Planta
Planta (1987) 171:32~-331
9 Springer-Verlag 1987
Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells Thomas C. Johnson, Ri Qiang Cao*, Jacob E. Kung and Bob B. Buchanan Division of Molecular Plant Biology, Hilgard Hall, University of California, Berkeley, CA 94720, U S A
Abstract. Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin s y s t e m NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-l,6-bisphosphatase (EC 3.1.3.11). NADPthioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADPthioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function. Key words: Cell culture (thioredoxin) - Daucus (thioredoxin) - NADP-thioredoxin reductase Thioredoxin.
Introduction Thioredoxins are low-molecular-weight, colorless disulfide proteins that participate in a variety of * Present address. Department of Biology, Nanjing University,
Nanjing, China D T N B = dithiolbis(2-nitrobenzoic acid) ; FBPase = fructose- 1,6-bisphosphatase; F T R = ferredoxin-thioredoxin reductase; N A D P - M D H = N A D P - m a l a t e dehydrogenase; N T R - NADP-thioredoxin reductase; SDS = sodium-dodecyl sulfate Abbreviations."
reactions by undergoing reversible oxidation and reduction (for a recent review, see Holmgren 1985). Thioredoxins are reduced enzymically with either N A D P H or reduced ferredoxin by NADP-thioredoxin reductase (NTR; EC 1.6.4.5) or ferredoxinthioredoxin reductase (FTR; no EC number), respectively. Ferredoxin-thioredoxin reductase and its associated ferredoxin and thioredoxin components constitute the ferredoxin/thioredoxin system, a system of enzyme regulation that occurs in higher plants, algae, cyanobacteria, and fermentative bacteria (Buchanan 1980, 1984; Crawford et al. 1984; Csbke and Buchanan 1986; Hammel et al. 1983; Huppe et al. 1987; Yee et al. 1981). The plant and cyanobacterial FTR is an iron sulfur protein that is composed of two dissimilar protein subunits and that reduces the two thioredoxins typically present in oxygenic photosynthetic systems, thioredoxins f and rn (Droux et al. 1987; Huppe et al. 1987; Jacquot 1984; Schfirmann 1981). These two thioredoxins can be distinguished by their ability, under certain conditions, to activate selectively chloroplast enzymes - thioredoxin f, fructose-l,6bisphosphatase (FBPase; EC 3.1.3.11), and thioredoxin m, NADP-malate dehydrogenase (NADPM D H ; EC 1.1.1.82). The NADP/thioredoxin system, consisting of N A D P H , N T R and a thioredoxin, has been extensively studied from aerobic bacteria, fungi, and animal cells (Holmgren 1985) as well as from anoxygenic photosynthetic bacteria (Johnson et al. 1984; Clement-Metral et al. 1986). In all cases, N T R has been found to be a flavoprotein composed of two identical subunits. The thioredoxin reduced by N A D P H and N T R has been shown to function in the in-vitro reduction of several proteins, but its in-vivo role as a hydrogen carrier is still under investigation (Holmgren 1985). An NADP/thiore-
322
doxin system has also been described for wheat kernels (flour) (Berstermann et al. 1983; Suske et al. 1979), soybeans (Berstermann et al. 1983), a cyanobacterium (Gleason 1986) and a green alga (Tsang 1981). However, the NADP/thioredoxin system has not been studied in higher-plant tissues other than seed, and nothing is known about the effect of greening on the abundance of its protein components, thioredoxin and NTR. We have, therefore, examined the NADP/thioredoxin system in cells capable of greening and, because of their relative structural simplicity, have selected cultured carrot cells as the experimental material. The thioredoxin characteristic of darkgrown cells, designated thioredoxin h (for heterotrophic), was identified, and purified to apparent homogeneity. NADP-thioredoxin reductase was partially purified also from dark-grown cells. Certain properties of both thioredoxin h and N T R were determined, including their response to greening of the cells. Materials and methods Growth of cells Carrot (Daucus carota L.) cells obtained from pith stock were grown as callus in Petri dishes on 1% (w/v) agar supplemented with Murashige and Skoog (1962) minimal organic medium, 3% (w/v)glucose, 10 mg.1-1 6-(3,3-methyl allyl)aminopurine (2-I-P), and I mg-1-1 1-naphthaleneacetic acid (NAA). For developmental studies, the carrot tissue was grown either under continuous white light applied from Sylvania (Danvers, Mass., USA) warm-white fluorescent lamp (F20T12-WW) [20 pmol (photons). m 2. s- 1 of 400-700 nm light] or in darkness. For growth of the larger quantities of tissue needed for preparation of N T R or thioredoxin h, cells were grown in a room with light from fluorescent lamps of about 1/20th this photon fluence rate on a rotary shaker in suspension in Murashige-Skoog minimal organic medium with 3% (w/v) sucrose and 0.1 mg-1-1 2,4-D. The suspension-grown cells, which did not green under these conditions, were harvested by vacuum filtration on Whatman (Clifton, N.J.) No. 1 filter paper and were either used fresh, or stored frozen as a cell paste at - 8 0 ~ C. Carrot leaves were purchased at a local market. Reagents, coupling enzymes, and related proteins Previously described methods were used for the preparation of corn chloroplast N A D P - M D H (Jacquot et al. 1981); spinach chloroplast FBPase (Nishizawa et al. 1982); spinach chloroplast thioredoxins m andf(Crawford et al. 1986). Spinach ferredoxin was a gift of R. K. Chain, of this Division: Escherichia coli thioredoxin was a gift of Dr. D. Le Master (Yale University, New Haven, Conn., USA) and E. eoli N T R was purchased from IMCO~ Stockholm, Sweden. Other bacterial thioredoxins were prepared in a homogeneous state as described in previous publications from this laboratory: Chromatium vinosum (Johnson et al. 1984); Clostridium pasteurianum (Hammel and Buchanan 1981) and a variation of this method for Chlorobium thiosulfatophilum (Mathews etal. 1987). Thi0redoxin h and
