Thioredoxm, Glutaredoxin, and Thioredoxm Reductase from Cultured Hela Cells (Thiol-Disulfide Exchange) MONICA LIK-SHING TSANG* and JAMES A

Home , Glutaredoxin, Glutathione reductase, Reductase, Thioredoxin reductase

Proc. Nati Acad. Sci. USA Vol. 78, No. 12, pp. 7478-7482, December-1981 Biochemistry

Thioredoxm, glutaredoxin, and thioredoxm reductase from cultured HeLa cells (thiol-disulfide exchange) MONICA LIK-SHING TSANG* AND JAMES A. WEATHERBEEti *Biolog Department, Clark University, Worcester, Massachusetts 01610; and tThe Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 Communicated by Martin Gibbs, September 8, 1981

ABSTRACT Thioredoxin and glutaredoxin may be important ATPase activity in intact chloroplasts (11); the reduction of di- in regulating cell metabolism by mediating interchanges between sulfides in proteins such as insulin, choriogonadotropin, and sulfhydryl and disulfide groups. Components of the thioredoxin/ fibrinogen (12-14); the functioning as an essential subunit for glutaredoxin system from cultured HeLa cells have been partially phage T7 DNA polymerase (15); and perhaps phosphate transfer purified and characterized by using Escherichia coli adenosine 3'- reactions (16). In many of the thioredoxin- and glutaredoxin- phosphate 5'-phosphosulfate reductase, a thioredoxin/glutare- dependent reactions studied, at least one function of thioredoxin-dependent enzyme on the pathway of sulfate, reduction, as an assay system. In HeLa cells, a NADPH-thioredoxin reductase doxin or glutaredoxin appears to' involve thiol-protein disulfide and three heat-labile proteins (designated PI, PH, and PHI) that interchange. have thioredoxin- or glutaredoxin-like properties are found. Both Even though E. coli thioredoxin and glutaredoxin are related PI and PIll have molecular masses of =w12,000 daltons and are functionally, there are differences in their physical as well as readily reduced by their homologous HeLa thioredoxin reductase. catalytic properties. Glutaredoxin has an amino acid composi- However, only PI can be reduced efficiently by the glutathione tion that is different from that of thioredoxin (3, 17). In agree- system and neither PI nor PHI has inherent glutathione-disulfide ment, the tryptic peptide maps ofthe two proteins are also dif- oxidoreductase activity. PH has a molecular mass of >30,000 dal- ferent, indicating that the two proteins are unrelated in their tons and appears to be associated with a reductase activity. The primary structure. Thioredoxin, but not glutaredoxin, is a sub- HeLa NADPH-thioredoxin reductase has been purified to near strate for E. coli thioredoxin reductase. Both E. coli thioredoxin homogeneity and found to be a 116,000-dalton flavoprotein com- and glutaredoxin can be reduced by dithiothreitol as well as posed of two 58,000-dalton subunits. The HeLa enzyme has low glutathione (GSH). Nevertheless, glutaredoxin is reduced species and substrate specificity and can reduce HeLa PIand' PIN, equally efficiently by dithiothreitol and GSH, thioredoxin is a E. coli thioredoxin and glutaredoxin, and the disulfide bond in better substrate for reduction by dithiothreitol than by GSH. 5,5'-dithiobis(2-nitrobenzoic acid). The exact in vito roles of the Since HeLa thioredoxin and glutaredoxin do not possess any HeLa thioredoxin/glutaredoxin system remain to be determined. enzyme activity by themselves, our observation that E. coli/ PAdoPS-reductase shows a high degree of crossreactivity to- Thiol-protein disulfide interchange reactions are components ward the HeLa thioredoxin and glutaredoxin system provides ofmany diverse cellular