. 1982; 710: 197-211Google Scholar). PHGPx has recently been shown to act as a structural component of the mitochondrial capsule and thus is needed in the formation of the flagellum (11Ursini F. Heim S. Kiess M. Maiorino M. Roveri A. Wissing J. Flohe L. Science. 1999; 285: 1393-1396Google Scholar), whereas in the nuclei, specific sperm nuclei GPx with properties similar to PHGPx and its processed products are involved in chromatin condensation and protection of the germline against oxidative damage (12Pfeifer H. Conrad M. Roethlein D. Kyriakopoulos A. Brielmeier M. Bornkamm G.W. Behne D. FASEB J. 2001; 15: 1236-1238Google Scholar). We recently reported that selenoprotein P (SeP), presumed to contain ten selenocysteine residues, reduces phospholipid hydroperoxide in a GPx-like manner (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar). SeP is an extracellular protein and is the major selenoprotein in rat and human plasma (14Burk R.F. Proc. Soc. Exp. Biol. Med. 1973; 143: 719-722Google Scholar, 15Burk R.F. Hill K.E. J. Nutr. 1994; 124: 1891-1897Google Scholar). The physiological function of SeP is currently unknown, but several reports have led to the hypothesis that it functions as an antioxidant (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar, 16Arteel G.E. Mostert V. Oubrahim H. Briviba K. Abel J. Sies H. Biol. Chem. 1998; 379: 1201-1205Google Scholar). It is generally known that all members of the GPx family reduce hydrogen peroxide at the expense of glutathione (GSH) and that PHGPx and eGPx reduce hydroperoxides of more complex lipids such as phosphatidylcholine hydroperoxide (17Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Google Scholar, 18Yamamoto Y. Takahashi K. Arch. Biochem. Biophys. 1993; 305: 541-545Google Scholar). However, the literature shows that the specific activity of each GPx was determined using a different kind of peroxide substrate at a different pH, temperature, and GSH concentration. Furthermore, as the enzyme was obtained from different origins (human, rat, and pig), a successful comparison of the enzyme activities of GPx family would appear impossible. We consider that to understand the physiological significance of GPx diversity, it is important to compare the specific activities and kinetic parameters of enzymes purified from the same origin determined under the same conditions. In this study, we first purified three ubiquitous forms of GPx together with human SeP and determined the specific activities and kinetic parameters of these enzymes using three kinds of synthetic peroxide substrates. We next compared the thiol (especially thioredoxin) specificity and determined the kinetic parameters of SeP against thioredoxin. Finally, we considered the physiological significance of GPx diversity. Tertiary butyl hydroperoxide (t-Bu-OOH), cumene hydroperoxide, and hydrogen peroxide were obtained from Nacalai, Kyoto. 1-Palmitoyl-2-linoleoyl-3-phosphatidylcholine (PC), cholesteryl linoleate, GSH, and GSH reductase were from Sigma. Soybean lipoxygenase was from Biozyme Laboratories Ltd. (Blaenavon, United Kingdom). Recombinant human thioredoxin (TRX) was prepared as described previously (19Mitsui A. Hirakawa T. Yodoi J. Biochem. Biophys. Res. Commun. 1992; 186: 1220-1226Google Scholar) and kindly provided by Ajinomoto, Co. Inc. (Kawasaki, Japan). Human out-dated red blood cells and frozen plasma were kindly donated by the Hokkaido Red Cross Blood Center. Human placenta was kindly donated by the Hokkaido Tonan Hospital. Distilled water was further purified by water purifier (Organo Puric model-S) for high-performance liquid chromatography (HPLC). Acetonitrile (HPLC grade) was purchased from Wako Pure Chemical Co. (Osaka, Japan). Methanol and 2-propanol were distilled before use. PC-OOH was prepared from PC by oxidation with soybean lipoxygenase as described previously (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar). Other chemicals were of the highest quality commercially available. cGPx was purified from human red blood cells as described previously (20Awasthi Y.C. Beutler E. Srivastava S.K. J. Biol. Chem. 1975; 250: 5144-5149Google Scholar) with a slight modification. eGPx and SeP were purified from human plasma as described previously (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar, 18Yamamoto Y. Takahashi K. Arch. Biochem. Biophys. 