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells N T R from wheat flour were prepared by following the procedure of Suske et al. (1979) through the DEAE-cellulose step and then were separated and further purified by following the procedure described below for their carrot counterparts. Horseradish-peroxidase color development reagent, goat anti-rabbit immunoglobulin G (whole molecule) peroxidase conjugate, protein dye-binding reagent and sodium dodecyl sulfate (SDS), electrophoresis grade, were purchased from Bio-Rad Laboratories, Richmond, Cal., USA.) Nitrocellulose paper, 0.1 gm pore size, was purchased from Schleicher and Schuell, Keene, N.H., USA. Biochemicals (including glutathione reductase, EC 1.6.4.2, from baker's yeast, spinach, and wheat germ), Nonidet P-40 and diethylaminoethyl (DEAE)-reactive blue 2 Agarose were purchased from Sigma Chemical Co., St. Louis, Mo., U S A ; DEAE-cellulose (DE52) and carboxymethyl cellulose (CM32) from Whatman; all other column materials from Pharmacia Fine Chemicals, Piscataway, N.J., USA. Acrylamide Gen A R was purchased from Mallinckrodt, St. Louis, Mo. Murashige-Skoog minimal medium, 2-I-P, 2,4-D and N A A were purchased from Gibco Laboratories, St. Lawrence, Mass., USA. Other reagents were obtained from commercial sources and were of the highest quality available. Analytical methods Protein concentrations were routinely determined by the dyebinding assay of Bradford (1976) with bovine serum albumin as standard. Sodiumdodecylsulfate-polyacrylamide gradient slab-gel electrophoresis was performed with 10-20% (w/v) gels as described by Laemmli (1970). Protein was stained with Coomassie blue R-250 (Johnson et al. 1984). Western blotting was carried out by the procedure previously described (Johnson et al. 1984) except: (i) the transfer solution contained 25 mM tris(hydroxymethyl) aminomethane (Trizma)/150 mM glycine buffer (pH 8.3) and 20% (v/v) methanol; (ii) transfer was made by applying 30 V for 14 h toward the anode; (iii) the solution used for blocking and for incubation with antibodies was 20 mM tris(hydroxymethyl) aminomethane (Tris)-HC1 buffer (pH 7.5), 150raM NaCI and 1% (w/v) bovine serum albumin; and (iv) the buffer used for wash between antibody incubations contained 20 mM Tris-HC1 buffer (pH7.5), ] 5 0 m M NaC1, and 0.95% (v/v) Nonidet P-40. Material for electron microscopy was glutaraldehyde- and osmium-fixed, dehydrated in acetone, stained with uranyl nitrate, embedded, and post-stained with lead citrate (Spurr and Harris 1968; Spurr 1969). Specimen were examined in a 100 CX electron microscope (Peabrody, Mass., USA) as in Appelquist et al. (1968). Assays For each of the assays described below, absorbance was measured in a Beckman model D U spectrophotometer equipped with a Gilford ,automatic sample changer and recorder; temperature, 22 ~ C. (i) Thioredoxin assays. Thioredoxins were routinely assayed by measuring their capability to promote the dithiothreitol (DTT)linked activation of chloroplast target enzymes, N A D P - M D H from corn or FBPase from spinach (Crawford et al. 1986). To limit distortion.which can be caused by variation in the assay, the purification table shown below for thioredoxin was generated by saving aliquots from each treatment and assaying them in parallel in the N A D P - M D H assay. (a) Thioredoxin m plus h. Carrot thioredoxin h, plant thioredoxin m, and bacterial thioredoxins were routinely assayed by
T.C. Johnson et al. : NADP/thioredoxin System in carrot cells the chloroplast N A D P - M D H assay in which N A D P - M D H (3 gg) was preincubated for 5 min with thioredoxin fractions in 0.2 ml (final volume) of a solution containing (in ~mol): Tris-HC1 buffer (pH 7.9), 10; and DTT, 2. An aliquot of the preincubation mixture (0.1 ml) was injected into a 1-ml cuvette containing the following in 0.85 ml (in gmol): Tris-HC1 buffer (pH 7.9), 100; and N A D P H , 0.025. The reaction was started by the addition of 0.05 ml of 50 m M oxalacetic acid, and N A D P - M D H activity followed by measuring the change in absorbance at 340 nm. (b) Thioredoxinf The complete reaction mixture contained thioredoxin as needed, FBPase (16 gg), and the following in gmol: N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH buffer (pH 8.0), 50; DTT, 2.5; and MgSO4, 0.5. The mixture was preincubated for 10 rain and the reaction started by the addition of 30 gmol of fructose-l,6-bisphosphate, final volume, 0.5 ml. The reaction was stopped after 20 min by the addition of 0.5 ml 12% (w/v) trichloroacetic acid, and centrifuged (5 rain, 3000.g). A 0.5-ml aliquot was analyzed for Pi by a modified Fiske-SubbaRow procedure (Wolosiuk et al. 1980). Absorbance at 660 nm was measured after 10 min. (c) Thioredoxin h. Carrot thioredoxin h was specifically assayed by using a modification of the NADP-thioredoxin reductase assay where a partly purified carrot N T R was held constant and the thioredoxin varied (see below). (ii) NADP-thioredoxin-reductase assay. Activity of N T R was determined by a dithiolbis(2-nitrobenzoic acid) (DTNB) assay (Johnson et al. 1984). The system contained N T R as needed, thioredoxin h equivalent to 2 5 gg E. coli thioredoxin as determined in the chloroplast N A D P - M D H assay and the following (in gmol): potassium-phosphate buffer, pH7.9, 100; NaEDTA, 10; N A D P H , 0.25; DTNB, 0.02. The reaction was started by the addition of thioredoxin, final volume, 1.0 ml. Increase in absorbance was followed at 412 nm. (iii) Glutathione-reductase assay. Activity of glutathione reductase was determined by following the oxidation of N A D P H at 340 nm in the presence of oxidized glutathione. The assay mixture was the same as the one described above for N T R except that thioredoxin and D T N B were omitted and the reaction was started by the addition of 0.05 ml of 50 m M oxidized glutathione.