processes, including tubulin assembly a convenient assay system for these proteins. into microtubules, development of mitotic spindle and astral rays, modulation ofthe specific activity ofcertain enzymes, etc. MATERIALS AND METHODS (for review, see refs. 1 and 2). Although a number of enzyme systems catalyzing thiol-protein disulfide interchange reactions Cell Growth and Preparation of Cell-Free Extract. HeLa have been studied in vitro, their relative physiological roles are cells (strain S-3) were grown in spinner bottles as described (18). still unclear. In this report, the partial purification and char- Cells (5-7 X 105/ml) were harvested by low-speed centrifu- acterization of the thioredoxin and glutaredoxin systems, en- gation. The cell pellet obtained was washed once with 2 vol of zyme systems that may be of importance in the reduction of phosphate-buffered saline (150 mM NaCV10 mM phosphate protein disulfides in vivo, from cultured HeLa cells are buffer, pH 7.0) and once with about the same volume of PM described. buffer [1 mM MgSOJ2 mM EDTA/2 mM dithioerythritol/ Thioredoxin and glutaredoxin are functionally related low 100 mM 1,4-piperazinediethanesulfonic acid, pH 6.9, contain- molecular weight acidic proteins containing an oxidation- ingTrasylol (aprotinin or kallikrein inactivator) at 100,000 units/ reduction-active disulfide bridge (3). In Escherichia coli, both ml]. The cell pellet obtained after the second wash was sus- proteins are cofactors that couple the reducing capacity from pended in an equal volume ofcold PM buffer and disrupted by various hydrogen donor systems to the in vitro reduction of sonication as described (19). After sonication, cells were also active sulfate (adenosine3'-phosphate 5'-phosphosulfate; PAdoPS) treated by five strokes in a glass Potter-Elvehjem homogenizer to sulfite (4-6) and of ribonucleotides to deoxyribonucleotides equipped with a'Teflon pestle. The pellet obtained after cen- (3, 7, 8). In recent years, aside from sulfate and ribonucleotide trifugation for 30 min at 35,000 x g (avg) was discarded. The reduction, thioredoxin or thioredoxin-like compounds have supernatant fraction, termed "crude extract," was used for all been implicated in numerous other cellular reactions-e.g., the subsequent protein purifications. For large-scale purification light activation of reductive pentose pathway enzymes in chlo- of thioredoxin, glutaredoxin, and thioredoxin reductase, the roplasts (9); the dark activation of oxidative pentose pathway combined supernatant fractions obtained from the first, second, enzyme in chloroplasts (10); the modulation of coupling factor Abbreviations: GSH, glutathione; DTNB, 5,5'-dithiobis(2-nitrobenzoic The publication costs ofthis article were defrayed in part by page charge acid); PAdoPS, adenosine 3'-phosphate 5'-phosphosulfate. payment. This article must therefore be hereby marked "advertise- t Present address: Dept. of Pharmacology, CMDNJ-Rutgers Medical ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. School, P.O. Box 101, Piscataway, NJ 08854. 7478 Downloaded by guest on September 30, 2021 Biochemistry: Tsang and Weatherbee Proc. NatL Acad. Sci. USA 78 (1981) 7479

