Identification of NADPH-thioredoxin reductase system

Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4233-4237, November 1975 Biochemistry Identification of NADPH-thioredoxin reductase system in Euglena gracilis* (ribonucleotide reduction) S. MUNAVALLIO, DOROTHEA V. PARKER, AND FRANKLIN D. HAMILTON* The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 Communicated by Richard B. Setlow, July 23,1975 ABSTRACT Euglena gracifis contains a protein system Euglena gracilis was grown in Hunt's organotrophic medium under constant illumination and aeration (15). Cells which can utilize the reducing power of NADPH in the ribonucleotide reductase-catalyzed reduction of CTP. The proteins required for this reaction are a flavoprotein with a molecular weight of approximately 185,000 which is functionalary growth phase. After washing with 0.2 M Tris-acetate were grown in 50 liter jars and harvested prior to the stationly similar to thioredoxin reductase (NADPH), EC 1.6.4.5, and buffer, ph 9.1, the packed cells could be stored at -50 another protein (Protein I) whose function in the reaction is until used for isolation of the thioredoxin reductase system. unknown. This new protein does not appear to contain a Gel Electrophoresis. Analytical gel electrophoresis was prosthetic group and has a molecular weight of approximately 240,000. In addition, the ribonucleotide reductase active in for 90 min at 3 ma per gel according to the procedure of the Euglena NADPH-thioredoxin reductase system is more Davis (16) using 7% polyacrylamide gels. complex than the protein reported in a previous publication For the estimation of molecular weights, samples were [(1974) J. Biol. Chem. 249, 4428-4434]. The enzyme preparation described in this report contains four different types of ing 7% acrylamide according to the procedure of Weber and run in sodium dodecyl sulfate-polyacrylamide gels contain- polypeptide chains which may complex to form the active Osborn (17). enzyme. Assays. Ribonucleotide reductase activity was determined The reduction of by ribonucleotides by measuring the reduction of ribonucleotide reductase requires a reduced dithiol [3H]CTP with dithiothreitol as a reducing agent (15). The thioredoxin reductase (1-5). In Escherichia coli this system was requirement can be assayed satisfied by NADPH and by coupling the oxidation of NADPH to the reduction of two proteins, thioredoxin and thioredoxin [3H]CTP reductase using ribonucleotide reductase. The (6, 7). Thioredoxin reductase, a protein of assays were performed as follows: The incubation 68,000 molecular mixture weight, contains tightly bound FAD contained which 0.2 M Tris-acetate can be reduced buffer by NADPH. The (ph 8.1), 2 mm magnesium hydrogens on FAD are then acetate, 0.46 mm transferred to thioredoxin, a [3H]CTP, 2 X 10-6 M datp, 1.0 mm low-molecular-weight NADPH, 6.4 X 10-6 M protein (about 12,000) containing a adenosylcobalamin, 0.25 mg of ribonucleotide cystine residue which is reduced to form the dithiol reductase, and 0.5-1.0 mg of total protein in a active in the volume of 0.25 ribonucleotide reductase ml. After system. Thioredoxin and thioredoxin reductase have been incubation at 370 for 15 min, the solution was identified in E. coli, Lactobacillus leichmannii chilled, carrier dcmp was added, and [3H]dCMP was (8), yeast (9), rat liver isolated on Dowex 50 H+ (10, 11), and columns (15). ascites hepatoma (12). A new thioredoxin is detectable Ribonucleotide Reductase. upon infection of E. coli with T4, T5, or T6 bacteriophage (13, Ribonucleotide reductase was purified from Euglena 14). gracilis as described previously except that the final two steps of the purification, DEAE-cellu- This report describes the thioredoxin reductase (NADPH), EC 1.6.4.5, system in Euglena gracilis. The lose NADPH-supported reduction of chromatography and preparative electrophoresis, were omitted. ribonucleotides with the Euglena Instead, the 0-50% proteins is shown saturated ammonium sulfate fraction to was require additional protein components, the chromatographed on a 2.5 X 55 cm column of functions of which are unknown at this Biogel time. In addition, (1.5 M, Bio-Rad Laboratories) and eluted with 0.05 M potassium the Euglena ribonucleotide reductase that reacts in this phosphate at ph 7.3 that contained 10-4 M dithiothreitol and 10-6 M NADPH-thioredoxin reductase system is shown to be larger datp. Fractions containing active and more complex than the enzyme protein were previously described pooled and concentrated by precipitation with (15). ammonium sulfate; then the concentrated sample was subjected to a second and a third chromatography