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Recycling of Vitamin C by Mammalian Thioredoxin Reductase
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[30] Recycling of Vitamin C by Mammalian Thioredoxin Reductase By JAMES M. MAY Introduction Mammalian thioredoxin reductase (TrxR) (EC 1.6.4.5) functions optimally to reduce disulfide bonds in conjunction with thioredoxin (Trx).l However, the enzyme alone has a broad substrate specificity, and can carry out NADPH-dependent reduction of a variety of redox-active molecules, including lipoic acid, vitamin K3, 5,51-dithiobis(2-nitrobenzoic acid) (DTNB), alloxan, and hydroperoxides. 1 This diversity may reflect the ability of the penultimate selenocysteine in the C-terminal portion of TrxR to function as a "super" thiol. Because thiols such as glutathione (GSH) 2 and lipoic acid 3 effectively reduce dehydroascorbic acid (DHA) to ascorbate, it is not surprising that TrxR can do the same. We found that purified rat liver TrxR reduces D H A to ascorbate, and that this reduction is enhanced by Trx. 4 Activity measured as either the disappearance of N A D P H or appearance of ascorbate is stoichiometric. The reaction of D H A with TrxR in the presence of Trx has an apparent Km of 0.7 mY/and a kcat of 71 min -1. Although the later is only about 2°7o of the rate at which the thioredoxin system reacts with disulfide substrates, 5 hydrophobic derivatives of D H A show up to 10-fold higher kca t v a l u e s . 4 The most likely mechanism for the reaction of TrxR with D H A is a two-step nucleophilic substitution as predicted for GSH reduction of DHA, especially because the ascorbate free radical (AFR) is not generated during the latter reaction. 6 More surprising from a mechanistic standpoint was the finding that the thioredoxin system (TrxR plus Trx) can also reduce the AFR to ascorbate. 7 Using purified rat liver TrxR and bacterial Trx, this activity has an apparent Km of 3 ttM for the AFR and a kcat of 135 rain -1. When measured either as loss of N A D P H or as a decrease in the steady state AFR concentration determined by electron paramagnetic resonance (EPR) spectroscopy, the reaction is inhibited by aurothioglueose (ATG) and stimulated by selenocystine. The latter has been shown to enhance the 1E. S. J. Arn6r and A. Holmgren, Eur. J. Biochem. 267, 6102 (2000). 2 B. S. Winkler, Biochim. Biophys. Aeta 1117, 287 (1992). 3 B. C. Scott, O. I. Aruoma, R J. Evans, C. O'Neill, A. Van der Vliet, C. E. Cross, H. Tritschler, and B. Halliwell, Free Radic. Res. 20, 119 (1994). 4 j. M. May, S. Mendiratta, K. E. Hill, and R. E Burk, J. Biol. Chem. 272, 22607 (1997). 5 M. Luthman and A. Holmgren,Biochemistry 21, 6628 (1982). 6 j. M. May, Z. C. Qu, R. R. Whitesell, and C. E. Cobb, Free Radic. BioL Med. 20, 543 (1996). 7 j. M. May, C. E. Cobb, S. Mendiratta, K. E. Hill, and R. E Burk, J. Biol. Chem. 273, 23039 (1998).
