- Email: [email protected]
[60] Glutathione peroxidase
490
GLUTATHIONE
[60]
Properties The purified enzyme from both sources is homogeneous in several electrophoretic and chromatographic systems. Pure glutathione reductase can be stored in the refrigerator for very long periods of time (without apparent change of its properties) provided that the enzyme is concentrated to 2 mg/ml (or more) and dialyzed against neutral Tris buffer. A crystal of thymol is added as an antibacterial agent. In the pure state glutathione reductase should preferably not be frozen, since freezing often leads to denaturation and loss of activity. Some of the molecular and kinetic properties are summarized in Table III. Glutathione reductase shows a broad pH optimum centered at about pH 7.0. Acknowledgment The work in our laboratorywas supportedby grants (to B.M.)fromthe SwedishNatural Science Research Council.
[60] G l u t a t h i o n e P e r o x i d a s e
By
BENGT MANNERVIK
Reactions Catalyzed Glutathione peroxidases 1-3 catalyze the reduction of hydroperoxides (ROOH) by glutathione (GSH): R O O H + 2 G S H - - * R O H + HzO + GSSG
R may be an aliphatic or aromatic organic group or, simply, hydrogen. The products are H20, an alcohol (ROH) (or a second HeO when H20~ serves as substrate) and glutathione disulfide (GSSG). Regeneration of GSH from GSSG in the cell is effected by the enzyme glutathione reductase. Assays of glutathione peroxidase activity are based on measurement of ROOH or GSH consumption. 4,5 Alternatively, GSSG production is I G. C. Mills, J. Biol. Chem. 229, 189 (1957). 2 A. Wendel, in " E n z y m a t i c Basis of Detoxication" (W. B. Jakoby, ed.), Vol. 1, p. 333. A c a d e m i c Press, N e w York, 1980. 3 L. Floh6, in " F r e e Radicals in Biology" (W. A. Pryor, ed.), Vol. 5, p. 223. A c a d e m i c Press, N e w York, 1982. 4 L. Floh~ and W. A. Giinzler, this series, Vol. 105, p. 114. 5 A. Wendel, this series, Vol. 77, p. 325.
METHODS IN ENZYMOLOGY,VOL. 113
Copyright © 1985by AcademicPress. Inc. All rights of reproduction in any form reserved.
[60]
GLUTATHIONEPEROXIDASE
491
monitored by coupling to the reaction catalyzed by glutathione reductase: GSSG + NADPH + H + ~ 2 GSH + NADP +
Oxidation of N A D P H is recorded spectrophotometrically or fluorometrically. A critical survey of assay methods has recently been publfshed. 4
Enzyme Proteins Two major types of glutathione peroxidase have been found. One type is distinguished by containing selenium in the form of covalently bound selenocysteine in its active site. 6,7 This selenium-dependent enzyme is active with both organic hydroperoxides and H202. The enzyme from bovine erythrocytes is a tetrameric protein of Mr - 80,000. 8 Its 3D structure has been determined by X-ray diffraction analysis, 9 and its amino acid sequence has been elucidated by conventional techniques of protein chemistry. Jo The second type of glutathione peroxidase consists of proteins that do not depend on selenium for catalysis and have negligible activity with H202.1~-~3 This class is constituted by glutathione transferases,14 first described as proteins catalyzing the conjugation of GSH with electrophilic compounds such as aryl halides.15,16 These enzymes are dimeric proteins that often occur in multiple forms in the same organ, and some of the isoenzymes have distinctly higher glutathione peroxidase activities than others. ~7-2° None of the transferases is yet characterized to the same 6 j. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973). 7 L. Floh6, W. A. Giinzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). s L. Floh6, B. Eisele, and A. Wendel, Hoppe-Seyler's Z. Physiol. Chem. 352, 151 (1971). 9 0 . Epp, R. Ladenstein, and A. Wendel, Eur. J. Biochem. 