1993; 305: 541-545Google Scholar). PHGPx was purified from human placental cytosol as described previously (21Roveri A. Maiorino M. Nisii C. Ursini F. Biochim. Biophys. Acta. 1994; 1208: 211-221Google Scholar) with a slight modification. TRX reductase was purified from human placenta as described previously (22Oblong J.E. Gasdaska P.Y. Sherrill K. Powis G. Biochemistry. 1993; 32: 7271-7277Google Scholar) with some modifications (23Yarimizu J. Nakamura H. Yodoi J. Takahashi K. Antioxid. Redox Signal. 2000; 2: 643-651Google Scholar). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) in slab gels (12.5% gel) under reducing conditions. After electrophoresis, proteins in the gel were stained with 0.1% Coomassie Brilliant Blue G-250 in 10% acetic acid and 30% methanol for 30 min, and then the gel was destained in 10% acetic acid and 30% methanol. The protein was dialyzed against distilled water and hydrolyzed in 6 m HCl for 24, 48, or 72 h or in 3 m mercaptoethanesulfonic acid for 24 h. After the removal of the solvent in vacuo, quantitative amino acid analysis was performed on a PICO-TAG system (Water, Millipore) (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar). GPx activities were examined by following the oxidation of NADPH in the presence of GSH reductase, which catalyzes the reduction of oxidized GSH formed by GPx as described previously (25Maiorino M. Gregolin C. Ursini F. Methods Enzymol. 1990; 186: 448-457Google Scholar) with a slight modification. Both samples and reference cuvettes contained 0.1 m Tris-HCl, pH 7.4, 0.2 mm NADPH, 0.5 mm EDTA, 2 mm GSH, and 1 unit of GSH reductase in a total volume of 1 ml. An aliquot of each enzyme was added to the sample cuvette only. The reaction mixture was preincubated at 37 °C for 2 min, after which the reaction was started by the addition of peroxide to both cuvettes. In the case of phospholipid hydroperoxide, Triton X-100 and deoxycholate were added to the reaction mixture at an appropriate concentration. The oxidation of NADPH was followed at 340 nm at 37 °C, and activity was expressed as micromoles of NADPH oxidized per minute. TRX peroxidase activity was determined analogously by replacing GSH with TRX (1–100 μm) and GSH reductase with TRX reductase (5 nm). To confirm the reduction of PC-OOH and examine the thiol specificity, a HPLC assay was also conducted (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar). After incubating the reaction mixture, a 9-fold volume of ice-cold 2-propanol was added to the reaction mixture. An aliquot of the reaction mixture was injected directly into the HPLC system. The HPLC conditions were basically those of Bao et al. (26Bao Y. Chambers S.J. Williamson G. Anal. Biochem. 1995; 224: 395-399Google Scholar) with a slight modification (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar). To examine the thiol specificity, the reduction of H2O2 by cGPx and eGPx was determined as described previously (13Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Google Scholar, 21Roveri A. Maiorino M. Nisii C. Ursini F. Biochim. Biophys. Acta. 1994; 1208: 211-221Google Scholar). Kinetic analysis was carried by following the of substrate in the with GPx as described previously (17Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Google Scholar). The substrate and the reaction at each from 1 to 3 thus obtained were then to the a as in the case of cGPx, eGPx, and The 1 and were from the as described previously (17Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Google Scholar, M. Aumann K.D. Brigelius-Flohe R. D. J. J. Roveri A. Ursini F. Flohe L. Biol. Chem. 1995; Scholar). The first is the oxidation of by hydroperoxide to a acid and The acid is then reduced by two molecules of GSH to a as shown in 1 and 1 2 forms of GPx eGPx, and and SeP were purified from human red blood human or human as described under shown in each of the four enzyme a stained on dodecyl sulfate-polyacrylamide gel electrophoresis with a to (cGPx), (eGPx), and under reducing conditions The of each enzyme was determined by quantitative amino acid activity was determined using three kinds of peroxide t-Bu-OOH, and PC-OOH, under the same conditions mm GSH, pH 7.4, at 37 cGPx and eGPx a with the and whereas PHGPx or SeP only slight or The enzyme assay for PC-OOH reduction a to PC-OOH in the and the activity of each enzyme was under the different conditions. PHGPx activity against the PC-OOH substrate was the highest among four enzymes at 0.1% Triton X-100 and mm whereas eGPx the same