METHODS IN ENZYMOLOGY, VOL. 347
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activity of TrxR in response to disulfide substrates. 8 The molecular mechanism by which the TrxR system can accomplish a one-electron reduction of the AFR has not been established, although there are several possible mechanisms. 7 The question arises as to whether reduction of either DHA or the AFR by TrxR or the thioredoxin system has physiologic relevance. About 80% of the NADPHdependent reduction of DHA to ascorbate in overnight-dialyzed rat liver cytosolic extracts can be attributed to TrxR, on the basis of inhibition by 10/xM ATG. 4 At this concentration ATG is specific for TrxR over glutathione peroxidase. 9 The remaining activity may be due to a 3ot-hydroxysteroid dehydrogenase in rat liver, which has a Km for DHA of 4 mM and a kcatof 59 rain -1.1° Further, dialysates from livers of selenium-deficient rats have only about 12% of the ATG-inhibitable DHA reductase activity of liver dialysates from control animals. 4 NADPH-dependent and ATG-sensitive DHA reductase activity is also present in dialyzed hemolysates prepared from human erythrocytes. 11 In liver dialysates, the rate of TrxR-mediated DHA reduction is about 25-30% that of GSH-dependent reduction, whereas in erythrocytes, it is only about 10%. NADPH-dependent AFR reductase activity is also present in overnightdialyzed rat liver cytosolic extracts. 7 Several features suggest that this activity is due to TrxR. It is (1) markedly decreased in dialysates from selenium-deficient rats, (2) inhibited by concentrations of ATG known to be selective for TrxR, and (3) stimulated by selenocystine. However, AFR reduction due to TrxR in dialysates is less than 2% of that of NADH-dependent AFR reduction in microsomes. The TrxR system can reduce both the AFR and DHA to ascorbate, and this activity is readily apparent in dialyzed cell extracts. Because the techniques developed in the studies noted above might be useful in comparing rates of TrxR-dependent DHA and AFR reduction with rates of ascorbate recycling by other routes in different cell types or tissues, the following outlines the methodological approaches that we have found most useful. Methods Tissue Preparation and Removal o f Low Molecular Weight Cofactors
TrxR-dependent reduction of either DHA or the AFR can be measured using crude extracts from various tissues, so long as low molecular weight cofactors for DHA reduction such as NADPH and GSH are removed. This is probably 8 M. Bj6rnstedt,M. Hamberg,S. Kumar,J. Xue, and A. Holmgren,J. Biol. Chem. 270,11761 (1995). 9 K. E. Hill, G. W. McCollum,M. E. Boeglin, and R. F. Burk,Biochem. Biophys. Res. Commun. 234, 293 (1997). lOB. Del Bello, E. Maellaro, L. Sugherini, A. Santucci, M. Comporti,and A. F. Casini, Biochem. J. 304, 385 (1994). 11S. Mendiratta, Z.-C. Qu, andJ. M. May, FreeRadic. Biol. Med. 25, 221 (1998).
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most easily accomplished by dialyzing cytosolic extracts of tissue or cells. For tissue, homogenize 100-400 nag in 1.0-1.5 ml of an ice-cold buffer consisting of 50 mM Tris-HC1 and 1 mM EDTA, pH 7.4 (Tris-EDTA). The type and duration of homogenization depend on the tissue, but for liver homogenization using a conical polytetrafluoroethylene (Teflon) pestle in microcentrifuge tubes is sufficient. The homogenate is centrifuged or microcentrifuged at 4000-6000g at 3 ° for 15 rain, and the resulting supernatant is centrifuged at 100,000g for 1 hr at 3 °. The clear supernatant is then dialyzed ovemight against three changes of 250 ml of the Tris-EDTA buffer at 3 °. The dialyzed sample (typically 5-25 mg of protein per milliliter) is saved on ice for assay and for protein determination.
Assay of Dehydroascorbic Acid Reduction by Thioredoxin Reductase The activity can be measured either by monitoring the appearance of ascorbate or the disappearance of NADPH. There is a one-to-one concordance between the two assays, using purified rat liver TrxR. 4 Measurement ofRate of NADPHLoss. The assay is more rapid than measuring the rate of ascorbate generation, and it confirms directly that the reaction is linear under the selected conditions. However, it is not specific and will detect NADPH oxidase activity. This can be controlled for by including a blank with NADPH, but no DHA. The reaction is carried out in a UV spectrophotometric semimicrocuvette at room temperature. Add 0.35 ml of Tris-EDTA buffer, 0.05 ml of enzyme, and 0.05 ml of 4 mM NADPH in the same buffer, and start the assay by adding 0.05 mt of freshly prepared 10 mM DHA. After mixing, record the change in absorption at 340 nm for 1-3 rain in a recording spectrophotometer. From the linear slope of this line calculate the amount of NADPH consumed, using an extinction coefficient of 6.22 x 103 M -1 cm -1 for NADPH. The rate of loss of NADPH equals the rate of ascorbate generated, corrected for the background rate of NADPH oxidation (the blank reaction). Commercial DHA (Sigma-Aldrich, St. Louis, MO) is not pure, but can be used if dissolved in the Tris-EDTA buffer just before addition to the assay (it is not stable at physiologic pH). The amount of DHA added in this assay is not saturating for TrxR, and therefore a decrease in the added DHA concentration during multiple assays over time may result. It is preferable to use DHA prepared by bromine oxidation of 10 mM ascorbic acid in water for most studies. 12 The resulting DHA is stabilized by the low pH of the reaction medium, so that multiple samples can be analyzed without significant loss of DHA. It is necessary to confirm that the pH of the reaction mixture is not affected by the addition.