third, and fourth microtubule polymerizations during microtubule preparations (19, 20) from crude extracts were also used. The combined supernatant fractions were essentially identical to the crude extracts except that tubulin and other microtubule- associated proteins had been removed. During purification, the 0 profiles ofthioredoxin, glutaredoxin, and thioredoxin reductase - 1.0 > obtained from the latter sources were similar to those obtained c- >20.2043 from crude cell extracts. ._ or re- 0 Enzyme Assays. Thioredoxin glutaredoxin activity, 0.5 1 -0.4 ferred to as thioredoxin/glutaredoxin activity, was assayed as 2 0.2 cn stimulation of the PAdoPS reductase-catalyzed reaction on the 1~0.1~ addition of thioredoxin or glutaredoxin using dithiothreitol as CIA the hydrogen donor (17). PAdoPS reductase activity was assayed by measuring the amount of acid-volatile radioactivity formed 10 20 30 40 50 60 70 80 after incubation with [3S]PAdoPS and thioredoxin/glutare- Fraction doxin in the presence ofan appropriate hydrogen donor system. Thioredoxin reductase activity was assayed by coupling with FIG. 1. Elution profile of thioredoxin/glutaredoxin activities from PAdoPS reductase, using NADPH (2.5 mmol) as the hydrogen a DEAE-cellulose column. donor for HeLa thioredoxin/glutaredoxin. Alternatively, thioredoxin reductase activity can be assaved in the presence of three, >80% of the activity loaded on the column initially was 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by following the in- recovered during this step. Whereas PI and PIII have similar crease in absorbance at 412 nm. The incubation mixture was elution volumes characteristic of proteins with molecular (total vol, 1.0 ml) 100 t&mol of Tris-HCl, pH 8.0, 10 Amol of masses of 12,000 daltons, PII eluted within the void volume of EDTA, 0.5 Amol of NADPH, 0.2 /Amol of DTNB. the column, indicating that this protein has a molecular mass GSH-disulfide oxidoreductase activity was assayed by using of >30,000 daltons. A typical purification of PI, PII, and PIII the method of Holmgren (3). The enzyme activity was coupled is summarized in Table 1. The final PI preparation appeared with glutathione reductase, which reduces oxidized glutathione as a single band with a molecular mass of 12,000 daltons in in the presence ofNADPH. The disappearance ofNADPH was NaDodSO4polyacrylamide gel electrophoresis. However, two followed at 340 nm using a Cary 210 spectrophotometer. One protein bands are apparent on the native polyacrylamide gel unit corresponds to 1 pmol of NADPH oxidized/min. electrophoretogram (Fig. 2). The PIII preparation showed mul- Protein Purifications. Unless otherwise noted, all steps were tiple bands on both native and NaDodSOJpolyacrylamide gel carried out at 40C and centrifugation was at 12,000 X g for 15 electrophoresis (data not shown). min. A typical purification ofa >90% pure thioredoxin reductase HeLa thioredoxin/glutaredoxin and NADPH-thioredoxin preparation from crude extracts of HeLa cells is summarized reductase were partially purified from either crude extracts or in Table 2. The isoelectric point of the enzyme was pH 5.37. the combined supernatant fractions. The protein solution, after During purification, the enzyme assay used was based on the exhaustive dialysis against several changes of buffer I (0.02 M finding that the HeLa enzyme, like thioredoxin reductase from phosphate buffer, pH 7.0/2 mM EDTA), was applied to a other mammalian (24, 25) sources, can catalyze direct reduction DEAE-cellulose column (75 ml ofpacked DEAE per g of pro- of the disulfide bonds in DTNB in the presence of NADPH. tein to be fractionated) previously equilibrated with buffer I. Similar to calfliver and thymus thioredoxin reductases (24), the After protein application, the column was washed with 2 bed HeLa enzyme activity was nonlinear with time. Thus, the initial vol ofbuffer I, and thioredoxin/glutaredoxin activities and thio- rates were used for the calculations ofenzyme specific activities. redoxin reductase activity were eluted with a