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 thiore- doxin-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 micro- tubule 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, thio- redoxin 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