Measurement of Dehydroascorbic Acid Reduction by Appearance of Ascorbate. For this assay, the reaction volume is scaled down to 100/zl and the reaction is carried out in 1.5-ml microcentrifuge tubes. Otherwise, the agent concentrations 12p. W. Washko,Y. Wang, and M. Levine,J. Biol. Chem.268, 15531 (1993).
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and conditions are the same as those described above. After adding DHA, incubate the reaction mixture for 10 rain at room temperature. Stop the reaction by adding 10 volumes of ice-cold 80% (v/v) methanol containing 1 mM EDTA. After 5 rain on ice, microcentrifuge the sample at 6000g at 3 ° for 15 rain and take aliquots of the supernatant for assay of ascorbate by high-performance liquid chromatography (HPLC). We use HPLC with electrochemical detection of ascorbate, 4 but UV detection should also have adequate sensitivity and low background in dialyzed samples. A blank sample with no enzyme should be run in assays using commercial DHA to correct for ascorbate contamination, which can vary depending on the lot of DHA. In liver dialysates, increases in ascorbate are linear over about 20 rain, but this should be confirmed in preliminary studies.
Assay of Thioredoxin Reductase Reduction of Ascorbate Free Radical It is important in this assay to generate stable steady state concentrations of the AFR in the reaction mixture. Because TrxR reduces DHA to ascorbate, it is not possible to use the reverse dismutation reaction of a mixture of DHA and ascorbate to generate the AFR. Stable concentrations of the AFR are generated for several minutes by using ascorbate oxidase (0.05-0.4 U/ml, from Cucurb#a species; Sigma-Aldrich) to oxidize 1-10 mM ascorbate. This reaction generates only the AFR, which then will dismutate to ascorbate and DHA. Although it is possible to monitor the disappearance of the AFR signal by EPR spectroscopy, the traditional indirect assay of NAD(P)H disappearance is much easier, can be quantified, and does not require an EPR spectrometer. In a UV semimicrocuvette containing 0.3 ml of Tris-EDTA buffer, add 0.05 ml of 2 mM NADPH, 0.05 ml of enzyme, 0.02 ml of ascorbate oxidase (1 U/ml), and, finally, to start the assay, 0.05 ml of freshly prepared 10 mM ascorbic acid in Tris-EDTA buffer. Mix and monitor the decrease in absorbance at 340 nm at room temperature for up to 3 rain. A blank sample without enzyme is used to correct for NADPH oxidation in the buffer, and the rate of AFR reduction is calculated as described above, with the caveat that 1 mol of NADPH will reduce 2 tool of the AFR. Activity then can be normalized to protein or to the amount of purified enzyme present, if known. Because DHA will be generated in this reaction mixture by dismutation of two molecules of the AFR to one molecule each of DHA and ascorbate, it is necessary to establish that this reaction is a small component of the AFR reduction for each system, and to correct for it. This is done by incubating ascorbate and the oxidase under the conditions of the assay in a UV cuvette and monitoring the loss of ascorbate over the time of the assay by the decrease in absorbance at 265 nm. On the basis of the dismutation reaction of the AFR, the amount of DHA generated will be half the amount of ascorbate lost over the short course of the assay. By including a sample containing that concentration of DHA as a control, the maximal
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component of DHA-dependent NADPH loss can be determined. In our previous studies, at 1 mM ascorbate and ascorbate oxidase at 0.05 U/ml, this rate was found to be less than 5% of the AFR-dependent rate]
Establishing Specificity for Thioredoxin Reductase in Crude Tissue Dialysates Because there may be other NADPH-dependent DHA or AFR reductases in crude tissue extracts, it is necessary to determine the fraction of measured activity that is specific for TrxR. This can be done by including an incubation containing 10 # M ATG. Activity dependent on TrxR is then calculated by subtracting the rate in the presence of ATG from that found in its absence. Other maneuvers to show specificity for TrxR are to include either 5 # M commercial Trx or 50 # M selenocystine. Both will increase the observed rates of DHA or AFR reduction. The effect of mammalian Trx is to increase activity by up to 2-fold, whereas selenocystine enhances ATG-sensitive activity by 3- to 5-fold. Conclusion Although TrxR-dependent reduction of both DHA and the AFR can be documented in tissue extracts, whether this contributes to ascorbate recycling within cells is yet to be determined. Optimal DHA reduction requires GSH in erythrocytes 6 lens epithelium, 13 and in several types of cultured cells. The latter include HepG2 cells, H4IIE cells, and primary cultures of bovine artery endothelial cells. 