convex gradient High DTNB concentrations (>0.1 mM) inhibited enzyme made with 5 bed vol of buffer I and 10 bed vol of 0.2 M phos- activity. phate buffer, pH 7.0/2 mM EDTA. Three distinct peaks ofthio- Based on the mobility of the HeLa enzyme on NaDodSOJ redoxin/glutaredoxin activities, PI, PII, and PIII, and a peak electrophoresis, the protein appears to have a subunit molec- of thioredoxin reductase activity that eluted slightly ahead of PI were found. Table 1. Purification of PI, PIT, and PER[ from cultured The pooled PI, PII, and PIII and thioredoxin reductase frac- HeLa cells tions were further purified as shown in Tables 1 and 2. For preparative isoelectric focusing ofthioredoxin reductase, carrier Chromatographic Protein, Activity Yield, ampholytes, pH 4-6, was used. step mg Cofactor* Specifict % Other Methods. Protein was determined by the Coomassie PI brilliant blue G-250 dye-binding method ofBradford (21) using DEAE-cellulose 100 1500 15 100 Bio-Rad dye reagent. Polyacrylamide gel electrophoresis (22) CM-cellulose 1.04 596 573 40 and [3S]PAdoPS preparation (23) were as described. Sephadex G-50 0.4 547 1367 36 P1[ RESULTS DEAE-cellulose 70 108 1.5 100 Three peaks of thioredoxin/glutaredoxin activities, designated Sephadex G-50 44 75 1.7 69 PI, PII, and PIII according to their order of elution with in- PEm creasing salt concentrations, were eluted from the DEAE-cel- DEAE-cellulose 66 198 3 100 lulose column (Fig. 1). Based on the heights ofthe three activity CM-cellulose 1.2 95 79 48 peaks, the relative amounts of PI/PII/PIII in crude HeLa ex- Sephadex G-50 0.07 67 956 34 tracts were estimated to be 100:3.6:8.0. PI, PI1, and PIII were The initial crude extract contained 2025 mg of protein. then analyzed by gel filtration on a calibrated Sephadex G-50 * Expressed as nmol of PAdoPS per hr. column in the presence of 10 mM 2-mercaptoethanol. For all t Expressed as nmol of PAdoPS per mg of protein. Downloaded by guest on September 30, 2021 7480 Biochemistry: Tsang and Weatherbee Proc. Nad Acad. Sci. USA 78 (1981) heat-stable proteins, HeLa PI, PII, and PIll appeared to be A 1 2 B1 2 rather heat labile. After heating at 750C for 3 min, PI, P11, and PIl retained 42%, 31%, and 32%, respectively, oftheir original activities. - The ability ofGSH to reduce PI, P11, and PIII was measured by the PAdoPS reductase coupled assay (Table 3). Since different PI, P11, and PI1 concentrations were used in these assays, the corresponding dithiothreitol-dependent PAdoPS reduction in the presence of identical amounts of thioredoxin/glutaredoxin was used as the basis for comparison. The effectiveness of GSH as a reductant is expressed as (PAdoPS reduced in the -~ ~ -_ presence of GSH/PAdoPS reduced in the presence of dithiothreitol) X 100. As shown in Table 3, GSH can reduce HeLa PI and P11 and E. coli glutaredoxin but not HeLa PIll or E. coli thioredoxin efficiently. The ability of HeLa thioredoxin reductase as well as E. coli thioredoxin reductase to reduce PI, PIT, and PITT in the pres- FIG. 2. (A) Polyacrylamide gel electrophoresis of PI. Lanes: 1, in the presence of NaDodSO4; 2, native gel. (B) NaDodSO4/polyacryl- ence of NADPH was also measured by the PAdoPS reductase amide gel electrophoresis of HeLa thioredoxin reductase. Lanes: 1, coupled assay (Table 4). Again, the corresponding dithiothrei- HeLa thioredoxin reductase; 2, molecular mass standards: a, phos- tol-dependent PAdoPS reduction was used as the basis for com- phorylaseb (94,000 daltons); b, bovine serum albumin (67,000 daltons); parison. Whereas the E. coli enzyme is species specific and uses c, ovalbumin (43,000 