133, 51 (1983). to L. Floh6, G. J. Steffens, W. A. Gtinzler, S.-M. A. Kim, F. 0tting, A. Grossman, and A. Wendel, Life Chem. Rep., Suppl. 2, Oxidative Damage Relat. Enzymes, 358 (1984). " R. A. Lawrence and R. F. Burk, Biochem. Biophys. Res. Commun. 71, 952 (1976). 12 j. R. Prohaska and H. E. Ganther, J, Neurochem. 27, 1379 (1976). 13 F. E. Hunter, Jr., F. Posadas del Rio, and A. A. Painter, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1529 (1976). 14 j. R. Prohaska and H. E. Ganther, Biochem. Biophys. Res. Commun. 76, 437 (1977). 15 B. Combes and G. S. Stakelum, J. Clin. Invest. 40, 981 (1961). ~6j. Booth, E. Boyland, and P. Sims, Biochem. J. 79, 516 (1961). i7 j. R. Prohaska, Biochim. Biophys. Acta 611, 87 (1980). IS B. Mannervik, C. Guthenberg, and K. ,~kerfeldt, in "Microsomes, Drug Oxidations and Chemical Carcinogenesis" (M. J. Coon, A. H. Conney, R. W. Estabrook, H. V. Gelboin, J. R. Gillette, and P. J. O'Brien, eds.), Vol. 2, p. 663. Academic Press, New York, 1980. 19 B. Mannervik and H. Jensson, J. Biol. Chem. 257, 9909 (1982). 20 B. Mannervik, C. Guthenberg, H. Jensson, M. Warholm, and P. A.lin, in "Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects" (A. Lars-
492
GLUTATH1ONE
[60]
extent with respect to molecular structure as the selenoprotein. It is currently not believed that the peroxidase activity of the glutathione transferases represents their major biological function, even though its significance is well d o c u m e n t e d . It is the p u r p o s e of the present chapter to delineate similarities and differences in the properties of the two types of protein exhibiting glutathione p e r o x i d a s e activity. Detailed descriptions of the preparation and characteristics of the selenium-dependent glutathione peroxidase 5,21 and various glutathione transferases glutathione peroxidase activity have been published in this series. 22-26 Occurrence
Glutathione peroxidase activity has been d e m o n s t r a t e d in all m a m m a lian tissues e x a m i n e d J 7 In m o s t animals the selenoprotein is responsible for a substantial fraction of the activity, but in the guinea pig (liver) the selenoprotein is absent or present in a very small amount. 28 The nonselenium-dependent activity is, in all case investigated in depth, ascribable to the glutathione transferases. The ratio between the selenium and non-selenium-dependent activities m a y vary not only between animal species but also f r o m tissue to tissue in the same species. In the rat, the contribution of the glutathione transferases to the total peroxidase activity is especially high in testis. 28 In selenium deficiency the relative importance of the peroxidase activity of the glutathione transferase increases. Subcellular Localization
The distribution of glutathione peroxidase has been most extensively studied in the rat. In h e p a t o c y t e s selenium-dependent glutathione peroxidase is localized primarily in the cytosol and in the matrix of mitochondria. 29 Glutathione transferases are present in the same c o m p a r t m e n t s , son, S. Orrenius, A. Holmgren, and B. Mannervik, eds.), p. 75. Raven Press, New York, 1983. 2~A. L. Tappel, this series, Vol. 52, p. 506. 2z W. H. Habig and W. B. Jakoby, this series, Vol. 77, p. 218. 23 B. Mannervik and C. Guthenberg, this series, Vol. 77, p. 231; P. C. Simons and D. L. Vander Jagt, ibid. p. 235. 24M. Warholm, C. Guthenberg, C. von Bahr, and B. Mannervik, this volume [62]. 2~H. Jensson, P. ,~,lin, and B. Mannervik, this volume [63]. 26C. Guthenberg, P. ~.lin, and B. Mannervik, this volume [64]. 27 L. Floh6, W. A. Giinzler, and G. Loschen, in "Trace Metals in Health and Disease" (N. Kharasch, ed.), p. 263. Raven Press, New York, 1979. 2s R. A. Lawrence and R. F. Burk, J. Nutr. 108, 211 (1978).