13a However, neither HL60 cells 14 nor skin keratinocytes15 appear to require GSH for DHA reduction. DHA reduction by the TrxR system could well play an important role in such cells. There is also a question of whether ascorbate is regenerated from DHA or the AFR in cells. DHA reduction is certainly important when ascorbate is oxidized outside cells to DHA, which enters cells by facilitated diffusion on GLUT-type glucose transporters and is rapidly reduced to ascorbate. On the other hand, it may be more efficient to recycle from the AFR stage when ascorbate is oxidized within cells by reactive oxygen species generated in mitochondria or by cellular metabolism. The AFR is relatively long-lived, and its reduction will prevent possible loss at the DHA stage due to irreversible ring opening. Coassin et al. 16 found greater AFR reductase activity than GSH-dependent DHA reductase activity in various pig tissues. Although they did not measure NADPH-dependent DHA reductase activity, 13 E J. Giblin, B. S. Winlder, H. Sasaki, B. Chakrapani, and V. Leverenz, Invest. Ophthalmol. Vis. Sci. 34, 1298 (1993). I3a j. M. May, Z.-C. Qu, and X. Li, Biochem. Pharmacol. 63, 873 (2001). ~4V. H. Guaiquil, C. M. Farber, D. W. Golde, and J. C. Vera, J. Biol. Chem. 272, 9915 (1997). 15 I. Savini, S. Duflot, and L. Avigliano, Biochem. J. 345, 665 (2000). !6 M. Coassin, A. Tomasi, V. Vannini, and E Ursilai, Arch. Biochem. Biophys. 290, 458 (1991).
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there was little evidence of NADH-dependent DHA reduction at DHA concentrations under 100/zM. Further, the measured affinities for DHA of glutaredoxin (0.26 mM),17and of the thioredoxin system (0.7 raM) 4 are not favorable for reducing the low concentrations of DHA expected to be generated within cells. On the other hand, the apparent Km of TrxR for the AFR (3/zM) 7 is in the range expected for the AFR in oxidatively stressed cellsJ 8'19 Although NADH-dependent mitochondrial and microsomal AFR reduction shows a low apparent Km and much greater activity when normalized to protein than does TrxR-mediated activity] sequestration of the NADH-dependent enzymes because of their membrane-bound nature may limit access to the AFR. The cytoplasmic location of the thioredoxin system in most cells might allow it to play a significant role in reducing the AFR, but this is yet to be demonstrated. 17 W. W. Wells, D. E Xu, and M. R Washburn, Methods EnzymoL 252, 30 (1995). 18 S. Pietri, M. Culcasi, L. Stella, and E J. Cozzone, Eur. J. Biochem. 193, 845 (1990). 19 G. R. Buettner and B. A. Jurkiewicz, Free Radic. Biol. Med. 14, 49 (1993).
[31] Thioredoxin Cytokine Action By YUMIKONISH1NAKA,HAJIME NAKAMURA, and JUNJI YODOI Introduction Human thioredoxin (TRX) is secreted from various types of cells including activated normal B lymphocytes and virus-transformed cells, and the secreted TRX exhibits multiple cytokine-like activities.l'2 In 1985, human TRX was first purified from the culture superuatant of an adult T cell leukemia (ATL) cell line, ATL-2, transformed by human T cell leukemia virus I (HTLV-I), as ATL-derived factor (ADF). 3 It induces interleukin 2 (IL-2) receptor u-chain (IL-2Rot) expression on a human large granular lymphocyte (LGL) cell line YT and it was thus considered a novel cytokine with IL-2R~-inducing activity.4 In 1989, cDNA sequencing revealed that ADF is a human homolog of TRX. 5 Expression and production of 1 j. Yodoi and T. Uchiyama, lmmunol. Today 13, 405 (1992). 2 H. Nakamura, K. Nakamura, and J. Yodoi, Annu. Rev. Immunol. 15, 351 (1997). 3 K. Teshigawara, M. Maeda, K. Nishino, T. Nikaido, T. Uchiyama, M. Tsudo, Y. Wano, and J. Yodoi, J. Mol. Cell. ImmunoL 2, 17 (1985). 4 j. Yodoi, K. Teshigawara, T. Nikaido, K. Fukui, T. Noma, T. Honjo, M. Takigawa, M. Sasaki, N. Minato, M. Tsudo, T. Uchiyama, and M. Moeda, J. lmmunol. 134, 1623 (1985). 5 y. Tagaya, Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K. Arai, T. Yokota, H. Wakasugi, and J. Yodoi, EMBO J. 8, 757 (1989).