323 suspension was processed for 1 min at medium speed in a commercial-size Waring blender and the cells were disrupted by sonic oscillation in lots of 500 ml by subjecting them to 2 rain continuous-duty cycle at power setting 7 with a Branson Model 200 Sonifier fitted with a large tip (Branson Sonic Power Co., Danbury, Conn., USA); temperature was maintained at 5~ with a salt-ice bath. The sonic extract was clarified by centrifugation (60 min, 13 000. g) and three volumes of cold acetone ( - 2 0 ~ C) were added slowly to the supernatant fraction while stirring. The mixture was allowed to stand for 1 h at - 2 0 ~ C and the resulting precipitate was collected by centrifugation (5 min, 1500-g), suspended in 200 ml of 30 m M Tris-HC1 buffer (pH 7.9) (buffer A), and dialyzed for 3 d against 6 1 buffer A (two changes). Insolubles formed during dialysis were removed by centrifugation (20 rain, 48 000 .g), and solid ammonium sulfate was added to the supernatant solution to 60% saturation. The pH was maintained at 7.9 by titration with I N ammonium hydroxide after addition of a m m o n i u m sulfate. After stirring for 1 h, the precipitate was collected by centrifugation (20 rain, 27000-g), resuspended in 100 ml buffer A, and dialyzed for 24 h against 6 1 of buffer A. (ii) DE52 chromatography. The dialyzed 0-60% ammoniumsulfate fraction was applied to a DE52 column (32 m m diameter, 320 m m long) pre-equilibrated in buffer A. The column was eluted with 3.9 volumes of buffer A, then with 7 volumes of a linear salt gradient from 0 to 500 m M NaC1 in buffer A. Fractions of 13 ml were collected. NADP-thioredoxin reductase and thioredoxin h activities eluted together in the salt gradient. The active fractions were combined and concentrated by ultrafiltration. (iii) Sephadex G-75 superfine filtration. The concentrated fraction from the DE52 column was divided in two parts and applied separately to a Sephadex G-75 superfine column (26 m m diameter, 850 m m long) that was equilibrated and developed in buffer A containing 200 m M NaC1. The N T R and thioredoxin h activities were separated at this step, N T R eluting near the void volume and thioredoxin h in the included volume. Active fractions were pooled separately, concentrated by dialysis against solid sucrose, and then dialyzed for 24 h against 3 1 of buffer A.
Thioredoxin h Purification procedures All preparative procedures were carried out at 4 ~ C unless otherwise stated. Fast protein liquid chromatography (FPLC) on M o n o Q and Mono S columns was carried out at 20 ~ C using a Pharmacia system. Spectropore dialysis membrane (VWR Scientific, San Francisco, Cal., USA) with a 6-8 kilodalton (kDa) cut-off was used for dialysis throughout. Unless indicated otherwise, ultrafiltration was accomplished with a Ym-5 membrane (Amicon Corp., Danvers, Mass.). When indicated, protein fractions were filtered with a 0.22-gm nylon syringe filter (Rainin Instrument, Woburn, Mass.). All buffers were adjusted to the indicated pH at 20 ~ C.
Thioredoxin h and NTR from carrot tissue culture (i) Preparation of cell-free extract. Frozen carrot cells ( - 8 0 ~ C, 430 g) were thawed and added to 660 g of freshly harvested cells in order to bring the total lot to about 1 kg. The combined material was suspended in two volumes (w/v) of a breaking buffer containing 30 m M Tris-HCl buffer (pH 7.9), 0.5 m M phenylmethylsulfonyl fluoride (PMSF), 0.2 m M Na-EDTA, and 1.0% (w/v) polyvinyl poly-pyrrolidone, insoluble form. The
(iv) Fast protein liquid chromatography with Mono Q HR 5/5. The pooled thioredoxin-h fraction from step (iii) above was applied separately in equal parts to an FPLC M o n o Q H R 5/5 column (5 m m diameter, 50 m m long) equilibrated in buffer A. Protein was eluted with two volumes of buffer A, then with 27 volumes of a linear salt gradient 0-500 m M NaCI in buffer A, and lastly with three volumes of a linear salt gradient 50(~1000 m M NaC1 in buffer A (flow rate 1 m l . m i n i). Fractions of 1 ml were collected. A single peak of thioredoxin-h activity was recovered at 150 m M NaC1. The fractions showing thioredoxin-h activity were combined, and concentrated to 2 ml by ultrafiltration. (v) Sephadex G-50 superfine filtration. The concentrated M o n o Q H R 5/5 fraction was applied to a Sephadex G-50 superfine column (16 m m diameter, 900 m m long) equilibrated and developed in buffer A containing 200 m M NaC1. Fractions of 1.8 ml were collected and those showing thioredoxin-h activity were combined, and concentrated to 2 ml by ultrafiltration. The concentrated fraction was applied a second time to the Sephadex G-50 superfine column, and fractions containing thioredoxin,h activity were combined and concentrated as before. The result-
324 ing sample was judged to be homogeneous by sodiumdodecylsulfate-polyacrylamide gel electrophoresis.