on Biogel. MATERIALS AND The METHODS enzyme preparation obtained by this procedure had a specific activity of 238 nmol of dctp/hr per mg of protein. Radioactive nucleoside triphosphates were purchased from During polyacrylamide gel electrophoresis of the sample, Schwarz/Mann Biochemicals. Diethylaminoethyl cellulose more than 80% of the protein migrated as a single band. (Brown Paper Co., Berlin, N.H.) was washed with acid and Protein was estimated from the absorbance at 280 nm assuming an absorbance of 1.0 for 1 mg/ml in a 1 cm light base (15). path. * A preliminary report of these data was given at the American Society for Cell Biology, November 1974, San Diego, Calif. Purification t Present address: Department of Chemistry, Livingstone College, Salisbury, N.C. All steps were performed at 40 unless indicated otherwise. $To whom reprint requests should be sent. The starting material for the isolation of the proteins needed Operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration. supernate of the ammonium sulfate fractionation described to obtain NADPH-stimulated [3H]CTP reduction was the 4233

4234 Biochemistry: Munavalli et al. in the purification procedure previously published for Euglena ribonucleotide reductase (15). After precipitation of the ribonucleotide reductase activity with ammonium sulfate, protein left in the supernate was further fractionated by adding 154 g of solid ammonium sulfate to the 700 ml of solution. The suspension was stirred for 30 min; then the precipitate was collected by centrifugation and dissolved in a small volume of buffer A. [Buffer A is a 0.05 M potassium phosphate (ph 7.3) buffer containing 1 mm EDTA and 1 mm mercaptoethanol.] Heat Treatment. The dissolved precipitate was desalted by passage through a column of Sephadex G-25 equilibrated with buffer A. The protein solution was heated to 600 during stirring and maintained at that temperature for 5 min. After rapid cooling the denatured protein was removed by centrifugation for 15 min at 27,000 X g, and the protein remaining in the supernate (305 ml) collected by adding 140 g of solid ammonium sulfate and centrifuging. The precipitate was dissolved in a small volume of buffer A. DEAE-Cellulose. The protein solution was desalted by passage through a column of Sephadex G-25 equilibrated with 5 mm potassium phosphate at ph 7.3, 1 mm EDTA, 1 mm mercaptoethanol. The desalted solution was then applied to a column (2.5 X 40 cm) of DEAE-cellulose previously equilibrated with the same buffer. The column was washed with the 5 mm buffer until the first protein peak was eluted, and the buffer was then changed to 50 mm potassium phosphate at ph 7.3. After elution of a second protein peak, the buffer was again changed to 0.2 M potassium phosphate at ph 7.3 which eluted two peaks of protein. Active fractions were pooled and concentrated in a Diaflo cell using a PM30 membrane. Preparative Electrophoresis. Final purification of active proteins was obtained by preparative electrophoresis. Electrophoresis was performed on 150 mg of protein as described by Gaertner and DeMoss (18), on an 80 ml 7% polyacrylamide gel and a 30 ml stacking gel. RESULTS [3HJtCTP reduction using NADPH A. To identify the Euglena thioredoxin reductase system, we tested protein fractions for their ability to couple NADPH oxidation with the reduction of [3H]CTP by ribonucleotide reductase. Table 1 lists the Euglena fractions needed to obtain the reduction of [3H]CTP under these conditions. No activity was obtained prior to ammonium sulfate fractionation of the Euglena extract. Little activity was obtained in either the 0-50% or 50-80% ammonium sulfate fraction when assayed separately, although the 50-80% ammonium sulfate fraction contained a small amount (<5%) of the ribonucleotide reductase activity. A substantial increase in activity was obtained, however, when the 0-50% ammonium sulfate fraction (ribonucleotide reductase) was supplemented with the 50-80% ammonium sulfate fraction. This activity was maintained if the 0-50% ammonium sulfate fraction was replaced by ribonucleotide reductase partially purified by DEAE-cellulose chromatography (Table 1). From these experiments it could be concluded that protein components needed for NADPH-supported [3H]CTP reduction, i.e., thioredoxin reductase were contained in the 50-80% ammonium sulfate fraction. However, further fractionation of ribonucleotide reductase by preparative electrophoresis gave an enzyme which was no longer active in the NADPH system (Table 1). The enzyme from preparative Table 1. Protein fractions required for [3H]CTP reduction with dithiothreitol or NADPH* Specific