daltons); d, carbonic anhydrase (30,000 daltons); E. coli thioredoxin but not E. coli glutaredoxin as a substrate, e, soybean trypsin inhibitor (20,000 daltons). the HeLa enzyme has a much wider substrate specificity and will reduce PI, PIII, E. coli thioredoxin, and, to acertain extent, ular mass of58,000 daltons (Fig. 2). The molecular mass of na- E. coli glutaredoxin. The P11 preparation used in these studies tive HeLa thioredoxin reductase was determined by elec- has an inherent reductase activity and can couple NADPH-de- trophoresis of the purified protein through nondenaturing pendent PAdoPS reduction in the absence ofadded thioredoxin polyacrylamide gels by the procedure of Hedrick and Smith reductase. (26). The protein migrated to a position corresponding to a One distinction between E. coli thioredoxin and glutaredoxin molecular mass 105,000 daltons as determined by comparison is that the glutaredoxin has an inherent GSH-disulfide oxido- with bovine serum albumin (monomer, dimer, and trimer). reductase activity (3). Assays ofthe HeLa proteins showed that Thus the HeLa enzyme is probably similar to the Novikoff rat this enzyme activity is not associated with either PI or PIII. Due tumor enzyme (25), which is a 116,000-dalton protein contain- to the fact that our P11 preparation has an inherent reductase ing two subunits of -58,000 daltons. The absorption spectrum activity, the presence of GSH-disulfide oxidoreductase activity of the purified HeLa thioredoxin reductase is similar to that of could not be measured. the E. coli enzyme and other typical flavoproteins. All three HeLa thioredoxin(s)/glutaredoxin(s), like E. coli DISCUSSION thioredoxin and glutaredoxin, are reduced efficiently by di- The thioredoxin and glutaredoxin systems are potentially im- thiothreitol and can couple the PAdoPS reductase-catalyzed re- portant enzyme systems that may be responsible for various duction of PAdoPS in the presence of dithiothreitol (Table 3). cellular processes involving thiol-protein disulfide interchange In all three cases, PAdoPS reductase activity was linearly de- reactions. The thioredoxin system has been studied in bacte- pendent on the concentrations of PI, PII, and PIII added (data riophage (e.g., T4) (27), microorganisms [e.g., E. coli (28) and not shown). Whereas E. colt thioredoxin and glutaredoxin are yeast (29)], algae [e.g., Scenedesmus obliquus (30)], higher plants [e.g., spinach (31)], and mammalian cells [e.g., calf thymus and liver, Novikoffrat tumor (32, 33)]. To date, besides E. Table 2. Purification of thioredoxin reductase from HeLa cells coli, the glutaredoxin system has been described only in calf thymus and bacteriophage T4 (27, 34). In the present study, Activity* components ofthe HeLa thioredoxin/glutaredoxin system were Specific, partially purified and characterized. The criterion used for clas- units/mg sifying a cellular protein as thioredoxin/glutaredoxin is that the Step Total, units of protein Yield, % protein can replace E. coli thioredoxin or E. coli glutaredoxin Crude extract 760 0.097 100 as cofactor for the E. coli PAdoPS reductase-catalyzed reaction DEAE-cellulose in the presence of an appropriate hydrogen donor system. In chromatography 352 0.0864 46 the absence of an unambiguous definition for thioredoxin and Phenyl-Sepharose glutaredoxin, the universality of this assay system to detect all chromatography 204 3.24 27 "thioredoxins" and "glutaredoxins," so classified based on their AcA-44 column interchangeability with or functional similarity to E. coli thio- chromatography 140 9.36 18 redoxin or E. coli glutaredoxin in other assay