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells Table 1. Purification of thioredoxin h from cultured carrot cells Step
Total activity a
Cell-free extract Acetone Ammonium-sulfate concentration (0-60%) DE52 chromatography Sephadex G-75 superfine filtration Mono Q chromatography Sephadex G-50 superfine filtration
62.1 n.d. ~ 40.1
12.0 n.d. 15.0
100 65
12.6 6.3
17.9 850
20 10
NADP-thioredoxin reductase Numerous attempts made to purify carrot NTR, in general, were unsuccessful because of low recovery of the enzyme during fractionation. The NTR was freed of interfering activities and purified about fourfold over the initial activity by subjecting the Sephadex G-75 superfine fraction from above (step iii) to three column steps: DEAE blue 2 Agarose, Mouo Q, and 2'-5' ADP-Sepharose affinity chromatography. The partially purified enzyme was stable when stored at - 2 0 ~ C, but was unstable when subjected to purification. A major reason for the loss of NTR activity during fractionation was traced to use of ultrafiltration as the method of concentration. Purified preparations of NTR had a high affinity for ultrafiltration membranes and could not be recovered following concentration. However, even after realizing this problem, it was not possible to purify NTR to homogeneity from cultured carrot cells despite repeated attempts. Thus only partially purified preparations could be used in the experiments below. Thioredoxin m from carrot leaves: Preparation of cell-free extract Thioredoxin m was partially purified from carrot leaves by following the DE52 chromatography and Sephadex G-75 superfine filtration as described in Crawford et al. (1986) except that MgClz, 2-mercaptoethanol and benzamidine-HC1 were omitted from the blending solution and the liquid-nitrogen treatment was eliminated. The thioredoxin-m fraction was free of interfering activities and could be stored at - 2 0 ~ C for several months without loss of activity. Preparation of antisera against NTR Antibodies to E. coli NTR were raised in a white female New Zealand rabbit injected with 0.15 mg enzyme that was previously emulsified in a 1 : 1 volume of Freund's complete adjuvant. Injections were subcutaneous and adjacent to the lymph nodes behind each leg. A 0.10-rag booster injection prepared in a similar manner with Freund's incomplete adjuvant was given after four weeks. Following one additional week, the rabbit was bled at weekly intervals for one month at the main ear artery after dilation by injection of droperidol-tentanyl (Tillman and Norman 1983). Approximately 30 ml of blood were collected at each bleeding; the serum was separated and stored at - 20 ~ C.
Results and discussion P u r i f i c a t i o n o f carrot t h i o r e d o x i n s T h i o r e d o x i n h. C a r r o t t h i o r e d o x i n h w a s p u r i f i e d to apparent homogeneity by the procedure outl i n e d in T a b l e I ( a c e t o n e p r e c i p i t a t i o n , a m m o n i um-sulfate fractionation, DE52 chromatography, S e p h a d e x G - 7 5 gel s u p e r f i n e f i l t r a t i o n , M o n o Q c h r o m a t o g r a p h y , a n d S e p h a d e x G - 5 0 s u p e r f i n e gel f i l t r a t i o n ) . A s d e t e r m i n e d in t h e c h l o r o p l a s t NADP-MDH assay, the procedure gave a 1 0 0 0 - f o l d p u r i f i c a t i o n a n d a y i e l d o f 0.46 m g t h i o r e d o x i n h f r o m 1090 g o f cells. T h i s y i e l d is l o w
a b c d
1.3 5.6
Specific activity (103) b
10060 12200
Recovery (%)
2 9d
gmol NADPH oxidized-min 1 lamol NADPH oxidized, min- 1. (mg protein) - 1 n.d.=not determined Represents 0.46 mg from 1090 g of cultured carrot cells
when compared with thioredoxins from bacterial s o u r c e s , s u c h as C h r o m a t i u m v i n o s u m , w h e r e 20-fold higher yields are common. A reason for t h e l o w y i e l d o f t h i o r e d o x i n h lies p e r h a p s in p a r t in t h e n a t u r e o f t h e p a r e n t tissue, w h i c h is e x t e n sively v a c u o l a t e d a n d r i c h in i n t e r f e r i n g l i p i d - l i k e materials. The latter consistently caused problems (i.e. f l o a t i n g p e l l e t s ) in i n i t i a l l y s e p a r a t i n g c e l l u l a r debris from soluble proteins by differential centrifu g a t i o n u n t i l t h e a c e t o n e - p r e c i p i t a t i o n s t e p w a s inc l u d e d (see a b o v e ) . A s seen p r e v i o u s l y w i t h C h r o m a t i u m v i n o s u m ( J o h n s o n et al. 1984), c a r r o t N T R a n d t h i o r e d o x i n copurified on DE52, but were easily separated on gel s i z i n g c o l u m n s . W i t h S e p h a d e x G - 7 5 s u p e r f i n e , NTR eluted with the bulk of the protein near the excluded volume whereas thioredoxin h eluted l a t e r , w e l l w i t h i n t h e i n c l u d e d v o l u m e ( F i g . 1). C a r rot thioredoxin h was further purified effectively b y c h r o m a t o g r a p h y o n a M o n o Q H R 5/5 c o l u m n (anion-exchange column) developed with a fast p r o t e i n l i q u i d c h r o m a t o g r a p h y s y s t e m a n d w a s fin a l l y p u r i f i e d to a p p a r e n t h o m o g e n e i t y (see b e l o w ) by serial filtration with a Sephadex G-50 superfine column. T h i o r e d o x i n m. C e l l - f r e e e x t r a c t s o f c a r r o t l e a v e s were found to require both the addition of polyvinyl poly-pyrrolidone and rapid processing to yield active thioredoxin-m preparations. When screened with the NADP-MDH assay, a single peak of thioredoxin activity that eluted with 125 m M N a C 1 w a s r e c o v e r e d f r o m t h e i n i t i a l D E 5 2 c o l u m n . T h e f r a c t i o n s c o n t a i n i n g this t h i o r e d o x i n rn a c t i v i t y w e r e a p p l i e d t o a S e p h a d e x G - 7 5 s u p e r -