[60]
GLUTATHIONEPEROXIDASE
493
but also in membrane-containing subcellular fractions. 3°-32 The six major glutathione transferase isoenzymes in rat cytosol all exhibit peroxidase activity.19 Transferase 2-2 (earlier named transferase AA o r B2323) has the highest specific activity, 17.19but transferase 1-2, in view of its high relative concentration, carries more peroxidase activity than any of the other isoenzymes. Judging from specific activities and relative abundance, transferase 1-1,3-4, and 3-3 also contribute substantially to the total activity (measured with cumene hydroperoxide); transferase 4-4 and the isoenzymes with lower isoelectric points j9 have significant specific activities as well. The specific peroxidase activities of the three groups of human transferases characterized differ more than those of the rat isoenzymes. The glutathione transferases with high isoelectric points have the highest peroxidase activity. 2°,33 The microsomal glutathione transferase in rat liver, which is distinct from the cytosolic isoenzymes, also has glutathione peroxidase activity with cumene hydroperoxide. 34,35This peroxidase activity, like the transferase activity with 1-chloro-2,4-dinitrobenzene, is activatable severalfold by pretreatment of the microsomal enzyme with N-ethylmaleimide. 34,35 The microsomes also contain the major "cytosolic" isoenzymes and quantitative determinations of the relative amounts of the different transferases in this subcellular fraction have been m a d e . 36 Judging from the specific activities with cumene hydroperoxide, it can be calculated that the peroxidase activity of the "cytosolic" isoenzymes in the microsomes are quantitatively at least as important as the "microsomal" isoenzyme, even after activation with N-ethylmaleimide. The "microsomal" glutathione transferase is also present in the outer mitochondrial membrane in which it constitutes 5% of the total protein content (the value for microsomes is 3%). 37 Less is known about the 29 L. Floh6 and W. Schlegel, Hoppe-Seyler's Z. Physiol. Chem. 352, 1401 (1971). 30 p. Kraus, in "Conjugation Reactions in Drug Biotransformation" (A. Aitio, ed.), p. 503. Elsevier/North-Holland, Amsterdam, 1978. 3i A. Wahll~nder, S. Soboll, and H. Sies, FEBS Lett. 97, 138 (1979). 32 T. Friedberg, P. Bentley, P. Stasiecki, H. R. Glatt, D. Raphael, and F. Oesch, J. Biol. Chem. 254, 12028 (1979). 323 W. B. Jakoby, B. Ketterer, and B. Mannervik, Biochem. Pharmacol. 33, 2539 (1984). 33 M. Warholm, C. Guthenberg, and B. Mannervik, Biochemistry 22, 3610 (1983). 34 C. C. Reddy, C.-P. D. Tu, J. R. Burgess, C.-Y. Ho, R. W. Scholz, and E. J. Massaro, Biochem, Biophys. Res. Commun. 101, 970 (1981). 35 R. Morgenstern and J. W. DePierre, Eur. J. Biochem. 134, 591 (1983). 36 R. Morgenstern, C. Guthenberg, B. Mannervik, and J. W. DePierre, FEBS Lett. 160, 264 (1983). 37 R. Morgenstern, Ph.D. dissertation, Univ. of Stockholm, 1983.