systems, remains Agarose-hexane- to be determined. adenosine 2',5'- In HeLa cells, a NADPH-thioredoxin reductase and three diphosphate proteins (designated PI, PII, and PIII) that have thioredoxin- chromatography 84 28 11 or glutaredoxin-like properties are found. Since some of these Electrofocusing in a proteins have characteristics that are hybrid between E. coli granulated gel 44 42 6 thioredoxin and E. coli glutaredoxin, they have not been cat- * One unit of activity corresponds to the formation of 1 ,umol of 5-thio- egorized as either thioredoxin or glutaredoxin but are referred 2-nitrobenzoic acid per min, E412,., = 13,600. to generally as thioredoxin/glutaredoxin proteins. Downloaded by guest on September 30, 2021 Biochemistry: Tsang and Weatherbee Proc. NatL Acad. Sci. USA 78 (1981) 7481

Table 3. Dithiothreitol and GSH-dependent PAdoPS facts that PIII is reduced by its homologous thioredoxin reduc- reductase activity tase and that it is a better substrate for reduction by dithio- PAdoPS reductase threitol than the glutathione system, it would appear that PIll activity* Relative is functionally related to E. coli thioredoxin. Whereas E. coli glutaredoxin and thioredoxin are present in a molar ratio of Type of thioredoxin/ Dithio- GSH/GR/ PAdoPS reductase HeLa ratio is =100:8. Although E. coli glutaredoxin threitol NADPH activityt 1:100 (17), the PI/PIII thioredoxin and glutaredoxin are structurally unrelated proteins None 0.04 0.01 evolved from and coded for by distinct genes, the structural 91 HeLa PI (8 pg) 10.95 10.00 relatedness among HeLa PI, PII, and E. coli thioredoxin and 1.02 1.05 102.9 HeLa PUI (1.002 mg) remains to be determined. HeLa PM (2.2 ,ug) 1.91 0.17 8.9 glutaredoxin E. coli Previously, another mammalian glutaredoxin protein from thioredoxin calfthymus (34) was identified based on its ability to couple the (1.52 pg) 5.78 1.33 23.0 glutathione-dependent ribonucleoside-diphosphate reductase E. coli reaction. Unlike HeLa PI, calfthymus glutaredoxin showed in- glutaredoxin herent activity as GSH-disulfide transhydrogenase and was not (1.92,ug) 2.03 1.87 92.1 reduced by its homologous thioredoxin reductase inthe pres- PAdoPS reductase activity was measured using dithiothreitol (1.5 ence of NADPH. At present, it is not clear whether the ob- p.tmol) or GSH (2 ,umol)/glutathione reductase (GR; 5 ,g)/NADPH (25 served qualitative and quantitative differences between HeLa pmol) as the hydrogen donor system. Incubation mixtures were (total and calfthymus thioredoxin/glutaredoxin simply reflect differ- vol, 500 Wd) Tris-HCl, pH 7.6 (25 ,&mol)/PAd&PS (30 umol; 5000-10,000 ences in specific functions between the cell types or are due to cpm/nmol)/EDTA (10 numol) containing PAdoPS reductase and re- the fact that HeLa cells are transformed. Furthermore, even doxin and hydrogen donor systems as indicated. the and glutaredoxin purified from E. coli * Expressed as nmol of PAdoPS converted per hr. though thioredoxin t GSH/dithiothreitol-dependent activity x 100. by using the coupled PAdoPS reductase assay are equivalent to those purified with the coupled ribonucleotide reductase assay, the same might not apply to other cell types. The three thioredoxin/glutaredoxin activities detected have It is interesting that HeLa PI is reminiscent ofbacteriophage similarities as well as dissimilarities to that ofE. coli thioredoxin T4 thioredoxin (27), which has mixed E. coli thioredoxin and and glutaredoxin. Unlike E. coli thioredoxin and glutaredoxin, glutaredoxin properties in that it can be reduced by the glu- which are heat stable, all three HeLa thioredoxin/glutare- tathione