METHODSIN ENZYMOLOGY,VOL.347
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[30] Recycling of Vitamin C by Mammalian Thioredoxin Reductase By JAMES M. MAY Introduction Mammalian thioredoxin reductase (TrxR) (EC 1.6.4.5) functions optimally to reduce disulfide bonds in conjunction with thioredoxin (Trx).l However, the enzyme alone has a broad substrate specificity, and can carry out NADPH-dependent reduction of a variety of redox-active molecules, including lipoic acid, vitamin K3, 5,51-dithiobis(2-nitrobenzoic acid) (DTNB), alloxan, and hydroperoxides. 1 This diversity may reflect the ability of the penultimate selenocysteine in the C-terminal portion of TrxR to function as a "super" thiol. Because thiols such as glutathione (GSH) 2 and lipoic acid 3 effectively reduce dehydroascorbic acid (DHA) to ascorbate, it is not surprising that TrxR can do the same. We found that purified rat liver TrxR reduces D H A to ascorbate, and that this reduction is enhanced by Trx. 4 Activity measured as either the disappearance of N A D P H or appearance of ascorbate is stoichiometric. The reaction of D H A with TrxR in the presence of Trx has an apparent Km of 0.7 mY/and a kcat of 71 min -1. Although the later is only about 2°7o of the rate at which the thioredoxin system reacts with disulfide substrates, 5 hydrophobic derivatives of D H A show up to 10-fold higher kca t v a l u e s . 4 The most likely mechanism for the reaction of TrxR with D H A is a two-step nucleophilic substitution as predicted for GSH reduction of DHA, especially because the ascorbate free radical (AFR) is not generated during the latter reaction. 6 More surprising from a mechanistic standpoint was the finding that the thioredoxin system (TrxR plus Trx) can also reduce the AFR to ascorbate. 7 Using purified rat liver TrxR and bacterial Trx, this activity has an apparent Km of 3 ttM for the AFR and a kcat of 135 rain -1. When measured either as loss of N A D P H or as a decrease in the steady state AFR concentration determined by electron paramagnetic resonance (EPR) spectroscopy, the reaction is inhibited by aurothioglueose (ATG) and stimulated by selenocystine. The latter has been shown to enhance the 1E. S. J. Arn6r and A. Holmgren, Eur. J. Biochem. 267, 6102 (2000). 2 B. S. Winkler, Biochim. Biophys. Aeta 1117, 287 (1992). 3 B. C. Scott, O. I. Aruoma, R J. Evans, C. O'Neill, A. Van der Vliet, C. E. Cross, H. Tritschler, and B. Halliwell, Free Radic. Res. 20, 119 (1994). 4 j. M. May, S. Mendiratta, K. E. Hill, and R. E Burk, J. Biol. Chem. 272, 22607 (1997). 5 M. Luthman and A. Holmgren,Biochemistry 21, 6628 (1982). 6 j. M. May, Z. C. Qu, R. R. Whitesell, and C. E. Cobb, Free Radic. BioL Med. 20, 543 (1996). 7 j. M. May, C. E. Cobb, S. Mendiratta, K. E. Hill, and R. E Burk, J. Biol. Chem. 273, 23039 (1998).