T.C. Johnson et al. : NADP/thioredoxinsystemin carrot cells
12.o
I"
I
0.25 E o_ 8.0
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325
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/ ]
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z
! jl
~ 0.20
th,oredox,nh0.04
,
=-
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Froction Number Fig. 1. Separation of thioredoxin h and NTR from cultured carrot cells by Sephadex G-75 superfine filtration. Aliquots of the indicated fractions, 0.12 ml, were assayed for thioredoxin activity by measuring their ability to activate corn chloroplast NADPMDH. The NTR activitywas determined by followingthe NADPH- and thioredoxin-dependentreduction of DTNB at 412 nm. The assay contained 20 gg of E. coli thioredoxin and a 0.02-ml aliquot of the indicated fractions. Details in Materials and methods fine column which proved to be effective in separating it from the bulk of the protein and from interfering activities.
thioredoxin, which is itself low in carrot cells when compared with bacteria (Holmgren 1977; Pigiet and Conley 1977).
Purification of carrot N T R
Properties of thioredoxin h
Cell-free extracts of cultured carrot cells were found to contain high levels of NADPH-dependent DTNB reduction activity without the addition of exogenous thioredoxin h or oxidized glutathione. However, both of these hydrogen accepters markedly enhanced the reduction of DTNB, indicating the presence of both N T R and glutathione reductase. We, therefore, embarked on a study to purify and characterize this putative carrot N T R activity. While this activity was found to be caused by NTR, we observed in numerous purification attempts that the activity of the enzyme decayed in a manner approximately proportional to its extent of purification, resulting in low yields of enzyme. Partially purified preparations of carrot N T R were obtained by using acetone precipitation, ammonium-sulfate fractionation, DE52 chromatography, Sephadex G-75 superfine gel filtration, DEAE reactive blue 2 Agarose chromatography, Mono Q chromatography, and 2"-5" ADP-Sepharose affinity chromatography. The most effective step in the purification sequence was the 2'-5' ADP-Sepharose column in which N T R binds and is subsequently eluted with 5 mM NADP. As for other sources, carrot N T R was found to represent a much smaller fraction of the total protein of cultured cells than
Carrot thioredoxin h was homogeneous in gradient SDS (10 to 20%) slab polyacrylamide gel electrophoresis when purified as described above (Fig. 2). The final purification step, filtration on Sephadex G-50 superfine, removed contaminants that consistently were present earlier in the preparation. This step was also successfully used in the purification of thioredoxin from Clostridium pasteurianum (Hammel and Buchanan 1981). Filtration of the homogeneous carrot preparation on a calibrated Sephadex G-50 superfine column yielded an M, (relative molecular mass) of 10500 (Fig. 3) - a value in the range typical of thioredoxins such as those from Escherichia coli and spinach chloroplasts (Laurent et al. 1964; Schfirmann et al. 1981; Tsugita et al. 1983; Maeda et al. 1986). Gradient SDS polyacrylamide gel electrophoresis gave a similar molecular weight for carrot thioredoxin h (11000; Fig. 2) although this value must be accepted with reservation as gradient gels do not resolve low-molecular-weight proteins linearly by size (Anderson et al. 1983). Thioredoxin h from carrot was much more active in the chloroplast N A D P - M D H assay (originally devised for thioredoxin m) than in the chloroplast FBPase assay (originally devised for thiore-
326
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells Table 2. Relative effectivenessof carrot thioredoxin h in activating chloroplast FBPase, compared with other plant thioredoxins Thioredoxin b Carrot thioredoxin h Carrot thioredoxin m Spinach thioredoxin f Spinach thioredoxin m None
FBPase a 1.8 0.0 60.0 0.6 0.0
nmol Pi released.min 1 b In each case, an amount of thioredoxin showing an activity of 16 nmol NADPH oxidized-min- 1in the NADP-MDH assay was used doxin f). Based on equal activities (determined in the N A D P - M D H assay) thioredoxin f effectively activated F B P a s e whereas thioredoxin h, like thioredoxin m, did not (Table 2). Similar results were shown previously for thioredoxins from several bacteria ( J a c q u o t et al. 1979; H a m m e l and Buc h a n a n 1981) and f r o m wheat seeds (Berstermann et al. 1983).