494
GLUTATHIONE
[60]
nature and amounts of the other transferases in the different subcellular fractions. Importance
Numerous chemical processes in aerobic cells lead to the production of peroxides by activated forms of oxygen. 38-4°The peroxides may cause oxidative damage in biological tissues as well as decompose to generate free radicals and other reactive chemical species. The simplest hydroperoxide, H202, can be detoxified by the selenium-dependent glutathione peroxidase. Catalase, which also decomposes H202, is primarily localized in peroxisomes of the hepatocytes, whereas the selenoprotein is found in the cytosol and the mitochondrial matrix. Thus, these two enzymes appear to have complementary intracellular localizations as well as complementary catalytic activities. 2 Organic hydroperoxides derived from polyunsaturated fatty acids such as linoleic and linolenic acid or certain prostaglandins can occur in vivo. Cholesterol 7/3-hydroperoxide as well as derivatives of some steroid hormones and vitamin K are also relevant in this context. These hydroperoxides, like thymine hydroperoxide and "peroxidized DNA," have all been reported to be reduced by glutathione under the influence of glutathione peroxidase (see refs. 3 and 39 for original references). It appears as if most organic hydroperoxides are substrates both for the seleniumdependent and the non-selenium-dependent enzymes, even though not all compounds have been tested with both types of enzyme. However, it should be noted that among the glutathione transferases the relative specific peroxidase activities between different isoenzymes may depend on the nature of the hydroperoxide. For example, linoleic acid hydroperoxide has been reported as a better substrate for a fraction of partially purified cytosolic kidney transferases than for corresponding liver transferases, in spite of the fact that cumene and t-butyl hydroperoxides are better substrates for the liver enzymes. 41 The explanation is that the sets of isoenzymes are different in the two tissues. A more complex chemical process of biological interest is lipid peroxidation, s9 This process has been studied in microsomal subcellular fractions and leads to degradation of the microsomal membranes. In this 38 B. Chance, H. Sies, and A. Boveris, Physiol. Rev. 59, 527 (1979). 39 H. Sies, A. Wendel, and W. Bors, in "Metabolic Basis of Detoxicalion" (W. B. Jakoby, J. R. Bend, and J. Caldwell, eds.L p. 307. Academic Press, New York, 1982. 40 K. Yagi, ed., "Lipid Peroxides in Biology and Medicine." Academic Press, New York, 1983. 41 C.-P. D. Tu, M. J. Weiss, N. Li, and C. C. Reddy, J. Biol. Chem. 258, 4659 (1983).
[61]
GLUTATHIONETRANSFERASES
495
experimental system, lipid peroxidation is inhibited by certain glutathione transferases in the presence of glutathione. 42-44 Very significant differences in inhibitory activity of the isoenzymes are apparent. Also other cellular membranes appear to be protected by the glutathione peroxidase activity. 3 That the glutathione peroxidase activity of the selenoprotein as well as of the glutathione transferases operates in vivo can be concluded from liver perfusion experiments. 45-~7 In normal rats glutathione disulfide is released into bile upon infusion of both H202 and t-butyl hydroperoxide. In selenium-deficient animals only t-butyl hydroperoxide is effective (owing to the lack of the H202-specific selenoprotein). 48 Thus, it appears evident that the glutathione peroxidase activity of both types of enzyme is important in cellular defense against a wide variety of hydroperoxides. Acknowledgments Work from the author's laboratory was supported by the Swedish Cancer Society, the Swedish Council for Planning and Coordination of Research, and the Swedish Natural Science Research Council. 4~ R. F. Burk, M. J. Trumble, and R. A. Lawrence, Biochim. Biophys. Acta 618, 35 (1980). 43 R. Morgenstern, J. W. DePierre, C. Lind, C. Guthenberg, B. Mannervik, and L. Ernster, Biochem. Biophys. Res. Commun. 99, 682 (1981). 44 K. H. Tan, D. J. Meyer, and B. Ketterer, Biochem. Soc. Trans. l l , 308 (1983). 4~ H. Sies, C. Gerstenecker, H. Menzel, and L. FIoh6, FEBS Lett. 27, 171 (1972). 46 N. Oshino and B. Chance, Biochem. J. 162, 509 (1977). 47 H. Sies, R. Brigelius, and T. P. M. Akerboom, in "Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects" (A. Larsson, S. Orrenius, A. Holmgren, and B. Mannervik, eds.), p. 51. Raven Press, New York, 1983. 48 R. F. Burk and R. A. Lawrence, in "'Functions of Glutathione in Liver and Kidney" (H. Sies and A. Wendel, eds.), p. 114. Springer-Verlag, Berlin and New York, 1978.
[61] G l u t a t h i o n e T r a n s f e r a s e s : A n O v e r v i e w B y W I L L I A M B . JAKOBY
The glutathione transferases (EC 2.5.1.18) are among the catalysts that participate in the process of detoxication,~ the means by which those compounds without nutritional value are eliminated, usually after metabolic processing. The glutathione transferases are normally present in W. B. Jakoby, ed., "Enzymatic Basis of Detoxication," Vols. 1 and 2. Academic Press, New York, 1980.