activity Protein fraction (nmol of dcmp/hr per mg) Thioredoxin Ribonucleotide reductase Dithioreductase [% (NH4)2S04] threitol NADPH ph 5 Supernate None 63 0.1 0-50% (NH4)2SO4 None 310 1.5 None [50-80] 22 0.4 0-50% (NH4)2SO4 [50-80] 336 21.4 DEAE-cellulose eluate [50-80] 960 17.3 Preparative electrophoresis eluate [50-80] 2460 0.4 * The reaction mixture contained 0.2 M Tris-acetate buffer (ph 8.1), 2 mm magnesium acetate, 0.46 mm [3H]CTP, 2 gm datp, 6 gm adenosylcobalamin, 0.5-1.5 mg of protein, and 70 mm dithiothreitol or 1 mm NADPH, as indicated, in a volume of 0.25 ml. Fractions containing ribonucleotide reductase activity and/or thioredoxin reductase activity were added as indicated. Samples were incubated for 15 min at 370 and the product was isolated as presented in the text. electrophoresis was, nevertheless, quite active when dithiothreitol was used as a reducing agent. It was evident, therefore, that proteins needed in the thioredoxin reductase system were located in both the 0-50% and 50-80% ammonium sulfate fractions. In addition, it was also apparent that some essential component of the reductase system was lost in the purification of ribonucleotide reductase. Therefore, the procedure for obtaining ribonucleotide reductase was changed as indicated in Materials and Methods in order to retain this component in the system. Ribonucleotide reductase obtained by the new protocol was used in all subsequent experiments. B. Further fractionation of the proteins in the 50-80% ammonium sulfate fraction resulted in the identification of two active components which could be separated by DEAEcellulose chromatography. One active fraction was obtained in the first protein peak from the column and a second was in the third protein peak. Both components were needed to obtain NADPH-supported [3H]CTP reduction (Table 2). Final purification of the active component in each of the fractions was achieved by preparative electrophoresis. A Table 2. Proc. Nat. Acad. Sci. USA 72 (1975) Purification of thioredoxin reductase system* Specific activity (nmol dcmp/hr Thioredoxin reductase per mg) preparation Dithiothreitol NADPH None 238 0.4 50-80% (NH4)2SO4 12.0 Heat-treated supernate 7.9 DEAE Peak I 1.4 DEAE Peak III 16.4 DEAE Peaks I & III 95.2 Protein I (preparative electrophoresis) 2.1 Thioredoxin reductase 16.4 Protein I + thioredoxin reductase 95.2 * All assays contained 0.25 mg of ribonucleotide reductase (Biogel concentrate).

Biochemistry: Munavalli et al. Proc. Nat. Acad. Scs. USA 72 (1975) 4235 1. Oni -Goloctosidose Serum albumin Pyruvote kinose j (a albumin E c ur 0. V5l WAVELENGTH (nm) FIG. 2. Relative fluorescence (A) and absorbance (B) of thioredoxin reductase. The fluorescence was recorded with an excitation wavelength of 450 nm. For spectra of the reduced protein, 50 nmol of NADPH was added to the cuvette. Thioredoxin reductase (-), thioredoxin reductase + NADPH (------ ). 0 II 0 1 2 3 4 5 6 7 8 9 LENGTH (cm) FIG. 1. An absorption scan of Protein I after 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein (50,g) was applied to the gel and electrophoresis performed as outlined in the text. Gels were stained with Coomassie blue and scanned at 575 nm. The standard proteins used were 0-galactosidase, serum albumin, pyruvate kinase, and ova albumin as indicated in the graph. characterization of the protein isolated from each is described. Protein I. Preparative electrophoresis of the first active fraction from the DEAE-cellulose column yielded a protein that migrated as a single band on a 7% polyacrylamide gel. This protein, referred to as Protein I, had an apparent molecular weight of 240,000 as determined by a semi-log plot of its elution from a Sephadex G-200 column. Protein I could be separated into two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1) with molecular weights of 135,000 and 42,000. Protein I appeared to contain no prosthetic group, since the spectrum showed absorbance only below 300 nm with a peak at 280 nm. The function of Protein I in the transfer of hydrogen from NADPH to the product dctp is unknown. Thioredoxin Reductase. Preparative electrophoresis of the second active fraction from DEAE-cellulose chromatography resulted in the isolation of a protein with spectral properties identical to thioredoxin reductase. It is a flavoprotein as determined from both its fluorescence and absorption spectra (Fig. 2). When a solution containing the protein is irradiated with light at 450 nm, a sharp fluorescence peak is observed at 525 nm. The absorption spectrum contained a sharp maximum at 275 nm and smaller peaks at 370 and 460 nm and showed an intense