Properties of N T R from carrot cells
Fig. 2. Photograph of gradient SDS-PAGE of thioredoxin h from cultured carrot cells. The standards (stds) in kDa were: bovine serum albumin (68), ovalbumin (43), chymotrypsinogen (25), cytochrome c (12.4), and bovine trypsin inhibitor (6.5). Carrot thioredoxin h (Thh) samples contained 5, 10, and 15 p.g of protein, from right to left
I
3.0
,-,,-
..c
I
I
,4~motrypsinogen "~oglobin
:~"~" 1.5
....
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0.7- Carrot Thioredo'xin h ~ Bovine (~0,500) -~ TrhYiPSl;r_ l 0.5I t .2
I 1.6
I 2.0
Ve/Vo Fig. 3. Molecular weight of thioredoxin h from cultured carrot cells estimated by Sephadex G-50 gel filtration
Early in this study, N T R was identified as a constituent o f c a r r o t callus g r o w n u n d e r h e t e r o t r o p h i c (dark) conditions and a partially purified preparation was obtained by using m e t h o d s described above. H o w e v e r , glutathione reductase, which typically is easily and completely separated f r o m N T R (Pigiet and Conley 1977), could be only partially eliminated f r o m our p r e p a r a t i o n s because o f the instability o f N T R . Thus, even t h o u g h the enzymes were partially separated by Sephadex G-100 superfine gel filtration, gluthathione reductase progressively b e c a m e relatively m o r e a b u n d a n t during the later purification steps in consequence o f the steady loss o f N T R activity. This was observed b o t h for p r e p a r a t i o n s f r o m c a r r o t cells and, as shown in Fig. 4, f r o m wheat seeds. The residual glutathione reductase interfered in repeated attempts to determine whether N T R c o n t r i b u t e d to the flavin present in the preparations. In other organisms, glutathione reductase is similar to N T R in that it is an N A D P - s p e c i f i c flavoprotein dimer c o m p o s e d o f two identical subunits but its subunit molecular weight is higher t h a n that o f N T R , 52000 versus 35000 ( H o l m g r e n 1981; Meister and A n d e r s o n 1983). Like its c o u n t e r p a r t f r o m o t h e r sources ( H o l m gren 1981), c a r r o t N T R was arsenite-sensitive, activity in the D T N B assay being reduced 74% by 0.5 m M sodium arsenite. T h e partially purified en-
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells
"~ I t
o EL
or) O r-
2f~Protein "~Z:
or"
327
Glutathione Reductose
,-!-I
0.100 '-o~
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0.075 .c_X oJ ",-" o "r 9o
Planta (1987) 171:32~-331
9 Springer-Verlag 1987
Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells Thomas C. Johnson, Ri Qiang Cao*, Jacob E. Kung and Bob B. Buchanan Division of Molecular Plant Biology, Hilgard Hall, University of California, Berkeley, CA 94720, U S A
Abstract. Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin s y s t e m NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-l,6-bisphosphatase (EC 3.1.3.11). NADPthioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADPthioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function. Key words: Cell culture (thioredoxin) - Daucus (thioredoxin) - NADP-thioredoxin reductase Thioredoxin.
Introduction Thioredoxins are low-molecular-weight, colorless disulfide proteins that participate in a variety of * Present address. Department of Biology, Nanjing University,
Nanjing, China D T N B = dithiolbis(2-nitrobenzoic acid) ; FBPase = fructose- 1,6-bisphosphatase; F T R = ferredoxin-thioredoxin reductase; N A D P - M D H = N A D P - m a l a t e dehydrogenase; N T R - NADP-thioredoxin reductase; SDS = sodium-dodecyl sulfate Abbreviations."