METHODS IN ENZYMOLOGY, VOL. 347
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activity of TrxR in response to disulfide substrates. 8 The molecular mechanism by which the TrxR system can accomplish a one-electron reduction of the AFR has not been established, although there are several possible mechanisms. 7 The question arises as to whether reduction of either DHA or the AFR by TrxR or the thioredoxin system has physiologic relevance. About 80% of the NADPHdependent reduction of DHA to ascorbate in overnight-dialyzed rat liver cytosolic extracts can be attributed to TrxR, on the basis of inhibition by 10/xM ATG. 4 At this concentration ATG is specific for TrxR over glutathione peroxidase. 9 The remaining activity may be due to a 3ot-hydroxysteroid dehydrogenase in rat liver, which has a Km for DHA of 4 mM and a kcatof 59 rain -1.1° Further, dialysates from livers of selenium-deficient rats have only about 12% of the ATG-inhibitable DHA reductase activity of liver dialysates from control animals. 4 NADPH-dependent and ATG-sensitive DHA reductase activity is also present in dialyzed hemolysates prepared from human erythrocytes. 11 In liver dialysates, the rate of TrxR-mediated DHA reduction is about 25-30% that of GSH-dependent reduction, whereas in erythrocytes, it is only about 10%. NADPH-dependent AFR reductase activity is also present in overnightdialyzed rat liver cytosolic extracts. 7 Several features suggest that this activity is due to TrxR. It is (1) markedly decreased in dialysates from selenium-deficient rats, (2) inhibited by concentrations of ATG known to be selective for TrxR, and (3) stimulated by selenocystine. However, AFR reduction due to TrxR in dialysates is less than 2% of that of NADH-dependent AFR reduction in microsomes. The TrxR system can reduce both the AFR and DHA to ascorbate, and this activity is readily apparent in dialyzed cell extracts. Because the techniques developed in the studies noted above might be useful in comparing rates of TrxR-dependent DHA and AFR reduction with rates of ascorbate recycling by other routes in different cell types or tissues, the following outlines the methodological approaches that we have found most useful. Methods Tissue Preparation and Removal o f Low Molecular Weight Cofactors
TrxR-dependent reduction of either DHA or the AFR can be measured using crude extracts from various tissues, so long as low molecular weight cofactors for DHA reduction such as NADPH and GSH are removed. This is probably 8 M. Bj6rnstedt,M. Hamberg,S. Kumar,J. Xue, and A. Holmgren,J. Biol. Chem. 270,11761 (1995). 9 K. E. Hill, G. W. McCollum,M. E. Boeglin, and R. F. Burk,Biochem. Biophys. Res. Commun. 234, 293 (1997). lOB. Del Bello, E. Maellaro, L. Sugherini, A. Santucci, M. Comporti,and A. F. Casini, Biochem. J. 304, 385 (1994). 11S. Mendiratta, Z.-C. Qu, andJ. M. May, FreeRadic. Biol. Med. 25, 221 (1998).
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most easily accomplished by dialyzing cytosolic extracts of tissue or cells. For tissue, homogenize 100-400 nag in 1.0-1.5 ml of an ice-cold buffer consisting of 50 mM Tris-HC1 and 1 mM EDTA, pH 7.4 (Tris-EDTA). The type and duration of homogenization depend on the tissue, but for liver homogenization using a conical polytetrafluoroethylene (Teflon) pestle in microcentrifuge tubes is sufficient. The homogenate is centrifuged or microcentrifuged at 4000-6000g at 3 ° for 15 rain, and the resulting supernatant is centrifuged at 100,000g for 1 hr at 3 °. The clear supernatant is then dialyzed ovemight against three changes of 250 ml of the Tris-EDTA buffer at 3 °. The dialyzed sample (typically 5-25 mg of protein per milliliter) is saved on ice for assay and for protein determination.
Assay of Dehydroascorbic Acid Reduction by Thioredoxin Reductase The activity can be measured either by monitoring the appearance of ascorbate or the disappearance of NADPH. There is a one-to-one concordance between the two assays, using purified rat liver TrxR. 4 Measurement ofRate of NADPHLoss. The assay is more rapid than measuring the rate of ascorbate generation, and it confirms directly that the reaction is linear under the selected conditions. However, it is not specific and will detect NADPH oxidase activity. This can be controlled for by including a blank with NADPH, but no DHA. The reaction is carried out in a UV spectrophotometric semimicrocuvette at room temperature. Add 0.35 ml of Tris-EDTA buffer, 0.05 ml of enzyme, and 0.05 ml of 4 mM NADPH in the same buffer, and start the assay by adding 0.05 mt of freshly prepared 10 mM DHA. After mixing, record the change in absorption at 340 nm for 1-3 rain in a recording spectrophotometer. From the linear slope of this line calculate the amount of NADPH consumed, using an extinction coefficient of 6.22 x 103 M -1 cm -1 for NADPH. The rate of loss of NADPH equals the rate of ascorbate generated, corrected for the background rate of NADPH oxidation (the blank reaction). Commercial DHA (Sigma-Aldrich, St. Louis, MO) is not pure, but can be used if dissolved in the Tris-EDTA buffer just before addition to the assay (it is not stable at physiologic pH). The amount of DHA added in this assay is not saturating for TrxR, and therefore a decrease in the added DHA concentration during multiple assays over time may result. It is preferable to use DHA prepared by bromine oxidation of 10 mM ascorbic acid in water for most studies. 12 The resulting DHA is stabilized by the low pH of the reaction medium, so that multiple samples can be analyzed without significant loss of DHA. It is necessary to confirm that the pH of the reaction mixture is not affected by the addition.