yellow color. The fluorescence peak at 525 nm is diminished and the absorbance peaks at 370 and 460 nm disappear upon anaerobic addition of NADPH (Fig. 2). The flavine was identified as FAD by boiling an aliquot of the protein, centrifuging it to remove denatured protein, and chromatographing the supernate on polyethyleneimine-cellulose thin-layer chromatography plates developed in 0.5 M LiCl. FAD and FMN were used as standards. Hence, this protein is referred to as Euglena thioredoxin reductase. After preparative electrophoresis, the fraction containing thioredoxin reductase activity eluted from Sephadex G-200 chromatography as a single peak, and an apparent molecular weight of 185,000 was estimated for the protein by this procedure. When the protein was subjected to electrophoresis on 7% polyacrylamide gel, two bands were observed. It remains to be determined whether thioredoxin reductase dissociates when electrophoresed on analytical gel or if the second protein is a contaminate. Requirements for CIP reduction NADPH is the only pyridine nucleotide tested that served as a hydrogen donor in the Euglena thioredoxin reductase-ribonucleotide reductase system; NADH is inactive. The ribonucleotide reductase activity obtained with the NADPHthioredoxin reductase system was only 10-20% of that obtained when dithiothreitol was used as a reducing agent (see Table 2). Neither FAD or FMN (10-6 M), when added to the incubation mixture, stimulated the reaction. The activity obtained with the NADPH-thioredoxin system varied among ribonucleotide reductase preparations, even though special care was taken to maintain constant conditions. Ribonucleotide Reductase. Final purification of the ribonucleotide reductase used in these studies was achieved by gel filtration. As shown in another section of this report, preparative electrophoresis of the enzyme resulted in a loss of activity in the NADPH-thioredoxin reductase assay. To compare the proteins obtained by the two procedures, we subjected a sample of each to 7% polyacrylamide gel electrophoresis. The results (Fig. 3) indicated that the protein ob-

4236 Biochemistry: Munavalli et al. P Proc. Nat. Acad. Sci. USA 72 (1975) E U-) 1 2 FIG. 3. Seven percent polyacrylamide gels of ribonucleotide reductase obtained by Biogel chromatography (1) and preparative electrophoresis (2). Electrophoresis was performed as indicated in the text. tained by Biogel chromatography migrated at a slower rate than the protein purified by preparative electrophoresis. Gel filtration on Sephadex G-200 indicated that the slower moving protein had a molecular weight greater than 500,000 as compared with 440,000 for the enzyme from preparative electrophoresis (15). Sodium dodecyl sulfate-gel electrophoresis of this protein resulted in the resolution of four major bands with molecular weights of approximately 100,000, 76,000, 63,000, and 15,000 as well as other minor bands (Fig. 4). By comparison, the 440,000-weight enzyme gave only one band on sodium dodecyl sulfate-gel electrophoresis (15). It'is apparent, therefore, that the less rigorous procedure produced an enzyme of greater complexity than reported earlier. Furthermore, only the more complex species is active in the thioredoxin reductase assay, suggesting that it is functional under in vivo conditions. However, it remains to be determined which of the four polypeptides observed on sodium dodecyl sulfate-gels are essential components of the complete system. DISCUSSION These experiments show that Euglena contain a protein system capable of utilizing NADPH for the reduction of ribo,- nucleoside triphosphates. When it was established that the active components of this system were located in the 50-80% ammonium sulfate fraction of the Euglena extracts, it became necessary to identify the essential proteins. This process was complicated somewhat, however, by the loss of components essential for the NADPH-thioredoxin reductase reaction after preparative electrophoresis of ribonucleotide reductase. The ribonucleotide reductase protein obtained from preparative electrophoresis was completely void of activity in the NADPH system, although it had a specific activity of 2460 nmol/hr per mg protein when dithiothreitol was used as a reducing agent. A ribonucleotide reductase enzyme active in the NADPH system was obtained by a more gentle isolation procedure. The enzyme obtained by Biogel chromatography was approximately 80% homogenous, was considerably larger (>500,000 molecular weight) as determined by gel filtration LENGTH (cm) FIG. 4. An absorbance scan of ribonucleotide reductase which was obtained by Biogel chromatography