activity at mm SeP reduced PC-OOH at Triton X-100 and mm The specific activity of SeP to PC-OOH was than that of PHGPx or in the case of eGPx the presence of Triton X-100 the enzyme activity for PC-OOH but for the other cGPx with PC-OOH under of the GPx family and SeP to peroxide conditions as mm 0.1% Triton X-100 and mm and Triton X-100 and mm conditions as mm 0.1% Triton X-100 and mm and Triton X-100 and mm conditions as mm 0.1% Triton X-100 and mm and Triton X-100 and mm activities were as and for the was determined by quantitative amino acid is by conditions as mm 0.1% Triton X-100 and mm and Triton X-100 and mm in a The activities were as and for the was determined by quantitative amino acid is by The kinetic for the reaction by SeP was following the of substrate at the various GSH these the of hydroperoxide were against the at and different GSH led to The other three enzymes also similar kinetic as is for a L. G. Gunzler W.A. E. Physiol. Chem. Scholar) as described in the of 1 for the oxidative of the enzyme and of for the of the two of the of the oxidized enzymes by The of 1 and for each enzyme determined under the same conditions are shown in 1 for and H2O2 were obtained from both cGPx and 1 for PC-OOH obtained from eGPx and SeP were and than that from PHGPx, that eGPx and SeP are to phospholipid hydroperoxide than is cGPx the highest followed by eGPx, PHGPx, and SeP, that cGPx is more to GSH than is other for and glutathione 1 1 for the oxidative reaction for the of the two can be from the and of a described under for each GPx were from all of the of the peroxide determined. in a The 1 for the oxidative reaction for the of the two can be from the and of a described under for each GPx were from all of the of the peroxide the among the four enzymes, we next the thiol specificity has been reported cGPx used only glutathione as an electron The other three enzymes or as a reducing at of 2 specificities of the GPx family and enzyme activity was using or at 2 activities of cGPx and eGPx were by and the PC-OOH reduction activities of PHGPx and SeP were by HPLC activity was to a of that shown using in a enzyme activity was using or at 2 activities of cGPx and eGPx were by and the PC-OOH reduction activities of PHGPx and SeP were by HPLC activity was to a of that shown using TRX is reported to be a thiol donor for eGPx M. J. W. B. A. J. Biol. Chem. 1994; Scholar). To the of TRX as a thiol donor for SeP, the reduction of PC-OOH by SeP was examined TRX reductase or SeP TRX reductase containing SeP or TRX some The activity was all three proteins were HPLC analysis of the reaction products the of PC-OOH to by SeP with TRX of PC-OOH and the formation of 3 These that SeP reduces the peroxide at the expense of We next examined the of GSH or TRX on the activity of the four enzymes cGPx the same to GSH and the other eGPx reduced peroxide at of GSH than those of In the case of PHGPx and SeP, TRX was more than was SeP was TRX was more than of TRX on the reduction activities of the GPx family and The of GSH and TRX (1–100 μm) on the reduction activity of each enzyme was activities to were for cGPx and eGPx whereas the reduction activities to PC-OOH were for PHGPx and SeP The kinetic for the and by SeP were compared as shown in 3 In the L. G. Gunzler W.A. E. Physiol. Chem. Scholar, K. Chem. Scholar) shown is as the of the is as the 1 for the oxidative and is as the for the of the two of the of the reduced enzyme by The PC-OOH reduction of SeP using GSH a as described the GSH against the for of PC-OOH a line the the 2 and m were obtained The of SeP with PC-OOH and TRX were in SeP also a 2 However, the TRX against the a in that the the at 2 with m The 1 and were determined as in the case of The 1 obtained by TRX was the same as that obtained by GSH, but the for TRX was than that for GSH of the kinetic for SeP with GSH and TRX as the thiol or 1 obtained for GSH and differ The or for GSH the of the of SeP by 2 whereas those for TRX the by the the of the two are to be in a The or 1 obtained for GSH and differ The or for GSH the of the of SeP by 2 whereas those for TRX the by the the of the two are to be To this was the first comparative study on the peroxide substrate specificity of the three ubiquitous GPx types cGPx, PHGPx, and eGPx and of SeP purified from human we a protein from an system such the of selenocysteine and R.F. Hill K.E. Nutr. 1993; Scholar). we have these four enzymes from human shown in all of the enzymes were shown in it is that cGPx and H2O2 but reduce To the activity of eGPx, PHGPx, and SeP for PC-OOH, the conditions were and were found to differ for each These be to the of the lipid that these three enzymes exhibit different to such as lipid on and each PHGPx and eGPx the same specific activity to in the case of eGPx the presence of Triton X-100 the enzyme activity for PC-OOH but for R.S. F.F. P. Doroshow J.H. Arch. Biochem. Biophys. 