system as well as by the T4 thioredoxin reductase. doxin proteins are heat labile. PII, with a molecular mass of Nevertheless, although T4 thioredoxin, like E. coli glutare- >30,000 daltons, is much larger than either E. coli thioredoxin doxin, has inherent glutathione-disulfide oxidoreductase activ- or glutaredoxin. Our PII preparation has endogenous reductase ity, the same is not true for HeLa PI. activity. However, since PII is not a homogeneous preparation, In contrast to E. coli NADPH-thioredoxin reductase (36, 37), it is not clear whether the reductase activity is a contaminant a 68,000-dalton flavoprotein composed of two 34,000-dalton or an inherent component. Although an equivalent of PII has subunits that is highly species specific, HeLa NADPH-thio- not been observed in E. coli, ferralterin (35), a recently dis- redoxin reductase, like the Novikoff rat tumor enzyme (25), is covered 30,000-dalton soluble chloroplast protein from spinach most probably a 116,000-dalton flavoprotein composed of two that can replace thioredoxin and ferredoxin-thioredoxin reduc- 58,000-dalton subunits that exhibit low species and substrate tase in the light-dependent activation of chloroplast fructose specificity. The HeLa enzyme can reduce HeLa PI and PIII, 1,6-biphosphatase, might be a functionally related protein. E. coli thioredoxin and glutaredoxin, and the disulfide bond in PI and PIII, like E. coli thioredoxin and glutaredoxin, have DTNB. Although these results are generally consistent with the molecular masses of12,000 daltons. Except forbeing a substrate known wider substrate specificities of mammalian thioredoxin for its homologous thioredoxin reductase, PI is functionally sim- reductases, it should be pointed out that, in previous studies, ilar to E. coli glutaredoxin since it is reduced equally efficiently E. coli glutaredoxin was never tested as a substrate for the mam- by either dithiothreitol or the glutathione system. Based on the malian enzyme. Furthermore, our result that HeLa thioredoxin Table 4. Thioredoxin reductase-coupled PAdoPS reductase activities Relative PAdoPS reductase activity PAdoPS reductase activity* HeLa TR/ E. coli TR/ Type of thioredoxin/ Dithio- HeLa TR/ E. coli TR/ dithio- dithio- glutaredoxin used threitol NADPH NADPH threitolt threitolt None 0.04 0.01 0.01 PI (8 pig) 10.95 13.00 0.14 119 1.3 P1 (1.002 mg) 1.02 0.85* 0.54V PH (2.2 1Ag) 1.91 1.84 0.05 97 2.4 E. coli thioredoxin (3.45 pg) 11.20 11.10 14.07 99 125.0 E. coli glutaredoxin (5.65 /g) 3.45 2.18 0.41 63 12.0 PAdoPS reductase activity was measured using dithiothreitol (1.5 ,umol), HeLa thioredoxin reductase (TR; 10 pug)/NADPH (25 /Lmol), or E. coli TR (20 /Ag)/NADPH as hydrogen donor system. Incubation mixtures were as in Table 3. * Expressed as nmol of PAdoPS converted per hr. tDependent activity x 100. t PII has an inherent reductase activity. In the absence of added HeLa TR orE. coli TR, 0.5 nmol ofPAdoPS were reduced per hr in the presence of NADPH. Downloaded by guest on September 30, 2021 7482 Biochemistry: Tsang and Weatherbee Proc. Nad Acad. Sci. USA 78 (1981) reductase can reduce HeLa PI, a protein similar to E. coli glu- 13. Holmgren, A. & Morgan, F. J. (1976) Eur. J. Biochem. 70, taredoxin, is in disagreement with the finding that a glutare- 377-383. 14. Blomback, B., Blombdck, M., Finkbeiner, W., Holmgren, A., doxin-like protein in calfthymus was not a substrate for calfthy- Kowalska-Loth, B. & Olovson, G. (1974) Thromb. Res. 5, 55-75. mus thioredoxin reductase. 