reactions by undergoing reversible oxidation and reduction (for a recent review, see Holmgren 1985). Thioredoxins are reduced enzymically with either N A D P H or reduced ferredoxin by NADP-thioredoxin reductase (NTR; EC 1.6.4.5) or ferredoxinthioredoxin reductase (FTR; no EC number), respectively. Ferredoxin-thioredoxin reductase and its associated ferredoxin and thioredoxin components constitute the ferredoxin/thioredoxin system, a system of enzyme regulation that occurs in higher plants, algae, cyanobacteria, and fermentative bacteria (Buchanan 1980, 1984; Crawford et al. 1984; Csbke and Buchanan 1986; Hammel et al. 1983; Huppe et al. 1987; Yee et al. 1981). The plant and cyanobacterial FTR is an iron sulfur protein that is composed of two dissimilar protein subunits and that reduces the two thioredoxins typically present in oxygenic photosynthetic systems, thioredoxins f and rn (Droux et al. 1987; Huppe et al. 1987; Jacquot 1984; Schfirmann 1981). These two thioredoxins can be distinguished by their ability, under certain conditions, to activate selectively chloroplast enzymes - thioredoxin f, fructose-l,6bisphosphatase (FBPase; EC 3.1.3.11), and thioredoxin m, NADP-malate dehydrogenase (NADPM D H ; EC 1.1.1.82). The NADP/thioredoxin system, consisting of N A D P H , N T R and a thioredoxin, has been extensively studied from aerobic bacteria, fungi, and animal cells (Holmgren 1985) as well as from anoxygenic photosynthetic bacteria (Johnson et al. 1984; Clement-Metral et al. 1986). In all cases, N T R has been found to be a flavoprotein composed of two identical subunits. The thioredoxin reduced by N A D P H and N T R has been shown to function in the in-vitro reduction of several proteins, but its in-vivo role as a hydrogen carrier is still under investigation (Holmgren 1985). An NADP/thiore-
322
doxin system has also been described for wheat kernels (flour) (Berstermann et al. 1983; Suske et al. 1979), soybeans (Berstermann et al. 1983), a cyanobacterium (Gleason 1986) and a green alga (Tsang 1981). However, the NADP/thioredoxin system has not been studied in higher-plant tissues other than seed, and nothing is known about the effect of greening on the abundance of its protein components, thioredoxin and NTR. We have, therefore, examined the NADP/thioredoxin system in cells capable of greening and, because of their relative structural simplicity, have selected cultured carrot cells as the experimental material. The thioredoxin characteristic of darkgrown cells, designated thioredoxin h (for heterotrophic), was identified, and purified to apparent homogeneity. NADP-thioredoxin reductase was partially purified also from dark-grown cells. Certain properties of both thioredoxin h and N T R were determined, including their response to greening of the cells. Materials and methods Growth of cells Carrot (Daucus carota L.) cells obtained from pith stock were grown as callus in Petri dishes on 1% (w/v) agar supplemented with Murashige and Skoog (1962) minimal organic medium, 3% (w/v)glucose, 10 mg.1-1 6-(3,3-methyl allyl)aminopurine (2-I-P), and I mg-1-1 1-naphthaleneacetic acid (NAA). For developmental studies, the carrot tissue was grown either under continuous white light applied from Sylvania (Danvers, Mass., USA) warm-white fluorescent lamp (F20T12-WW) [20 pmol (photons). m 2. s- 1 of 400-700 nm light] or in darkness. For growth of the larger quantities of tissue needed for preparation of N T R or thioredoxin h, cells were grown in a room with light from fluorescent lamps of about 1/20th this photon fluence rate on a rotary shaker in suspension in Murashige-Skoog minimal organic medium with 3% (w/v) sucrose and 0.1 mg-1-1 2,4-D. The suspension-grown cells, which did not green under these conditions, were harvested by vacuum filtration on Whatman (Clifton, N.J.) No. 1 filter paper and were either used fresh, or stored frozen as a cell paste at - 8 0 ~ C. Carrot leaves were purchased at a local market. Reagents, coupling enzymes, and related proteins Previously described methods were used for the preparation of corn chloroplast N A D P - M D H (Jacquot et al. 1981); spinach chloroplast FBPase (Nishizawa et al. 1982); spinach chloroplast thioredoxins m andf(Crawford et al. 1986). Spinach ferredoxin was a gift of R. K. Chain, of this Division: Escherichia coli thioredoxin was a gift of Dr. D. Le Master (Yale University, New Haven, Conn., USA) and E. eoli N T R was purchased from IMCO~ Stockholm, Sweden. Other bacterial thioredoxins were prepared in a homogeneous state as described in previous publications from this laboratory: Chromatium vinosum (Johnson et al. 1984); Clostridium pasteurianum (Hammel and Buchanan 1981) and a variation of this method for Chlorobium thiosulfatophilum (Mathews etal. 1987). Thi0redoxin h and
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells N T R from wheat flour were prepared by following the procedure of Suske et al. (1979) through the DEAE-cellulose step and then were separated and further purified by following the procedure described below for their carrot counterparts. Horseradish-peroxidase color development reagent, goat anti-rabbit immunoglobulin G (whole molecule) peroxidase conjugate, protein dye-binding reagent and sodium dodecyl sulfate (SDS), electrophoresis grade, were purchased from Bio-Rad Laboratories, Richmond, Cal., USA.) Nitrocellulose paper, 0.1 gm pore size, was purchased from Schleicher and Schuell, Keene, N.H., USA. Biochemicals (including glutathione reductase, EC 1.6.4.2, from baker's yeast, spinach, and wheat germ), Nonidet P-40 and diethylaminoethyl (DEAE)-reactive blue 2 Agarose were purchased from Sigma Chemical Co., St. Louis, Mo., U S A ; DEAE-cellulose (DE52) and carboxymethyl cellulose (CM32) from Whatman; all other column materials from Pharmacia Fine Chemicals, Piscataway, N.J., USA. Acrylamide Gen A R was purchased from Mallinckrodt, St. Louis, Mo. Murashige-Skoog minimal