METHODS IN ENZYMOLOGY, VOL. 113
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
GLUTATHIONE
[60]
Properties The purified enzyme from both sources is homogeneous in several electrophoretic and chromatographic systems. Pure glutathione reductase can be stored in the refrigerator for very long periods of time (without apparent change of its properties) provided that the enzyme is concentrated to 2 mg/ml (or more) and dialyzed against neutral Tris buffer. A crystal of thymol is added as an antibacterial agent. In the pure state glutathione reductase should preferably not be frozen, since freezing often leads to denaturation and loss of activity. Some of the molecular and kinetic properties are summarized in Table III. Glutathione reductase shows a broad pH optimum centered at about pH 7.0. Acknowledgment The work in our laboratorywas supportedby grants (to B.M.)fromthe SwedishNatural Science Research Council.
[60] G l u t a t h i o n e P e r o x i d a s e
By
BENGT MANNERVIK
Reactions Catalyzed Glutathione peroxidases 1-3 catalyze the reduction of hydroperoxides (ROOH) by glutathione (GSH): R O O H + 2 G S H - - * R O H + HzO + GSSG
R may be an aliphatic or aromatic organic group or, simply, hydrogen. The products are H20, an alcohol (ROH) (or a second HeO when H20~ serves as substrate) and glutathione disulfide (GSSG). Regeneration of GSH from GSSG in the cell is effected by the enzyme glutathione reductase. Assays of glutathione peroxidase activity are based on measurement of ROOH or GSH consumption. 4,5 Alternatively, GSSG production is I G. C. Mills, J. Biol. Chem. 229, 189 (1957). 2 A. Wendel, in " E n z y m a t i c Basis of Detoxication" (W. B. Jakoby, ed.), Vol. 1, p. 333. A c a d e m i c Press, N e w York, 1980. 3 L. Floh6, in " F r e e Radicals in Biology" (W. A. Pryor, ed.), Vol. 5, p. 223. A c a d e m i c Press, N e w York, 1982. 4 L. Floh~ and W. A. Giinzler, this series, Vol. 105, p. 114. 5 A. Wendel, this series, Vol. 77, p. 325.
METHODS IN ENZYMOLOGY,VOL. 113
Copyright © 1985by AcademicPress. Inc. All rights of reproduction in any form reserved.
[60]
GLUTATHIONEPEROXIDASE
491
monitored by coupling to the reaction catalyzed by glutathione reductase: GSSG + NADPH + H + ~ 2 GSH + NADP +
Oxidation of N A D P H is recorded spectrophotometrically or fluorometrically. A critical survey of assay methods has recently been publfshed. 4
Enzyme Proteins Two major types of glutathione peroxidase have been found. One type is distinguished by containing selenium in the form of covalently bound selenocysteine in its active site. 6,7 This selenium-dependent enzyme is active with both organic hydroperoxides and H202. The enzyme from bovine erythrocytes is a tetrameric protein of Mr - 80,000. 8 Its 3D structure has been determined by X-ray diffraction analysis, 9 and its amino acid sequence has been elucidated by conventional techniques of protein chemistry. Jo The second type of glutathione peroxidase consists of proteins that do not depend on selenium for catalysis and have negligible activity with H202.1~-~3 This class is constituted by glutathione transferases,14 first described as proteins catalyzing the conjugation of GSH with electrophilic compounds such as aryl halides.15,16 These enzymes are dimeric proteins that often occur in multiple forms in the same organ, and some of the isoenzymes have distinctly higher glutathione peroxidase activities than others. ~7-2° None of the transferases is yet characterized to the same 6 j. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, Science 179, 588 (1973). 7 L. Floh6, W. A. Giinzler, and H. H. Schock, FEBS Lett. 32, 132 (1973). s L. Floh6, B. Eisele, and A. Wendel, Hoppe-Seyler's Z. Physiol. Chem. 352, 151 (1971). 9 0 . Epp, R. Ladenstein, and A. Wendel, Eur. J. Biochem. 