Measurement of Dehydroascorbic Acid Reduction by Appearance of Ascorbate. For this assay, the reaction volume is scaled down to 100/zl and the reaction is carried out in 1.5-ml microcentrifuge tubes. Otherwise, the agent concentrations 12p. W. Washko,Y. Wang, and M. Levine,J. Biol. Chem.268, 15531 (1993).
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and conditions are the same as those described above. After adding DHA, incubate the reaction mixture for 10 rain at room temperature. Stop the reaction by adding 10 volumes of ice-cold 80% (v/v) methanol containing 1 mM EDTA. After 5 rain on ice, microcentrifuge the sample at 6000g at 3 ° for 15 rain and take aliquots of the supernatant for assay of ascorbate by high-performance liquid chromatography (HPLC). We use HPLC with electrochemical detection of ascorbate, 4 but UV detection should also have adequate sensitivity and low background in dialyzed samples. A blank sample with no enzyme should be run in assays using commercial DHA to correct for ascorbate contamination, which can vary depending on the lot of DHA. In liver dialysates, increases in ascorbate are linear over about 20 rain, but this should be confirmed in preliminary studies.
Assay of Thioredoxin Reductase Reduction of Ascorbate Free Radical It is important in this assay to generate stable steady state concentrations of the AFR in the reaction mixture. Because TrxR reduces DHA to ascorbate, it is not possible to use the reverse dismutation reaction of a mixture of DHA and ascorbate to generate the AFR. Stable concentrations of the AFR are generated for several minutes by using ascorbate oxidase (0.05-0.4 U/ml, from Cucurb#a species; Sigma-Aldrich) to oxidize 1-10 mM ascorbate. This reaction generates only the AFR, which then will dismutate to ascorbate and DHA. Although it is possible to monitor the disappearance of the AFR signal by EPR spectroscopy, the traditional indirect assay of NAD(P)H disappearance is much easier, can be quantified, and does not require an EPR spectrometer. In a UV semimicrocuvette containing 0.3 ml of Tris-EDTA buffer, add 0.05 ml of 2 mM NADPH, 0.05 ml of enzyme, 0.02 ml of ascorbate oxidase (1 U/ml), and, finally, to start the assay, 0.05 ml of freshly prepared 10 mM ascorbic acid in Tris-EDTA buffer. Mix and monitor the decrease in absorbance at 340 nm at room temperature for up to 3 rain. A blank sample without enzyme is used to correct for NADPH oxidation in the buffer, and the rate of AFR reduction is calculated as described above, with the caveat that 1 mol of NADPH will reduce 2 tool of the AFR. Activity then can be normalized to protein or to the amount of purified enzyme present, if known. Because DHA will be generated in this reaction mixture by dismutation of two molecules of the AFR to one molecule each of DHA and ascorbate, it is necessary to establish that this reaction is a small component of the AFR reduction for each system, and to correct for it. This is done by incubating ascorbate and the oxidase under the conditions of the assay in a UV cuvette and monitoring the loss of ascorbate over the time of the assay by the decrease in absorbance at 265 nm. On the basis of the dismutation reaction of the AFR, the amount of DHA generated will be half the amount of ascorbate lost over the short course of the assay. By including a sample containing that concentration of DHA as a control, the maximal
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component of DHA-dependent NADPH loss can be determined. In our previous studies, at 1 mM ascorbate and ascorbate oxidase at 0.05 U/ml, this rate was found to be less than 5% of the AFR-dependent rate]
Establishing Specificity for Thioredoxin Reductase in Crude Tissue Dialysates Because there may be other NADPH-dependent DHA or AFR reductases in crude tissue extracts, it is necessary to determine the fraction of measured activity that is specific for TrxR. This can be done by including an incubation containing 10 # M ATG. Activity dependent on TrxR is then calculated by subtracting the rate in the presence of ATG from that found in its absence. Other maneuvers to show specificity for TrxR are to include either 5 # M commercial Trx or 50 # M selenocystine. Both will increase the observed rates of DHA or AFR reduction. The effect of mammalian Trx is to increase activity by up to 2-fold, whereas selenocystine enhances ATG-sensitive activity by 3- to 5-fold. Conclusion Although TrxR-dependent reduction of both DHA and the AFR can be documented in tissue extracts, whether this contributes to ascorbate recycling within cells is yet to be determined. Optimal DHA reduction requires GSH in erythrocytes 6 lens epithelium, 13 and in several types of cultured cells. The latter include HepG2 cells, H4IIE cells, and primary cultures of bovine artery endothelial cells. 