15. Mark, D. F. & Richardson, C. C. (1976) Proc. Nate Acad. Sci. Based on our results, it appears that the glutaredoxin system, USA 73, 780-784. like the thioredoxin system, may have an ubiquitous distribu- 16. Pigiet, V. & Conley, R. R. (1978) J. Biol Chem. 253, 1910-1920. tion and therefore may play afundamental role in all living cells. 17. Tsang, M. L.-S. (1981)J. Bacteriol. 146, 1059-1066. Nevertheless, its exact physiological role(s) has yet to be estab- 18. Gruenstein, E., Rich, A. & Weihing, R. R. (1975)J. Cell Biol 64, lished. Many of the functions proposed for thioredoxin/glu- 223-234. 19. Weatherbee, J. A., Luftig, R. B. & Weihing, R. R. (1978)J. Cell taredoxin systems, especially those involving thiol-disulfide BioL 78, 47-57. exchange reactions, overlap those previously proposed for glu- 20. Weatherbee, J. A., Luftig, R. B. & Weihing, R. R. (1980) Bio- tathione (38). Undoubtedly, many ofthese proposed glutathione chemistry 19, 4116-4123. functions could actually be mediated by thioredoxin/glutare- 21. Bradford, M. (1976) Anal Biochem. 72, 248-254. doxin systems. 22. Tsang, M. L.-S. & Schiff, J. A. (1976) Eur. J. Biochem. 75, 113-121. 23. Tsang, M. L.-S., Lemieux, J., Schiff, J. A. & Bogarski, T. B. (1976) Anal Biochem. 74, 623-626. 24. Holmgren, A. (1977) J. Biol Chem. 252, 4600-4606. We thank Dr. Robert R. Weihing for providing the HeLa cells and 25. Chen, C. C., Borns McCall, B. L. & Moore, E. C. (1977) Prep. Ms. Hedy Wald Weinerfor excellent technical assistance. This research Biochem. 7, 165-177. was supported in part by Grant PCM-78-15249 from the National Sci- 26. Hedrick, J. L. & Smith, A. J. (1968) Arch. Biochem. Biophys. ence Foundation and by Grant CA-28078 (to R. B. Luftig) from the 126, 155-164. U.S. Public Health Service. 27. Holmgren, A. (1978) J. Biol Chem. 253, 7424-7430. 28. Moore, E. C., Reichard, P. & Thelander, L. (1974)J. Biol Chem. 239, 3443-3452. 29. Porque, P. G., Baldsten, A. & Reichard, P. (1970) J. Biol Chem. 245, 2363-2370. 1. Jocelyn, P. C. (1972) Biochemistry of the SH Group (Academic, 30. Wagner, W. & Follmann, H. (1977) Biochem. Biophys. Res. Com- New York). mun. 77, 1044-1050. 2. Freedman, R. B. (1979) FEBS Lett. 97, 201-210. 31. Wolosiuk, R. A., Crawford, N. A., Yee, B. C. & Buchanan, B. B. 3. Holmgren, A. (1979) J. Biol, Chem. 254, 3664-3671. (1979) J. Biol. Chem. 254, 1627-1632. 4. Tsang, M. L.-S. & Schiff, J. A. (1976)J. Bacteriol 125, 923-933. 32. Engstrbm, N. E., Holmgren, A., Larsson, A. & S6derhdll, S. 5. Tsang, M. L.-S. & Schiff, J. A. (1978)J. Bacteriol 134, 131-138. (1974) J. Biol. Chem. 249, 205-210. 6. Porqu6, P. G. & Baldsten, A. (1970) J. Biol Chem. 245, 33. Herrmann, E. C. & Moore, E. C. (1973) J. Biol Chem. 248, 2271-2375. 1219-1223. 7. Holmgren, A. (1979)J. Biol. Chem. 254, 3672-2678. 34. Luthman, M., Ericksson, S., Holmgren, A. & Thelander, L. 8. Thelander, L. & Reichard, P. (1979) Annu. Rev. Biochem. 48, (1979) Proc. Nate Acad. Sci. USA 76, 2158-2162. 133-158. 35. Lara, C., de la Toree, A. & Buchanan, B. B. (1980) Biochem. Bio- 9. Buchanan, B. B., Wolosiuk, R. & Schflrmann, P. (1979) Trends phys. Res. Commun. 94, 1337-1364. Biochem. Sci. 4, 93-96. 36. Thelander, L. (1967)J. Biol Chem. 252, 852-859. 10. Scheibe, R. & Anderson, L. E. (1980) Proceedings, Fifth Inter- 37. Pigiet, V. P. & Conley, R. R. (1977) J. Biol. Chem. 252, national Photosynthesis Congress. 6267-6372. 11. Mills, J. D., Mitchell, P. M. & Schfirmann, P. (1980) FEBS Lett. 38. Meister, A. (1975) in Biochemistry of Glutathione in Metabolic 112, 173-177. Pathways, Metabolism ofSulfur Compounds, ed. Greenberg, D. 12. Holmgren, A. (1979)J. Biol Chem. 254, 9113-9119. M. (Academic, New York), Vol. 7, pp. 101-188. Downloaded by guest on September 30, 2021