133, 51 (1983). to L. Floh6, G. J. Steffens, W. A. Gtinzler, S.-M. A. Kim, F. 0tting, A. Grossman, and A. Wendel, Life Chem. Rep., Suppl. 2, Oxidative Damage Relat. Enzymes, 358 (1984). " R. A. Lawrence and R. F. Burk, Biochem. Biophys. Res. Commun. 71, 952 (1976). 12 j. R. Prohaska and H. E. Ganther, J, Neurochem. 27, 1379 (1976). 13 F. E. Hunter, Jr., F. Posadas del Rio, and A. A. Painter, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 1529 (1976). 14 j. R. Prohaska and H. E. Ganther, Biochem. Biophys. Res. Commun. 76, 437 (1977). 15 B. Combes and G. S. Stakelum, J. Clin. Invest. 40, 981 (1961). ~6j. Booth, E. Boyland, and P. Sims, Biochem. J. 79, 516 (1961). i7 j. R. Prohaska, Biochim. Biophys. Acta 611, 87 (1980). IS B. Mannervik, C. Guthenberg, and K. ,~kerfeldt, in "Microsomes, Drug Oxidations and Chemical Carcinogenesis" (M. J. Coon, A. H. Conney, R. W. Estabrook, H. V. Gelboin, J. R. Gillette, and P. J. O'Brien, eds.), Vol. 2, p. 663. Academic Press, New York, 1980. 19 B. Mannervik and H. Jensson, J. Biol. Chem. 257, 9909 (1982). 20 B. Mannervik, C. Guthenberg, H. Jensson, M. Warholm, and P. A.lin, in "Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects" (A. Lars-
492
GLUTATH1ONE
[60]
extent with respect to molecular structure as the selenoprotein. It is currently not believed that the peroxidase activity of the glutathione transferases represents their major biological function, even though its significance is well d o c u m e n t e d . It is the p u r p o s e of the present chapter to delineate similarities and differences in the properties of the two types of protein exhibiting glutathione p e r o x i d a s e activity. Detailed descriptions of the preparation and characteristics of the selenium-dependent glutathione peroxidase 5,21 and various glutathione transferases glutathione peroxidase activity have been published in this series. 22-26 Occurrence
Glutathione peroxidase activity has been d e m o n s t r a t e d in all m a m m a lian tissues e x a m i n e d J 7 In m o s t animals the selenoprotein is responsible for a substantial fraction of the activity, but in the guinea pig (liver) the selenoprotein is absent or present in a very small amount. 28 The nonselenium-dependent activity is, in all case investigated in depth, ascribable to the glutathione transferases. The ratio between the selenium and non-selenium-dependent activities m a y vary not only between animal species but also f r o m tissue to tissue in the same species. In the rat, the contribution of the glutathione transferases to the total peroxidase activity is especially high in testis. 28 In selenium deficiency the relative importance of the peroxidase activity of the glutathione transferase increases. Subcellular Localization
The distribution of glutathione peroxidase has been most extensively studied in the rat. In h e p a t o c y t e s selenium-dependent glutathione peroxidase is localized primarily in the cytosol and in the matrix of mitochondria. 29 Glutathione transferases are present in the same c o m p a r t m e n t s , son, S. Orrenius, A. Holmgren, and B. Mannervik, eds.), p. 75. Raven Press, New York, 1983. 2~A. L. Tappel, this series, Vol. 52, p. 506. 2z W. H. Habig and W. B. Jakoby, this series, Vol. 77, p. 218. 23 B. Mannervik and C. Guthenberg, this series, Vol. 77, p. 231; P. C. Simons and D. L. Vander Jagt, ibid. p. 235. 24M. Warholm, C. Guthenberg, C. von Bahr, and B. Mannervik, this volume [62]. 2~H. Jensson, P. ,~,lin, and B. Mannervik, this volume [63]. 26C. Guthenberg, P. ~.lin, and B. Mannervik, this volume [64]. 27 L. Floh6, W. A. Giinzler, and G. Loschen, in "Trace Metals in Health and Disease" (N. Kharasch, ed.), p. 263. Raven Press, New York, 1979. 2s R. A. Lawrence and R. F. Burk, J. Nutr. 108, 211 (1978).