13a However, neither HL60 cells 14 nor skin keratinocytes15 appear to require GSH for DHA reduction. DHA reduction by the TrxR system could well play an important role in such cells. There is also a question of whether ascorbate is regenerated from DHA or the AFR in cells. DHA reduction is certainly important when ascorbate is oxidized outside cells to DHA, which enters cells by facilitated diffusion on GLUT-type glucose transporters and is rapidly reduced to ascorbate. On the other hand, it may be more efficient to recycle from the AFR stage when ascorbate is oxidized within cells by reactive oxygen species generated in mitochondria or by cellular metabolism. The AFR is relatively long-lived, and its reduction will prevent possible loss at the DHA stage due to irreversible ring opening. Coassin et al. 16 found greater AFR reductase activity than GSH-dependent DHA reductase activity in various pig tissues. Although they did not measure NADPH-dependent DHA reductase activity, 13 E J. Giblin, B. S. Winlder, H. Sasaki, B. Chakrapani, and V. Leverenz, Invest. Ophthalmol. Vis. Sci. 34, 1298 (1993). I3a j. M. May, Z.-C. Qu, and X. Li, Biochem. Pharmacol. 63, 873 (2001). ~4V. H. Guaiquil, C. M. Farber, D. W. Golde, and J. C. Vera, J. Biol. Chem. 272, 9915 (1997). 15 I. Savini, S. Duflot, and L. Avigliano, Biochem. J. 345, 665 (2000). !6 M. Coassin, A. Tomasi, V. Vannini, and E Ursilai, Arch. Biochem. Biophys. 290, 458 (1991).
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there was little evidence of NADH-dependent DHA reduction at DHA concentrations under 100/zM. Further, the measured affinities for DHA of glutaredoxin (0.26 mM),17and of the thioredoxin system (0.7 raM) 4 are not favorable for reducing the low concentrations of DHA expected to be generated within cells. On the other hand, the apparent Km of TrxR for the AFR (3/zM) 7 is in the range expected for the AFR in oxidatively stressed cellsJ 8'19 Although NADH-dependent mitochondrial and microsomal AFR reduction shows a low apparent Km and much greater activity when normalized to protein than does TrxR-mediated activity] sequestration of the NADH-dependent enzymes because of their membrane-bound nature may limit access to the AFR. The cytoplasmic location of the thioredoxin system in most cells might allow it to play a significant role in reducing the AFR, but this is yet to be demonstrated. 17 W. W. Wells, D. E Xu, and M. R Washburn, Methods EnzymoL 252, 30 (1995). 18 S. Pietri, M. Culcasi, L. Stella, and E J. Cozzone, Eur. J. Biochem. 193, 845 (1990). 19 G. R. Buettner and B. A. Jurkiewicz, Free Radic. Biol. Med. 14, 49 (1993).
[31] Thioredoxin Cytokine Action By YUMIKONISH1NAKA,HAJIME NAKAMURA, and JUNJI YODOI Introduction Human thioredoxin (TRX) is secreted from various types of cells including activated normal B lymphocytes and virus-transformed cells, and the secreted TRX exhibits multiple cytokine-like activities.l'2 In 1985, human TRX was first purified from the culture superuatant of an adult T cell leukemia (ATL) cell line, ATL-2, transformed by human T cell leukemia virus I (HTLV-I), as ATL-derived factor (ADF). 3 It induces interleukin 2 (IL-2) receptor u-chain (IL-2Rot) expression on a human large granular lymphocyte (LGL) cell line YT and it was thus considered a novel cytokine with IL-2R~-inducing activity.4 In 1989, cDNA sequencing revealed that ADF is a human homolog of TRX. 5 Expression and production of 1 j. Yodoi and T. Uchiyama, lmmunol. Today 13, 405 (1992). 2 H. Nakamura, K. Nakamura, and J. Yodoi, Annu. Rev. Immunol. 15, 351 (1997). 3 K. Teshigawara, M. Maeda, K. Nishino, T. Nikaido, T. Uchiyama, M. Tsudo, Y. Wano, and J. Yodoi, J. Mol. Cell. ImmunoL 2, 17 (1985). 4 j. Yodoi, K. Teshigawara, T. Nikaido, K. Fukui, T. Noma, T. Honjo, M. Takigawa, M. Sasaki, N. Minato, M. Tsudo, T. Uchiyama, and M. Moeda, J. lmmunol. 134, 1623 (1985). 5 y. Tagaya, Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N. Brown, K. Arai, T. Yokota, H. Wakasugi, and J. Yodoi, EMBO J. 8, 757 (1989).
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