and subjected to sodium dodecyl sulfate-gel electrophoresis. Electrophoresis was performed as indicated in the text. than the protein obtained previously (440,000), and showed multiple bands on sodium dodecyl sulfate-gel electrophoresis. That the specific activity of the protein'prepared by gel filtration was 10-fold lower than that of the enzyme obtained by preparative electrophoresis may be due simply to the removal of fewer contaminates. However, it is possible that this larger enzyme may contain regulatory components as well as proteins essential for the NADPH-thioredoxin reductase system. Such a regulatory component could result in a lower specific activity for CTP reduction. An example of this type of regulation can be found in the aspartate transcarbamoylase enzyme (19), which is composed of two subunits, a catalytic protein and a regulatory protein. The specific activity of the catalytic protein is much greater when it is separated from the regulatory protein. An explanation of the lowered activity in the ribonucleotide reductase described in our report must await a more extensive examination of the polypeptides which appear to be associated with the enzyme. Euglena thioredoxin reductase appears to contain FAD and to be reducible by NADPH. It is of interest that the Euglena thioredoxin reductase appears to be a protein of a unique size (185,000. molecular weight). This difference cannot be assigned to the difference between prokaryotes and eukaryotes since both E. coli (7) and yeast (9) are reported to contain proteins with a molecular weight of approximately 68,000. Thioredoxin reductase activity has been identified in mammalian cells, but the molecular weight is not known (12). The role of Protein I in the thioredoxin reductase system 8

Biochemistry: Munavalli et al. has not been determined, nor has the function of other proteins which appear along with the larger ribonucleotide reductase molecule. These studies suggest, however, that the ribonucleotide reductase system in Euglena requires a number of different proteins for activity. It is significant that the present publication presents a report of a combined reaction of ribonucleotide reductase and the thioredoxin reductase system, both obtained from a eukaryotic cell. In other studies with eukaryotic thioredoxin reductase systems, the reaction was coupled to the reduction of CDP with E. coli ribonucleotide reductase (9-11). Studies in our laboratory have shown that Euglena thioredoxin reductase and Protein I can also function with the E. coli ribonucleotide reductase (manuscript in preparation). Therefore, successful reaction of thioredoxin and thioredoxin reductase obtained from another organism with the E. coil enzyme does not establish that it will also react with the ribonucleotide reductase of the same organism. It appears quite possible that eukaryotic cells might contain a thioredoxin reductase system of considerably greater complexity than that described for the prokaryotic cell. We thank Ed Phares and Mary Long for advice and assistance in growing large cultures of Euglena graclis. Partial support for this research was obtained from a grant by the Carnegie Corporation of New York to the University of Tennessee, Knoxville. (S.M. is supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.) Proc. Nat. Acad. Sci. USA 72 (1975) 4237 1. Reichard, P. (1962) J. Biol. Chem. 237,3513-3519. 2. Abrams, R. & Duraiswami, S. (1965) Biochem. Biophys. Res. Commun. 18,409-414. 3. Moore, E. C. & Reichard, P. (1964) J. Biol. Chem. 239, 3453-346. 4. Hooper, S. (1972) J. Biol. Chem. 247,3336-3340. 5. Gleason, F. K. & Hogenkamp, H. P. (1972) Biochim. Biophys. Acta 277,466-470. 6. Moore, E. C., Reichard, P. & Thelander, L. (1964) J. Biol. Chem. 239,3445-452. 7. Laurent, T. C., Moore, E. C. & Reichard, P. (1964) J. Biol. Chem. 239,3436-3444. 8. Orr, M. D. & Vitols, E. (1966) Biochem. Biophys. Res. Commun. 25,109-115. 9. Porque, P. G., Baldesten, A. & Reichard, P. (1970) J. Blol. Chem. 245,2363-2370. 10. Larson, G. & Larsson, A. (1972) Eur. J. Biochem. 26, 119-124. 11. Larsson, A. (1973) Eur. J. Biochem. 35,346-349. 12. Herrmann, E. C. & Moore, E. C. (1973) J. Biol. Chem. 248, 1219-1223. 13. Sjoberg, B.-M. (1972) J. Biol. Chem. 247,8058-8062. 14. Eriksson, S. & Ove, B. (1974) Eur. J. Biochem. 46, 271-278. 15. Hamilton, F. D. (1974) J. Biol. Chem. 249,4428-4434. 16. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427. 17. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. 18. Gaertner, F. H. & DeMoss, J. A. (1969) J. Biol. Chem. 244, 2716-2725. 19. Gerhart, J. C. & Schachman, H. K. (1965) Biochemistry 4, 1054-1062.