[60]
GLUTATHIONEPEROXIDASE
493
but also in membrane-containing subcellular fractions. 3°-32 The six major glutathione transferase isoenzymes in rat cytosol all exhibit peroxidase activity.19 Transferase 2-2 (earlier named transferase AA o r B2323) has the highest specific activity, 17.19but transferase 1-2, in view of its high relative concentration, carries more peroxidase activity than any of the other isoenzymes. Judging from specific activities and relative abundance, transferase 1-1,3-4, and 3-3 also contribute substantially to the total activity (measured with cumene hydroperoxide); transferase 4-4 and the isoenzymes with lower isoelectric points j9 have significant specific activities as well. The specific peroxidase activities of the three groups of human transferases characterized differ more than those of the rat isoenzymes. The glutathione transferases with high isoelectric points have the highest peroxidase activity. 2°,33 The microsomal glutathione transferase in rat liver, which is distinct from the cytosolic isoenzymes, also has glutathione peroxidase activity with cumene hydroperoxide. 34,35This peroxidase activity, like the transferase activity with 1-chloro-2,4-dinitrobenzene, is activatable severalfold by pretreatment of the microsomal enzyme with N-ethylmaleimide. 34,35 The microsomes also contain the major "cytosolic" isoenzymes and quantitative determinations of the relative amounts of the different transferases in this subcellular fraction have been m a d e . 36 Judging from the specific activities with cumene hydroperoxide, it can be calculated that the peroxidase activity of the "cytosolic" isoenzymes in the microsomes are quantitatively at least as important as the "microsomal" isoenzyme, even after activation with N-ethylmaleimide. The "microsomal" glutathione transferase is also present in the outer mitochondrial membrane in which it constitutes 5% of the total protein content (the value for microsomes is 3%). 37 Less is known about the 29 L. Floh6 and W. Schlegel, Hoppe-Seyler's Z. Physiol. Chem. 352, 1401 (1971). 30 p. Kraus, in "Conjugation Reactions in Drug Biotransformation" (A. Aitio, ed.), p. 503. Elsevier/North-Holland, Amsterdam, 1978. 3i A. Wahll~nder, S. Soboll, and H. Sies, FEBS Lett. 97, 138 (1979). 32 T. Friedberg, P. Bentley, P. Stasiecki, H. R. Glatt, D. Raphael, and F. Oesch, J. Biol. Chem. 254, 12028 (1979). 323 W. B. Jakoby, B. Ketterer, and B. Mannervik, Biochem. Pharmacol. 33, 2539 (1984). 33 M. Warholm, C. Guthenberg, and B. Mannervik, Biochemistry 22, 3610 (1983). 34 C. C. Reddy, C.-P. D. Tu, J. R. Burgess, C.-Y. Ho, R. W. Scholz, and E. J. Massaro, Biochem, Biophys. Res. Commun. 101, 970 (1981). 35 R. Morgenstern and J. W. DePierre, Eur. J. Biochem. 134, 591 (1983). 36 R. Morgenstern, C. Guthenberg, B. Mannervik, and J. W. DePierre, FEBS Lett. 160, 264 (1983). 37 R. Morgenstern, Ph.D. dissertation, Univ. of Stockholm, 1983.