1993; Scholar). Triton X-100 eGPx activity the with selenocysteine at the but the of the lipid The in the conditions for the PC-OOH reducing activity of PHGPx, eGPx, and SeP that these three enzymes be to different kinds of phospholipid hydroperoxides such as oxidized and oxidized lipids on the plasma of the cells. 1 for H2O2 were obtained from both cGPx and eGPx as with other forms of GPx (17Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Google Scholar, L. G. Gunzler W.A. E. Physiol. Chem. Scholar). 1 for PC-OOH was also obtained from PHGPx as reported previously for PHGPx (17Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Google Scholar). 1 and were in SeP compared with PHGPx, that SeP is to PC-OOH than of the GPx family an of and (1Ursini F. Maiorino M. Brigelius-Flohe R. Aumann K.D. Roveri A. Schomburg D. Flohe L. Methods Enzymol. 1995; 252: 38-53Google Scholar). SeP of the the of this amino acid be the SeP shows a specificity to peroxide substrates. cGPx the highest followed by eGPx, PHGPx, and that cGPx is more to GSH than are the other cGPx used only GSH as an electron The other three enzymes or as a reducing cGPx is a glutathione peroxidase, the other three enzymes are glutathione peroxidases but are thiol cGPx a GSH of and four R. A. J. Biochem. Scholar). eGPx three of the of the GSH PHGPx and SeP all of the GSH of these amino acid be the the other three enzymes a specificity to plasma GSH are to a reduction for eGPx and SeP, it has been that a substrate GSH It has been reported that eGPx is to TRX of GSH as a thiol substrate M. J. W. B. A. J. Biol. Chem. 1994; Scholar). TRX protein and the of GPx protein the same tertiary that the TRX and GSH be we to the other GPxs including SeP also reduce hydroperoxides at the expense of cGPx both GSH and TRX at the same on thiol specificity have that GSH is the only electron donor to we are to this the of eGPx, the for GSH were in to the specificity for TRX is more as an electron donor to SeP than comparison of kinetic and the obtained from kinetic of SeP using GSH and TRX as a reducing the following as 1 for and are the the reducing the enzyme reaction with the presence of that the formation of a complex of oxidized SeP and reduced TRX and that the for TRX is and at Finally, the high and together with the low m that TRX is a specific thiol substrate of the similar kinetic was reported in the case of TRX peroxidase H. B. Aumann K.D. C. K. Brigelius-Flohe R. Flohe L. J. Biol. Chem. 2001; Scholar). enzyme was previously considered to be a GPx and as TRX peroxidase by kinetic SeP be as an extracellular TRX TRX together with TRX reductase, which is also a to reduce the of proteins using NADPH L. A. Methods Enzymol. 1999; Scholar). TRX is in extracellular fluids N. Y. H. A. M. Yodoi J. T. Proc. S. A. 1990; Scholar), and of TRX at the of human cells is reported A. D. Biochem. Biophys. Res. Commun. 1997; Scholar). Furthermore, TRX reductase is also released into the plasma A. B. A. Res. 2000; Scholar). These that TRX and TRX reductase with SeP in the removal of phospholipid hydroperoxide in the extracellular fluids or on the with it was that the of SeP was with the of lipid R.F. Hill K.E. T. 1995; Scholar). with in described these in that SeP to protect the plasma from oxidative damage in the presence of Although the GSH in plasma is TRX a of for SeP The of SeP to cells R.F. Hill K.E. F.F. Biol. 1997; 108: Scholar) to a high of SeP and TRX on these and in the protection of cells from oxidative SeP and eGPx are in the extracellular and cGPx and PHGPx are found in the These enzymes exhibit different substrate and thiol specificities and collaborate to protect the biological molecules from oxidative stress inside and outside the However, further are to the of the GPx family and SeP against various hydroperoxides oxidized and oxidized lipids on the We the Hokkaido Red Cross Blood and Tonan for human blood and
Takebe et al. (Tue,) studied this question.