494
GLUTATHIONE
[60]
nature and amounts of the other transferases in the different subcellular fractions. Importance
Numerous chemical processes in aerobic cells lead to the production of peroxides by activated forms of oxygen. 38-4°The peroxides may cause oxidative damage in biological tissues as well as decompose to generate free radicals and other reactive chemical species. The simplest hydroperoxide, H202, can be detoxified by the selenium-dependent glutathione peroxidase. Catalase, which also decomposes H202, is primarily localized in peroxisomes of the hepatocytes, whereas the selenoprotein is found in the cytosol and the mitochondrial matrix. Thus, these two enzymes appear to have complementary intracellular localizations as well as complementary catalytic activities. 2 Organic hydroperoxides derived from polyunsaturated fatty acids such as linoleic and linolenic acid or certain prostaglandins can occur in vivo. Cholesterol 7/3-hydroperoxide as well as derivatives of some steroid hormones and vitamin K are also relevant in this context. These hydroperoxides, like thymine hydroperoxide and "peroxidized DNA," have all been reported to be reduced by glutathione under the influence of glutathione peroxidase (see refs. 3 and 39 for original references). It appears as if most organic hydroperoxides are substrates both for the seleniumdependent and the non-selenium-dependent enzymes, even though not all compounds have been tested with both types of enzyme. However, it should be noted that among the glutathione transferases the relative specific peroxidase activities between different isoenzymes may depend on the nature of the hydroperoxide. For example, linoleic acid hydroperoxide has been reported as a better substrate for a fraction of partially purified cytosolic kidney transferases than for corresponding liver transferases, in spite of the fact that cumene and t-butyl hydroperoxides are better substrates for the liver enzymes. 41 The explanation is that the sets of isoenzymes are different in the two tissues. A more complex chemical process of biological interest is lipid peroxidation, s9 This process has been studied in microsomal subcellular fractions and leads to degradation of the microsomal membranes. In this 38 B. Chance, H. Sies, and A. Boveris, Physiol. Rev. 59, 527 (1979). 39 H. Sies, A. Wendel, and W. Bors, in "Metabolic Basis of Detoxicalion" (W. B. Jakoby, J. R. Bend, and J. Caldwell, eds.L p. 307. Academic Press, New York, 1982. 40 K. Yagi, ed., "Lipid Peroxides in Biology and Medicine." Academic Press, New York, 1983. 41 C.-P. D. Tu, M. J. Weiss, N. Li, and C. C. Reddy, J. Biol. Chem. 258, 4659 (1983).
[61]
GLUTATHIONETRANSFERASES
495
experimental system, lipid peroxidation is inhibited by certain glutathione transferases in the presence of glutathione. 42-44 Very significant differences in inhibitory activity of the isoenzymes are apparent. Also other cellular membranes appear to be protected by the glutathione peroxidase activity. 3 That the glutathione peroxidase activity of the selenoprotein as well as of the glutathione transferases operates in vivo can be concluded from liver perfusion experiments. 45-~7 In normal rats glutathione disulfide is released into bile upon infusion of both H202 and t-butyl hydroperoxide. In selenium-deficient animals only t-butyl hydroperoxide is effective (owing to the lack of the H202-specific selenoprotein). 48 Thus, it appears evident that the glutathione peroxidase activity of both types of enzyme is important in cellular defense against a wide variety of hydroperoxides. Acknowledgments Work from the author's laboratory was supported by the Swedish Cancer Society, the Swedish Council for Planning and Coordination of Research, and the Swedish Natural Science Research Council. 4~ R. F. Burk, M. J. Trumble, and R. A. Lawrence, Biochim. Biophys. Acta 618, 35 (1980). 43 R. Morgenstern, J. W. DePierre, C. Lind, C. Guthenberg, B. Mannervik, and L. Ernster, Biochem. Biophys. Res. Commun. 99, 682 (1981). 44 K. H. Tan, D. J. Meyer, and B. Ketterer, Biochem. Soc. Trans. l l , 308 (1983). 4~ H. Sies, C. Gerstenecker, H. Menzel, and L. FIoh6, FEBS Lett. 27, 171 (1972). 46 N. Oshino and B. Chance, Biochem. J. 162, 509 (1977). 47 H. Sies, R. Brigelius, and T. P. M. Akerboom, in "Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects" (A. Larsson, S. Orrenius, A. Holmgren, and B. Mannervik, eds.), p. 51. Raven Press, New York, 1983. 48 R. F. Burk and R. A. Lawrence, in "'Functions of Glutathione in Liver and Kidney" (H. Sies and A. Wendel, eds.), p. 114. Springer-Verlag, Berlin and New York, 1978.
[61] G l u t a t h i o n e T r a n s f e r a s e s : A n O v e r v i e w B y W I L L I A M B . JAKOBY
The glutathione transferases (EC 2.5.1.18) are among the catalysts that participate in the process of detoxication,~ the means by which those compounds without nutritional value are eliminated, usually after metabolic processing. The glutathione transferases are normally present in W. B. Jakoby, ed., "Enzymatic Basis of Detoxication," Vols. 1 and 2. Academic Press, New York, 1980.
METHODS IN ENZYMOLOGY, VOL. 113
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