medium, 2-I-P, 2,4-D and N A A were purchased from Gibco Laboratories, St. Lawrence, Mass., USA. Other reagents were obtained from commercial sources and were of the highest quality available. Analytical methods Protein concentrations were routinely determined by the dyebinding assay of Bradford (1976) with bovine serum albumin as standard. Sodiumdodecylsulfate-polyacrylamide gradient slab-gel electrophoresis was performed with 10-20% (w/v) gels as described by Laemmli (1970). Protein was stained with Coomassie blue R-250 (Johnson et al. 1984). Western blotting was carried out by the procedure previously described (Johnson et al. 1984) except: (i) the transfer solution contained 25 mM tris(hydroxymethyl) aminomethane (Trizma)/150 mM glycine buffer (pH 8.3) and 20% (v/v) methanol; (ii) transfer was made by applying 30 V for 14 h toward the anode; (iii) the solution used for blocking and for incubation with antibodies was 20 mM tris(hydroxymethyl) aminomethane (Tris)-HC1 buffer (pH 7.5), 150raM NaCI and 1% (w/v) bovine serum albumin; and (iv) the buffer used for wash between antibody incubations contained 20 mM Tris-HC1 buffer (pH7.5), ] 5 0 m M NaC1, and 0.95% (v/v) Nonidet P-40. Material for electron microscopy was glutaraldehyde- and osmium-fixed, dehydrated in acetone, stained with uranyl nitrate, embedded, and post-stained with lead citrate (Spurr and Harris 1968; Spurr 1969). Specimen were examined in a 100 CX electron microscope (Peabrody, Mass., USA) as in Appelquist et al. (1968). Assays For each of the assays described below, absorbance was measured in a Beckman model D U spectrophotometer equipped with a Gilford ,automatic sample changer and recorder; temperature, 22 ~ C. (i) Thioredoxin assays. Thioredoxins were routinely assayed by measuring their capability to promote the dithiothreitol (DTT)linked activation of chloroplast target enzymes, N A D P - M D H from corn or FBPase from spinach (Crawford et al. 1986). To limit distortion.which can be caused by variation in the assay, the purification table shown below for thioredoxin was generated by saving aliquots from each treatment and assaying them in parallel in the N A D P - M D H assay. (a) Thioredoxin m plus h. Carrot thioredoxin h, plant thioredoxin m, and bacterial thioredoxins were routinely assayed by
T.C. Johnson et al. : NADP/thioredoxin System in carrot cells the chloroplast N A D P - M D H assay in which N A D P - M D H (3 gg) was preincubated for 5 min with thioredoxin fractions in 0.2 ml (final volume) of a solution containing (in ~mol): Tris-HC1 buffer (pH 7.9), 10; and DTT, 2. An aliquot of the preincubation mixture (0.1 ml) was injected into a 1-ml cuvette containing the following in 0.85 ml (in gmol): Tris-HC1 buffer (pH 7.9), 100; and N A D P H , 0.025. The reaction was started by the addition of 0.05 ml of 50 m M oxalacetic acid, and N A D P - M D H activity followed by measuring the change in absorbance at 340 nm. (b) Thioredoxinf The complete reaction mixture contained thioredoxin as needed, FBPase (16 gg), and the following in gmol: N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH buffer (pH 8.0), 50; DTT, 2.5; and MgSO4, 0.5. The mixture was preincubated for 10 rain and the reaction started by the addition of 30 gmol of fructose-l,6-bisphosphate, final volume, 0.5 ml. The reaction was stopped after 20 min by the addition of 0.5 ml 12% (w/v) trichloroacetic acid, and centrifuged (5 rain, 3000.g). A 0.5-ml aliquot was analyzed for Pi by a modified Fiske-SubbaRow procedure (Wolosiuk et al. 1980). Absorbance at 660 nm was measured after 10 min. (c) Thioredoxin h. Carrot thioredoxin h was specifically assayed by using a modification of the NADP-thioredoxin reductase assay where a partly purified carrot N T R was held constant and the thioredoxin varied (see below). (ii) NADP-thioredoxin-reductase assay. Activity of N T R was determined by a dithiolbis(2-nitrobenzoic acid) (DTNB) assay (Johnson et al. 1984). The system contained N T R as needed, thioredoxin h equivalent to 2 5 gg E. coli thioredoxin as determined in the chloroplast N A D P - M D H assay and the following (in gmol): potassium-phosphate buffer, pH7.9, 100; NaEDTA, 10; N A D P H , 0.25; DTNB, 0.02. The reaction was started by the addition of thioredoxin, final volume, 1.0 ml. Increase in absorbance was followed at 412 nm. (iii) Glutathione-reductase assay. Activity of glutathione reductase was determined by following the oxidation of N A D P H at 340 nm in the presence of oxidized glutathione. The assay mixture was the same as the one described above for N T R except that thioredoxin and D T N B were omitted and the reaction was started by the addition of 0.05 ml of 50 m M oxidized glutathione.
323 suspension was processed for 1 min at medium speed in a commercial-size Waring blender and the cells were disrupted by sonic oscillation in lots of 500 ml by subjecting them to 2 rain continuous-duty cycle at power setting 7 with a Branson Model 200 Sonifier fitted with a large tip (Branson Sonic Power Co., Danbury, Conn., USA); temperature was maintained at 5~ with a salt-ice bath. The sonic extract was clarified by centrifugation (60 min, 13 000. g) and three volumes of cold acetone ( - 2 0 ~ C) were added slowly to the supernatant fraction while stirring. The mixture was allowed to stand for 1 h at - 2 0 ~ C and the resulting precipitate was collected by centrifugation (5 min, 1500-g), suspended in 200 ml of 30 m M Tris-HC1 buffer (pH 7.9) (buffer A), and dialyzed for 3 d against 6 1 buffer A (two changes). Insolubles formed during dialysis were removed by centrifugation (20 rain, 48 000 .g), and solid ammonium sulfate was added to the supernatant solution to 60% saturation. The pH was maintained at 7.9 by titration with I N ammonium hydroxide after addition of a m m o n i u m sulfate. After stirring for 1 h, the precipitate was collected by centrifugation (20 rain, 27000-g), resuspended in 100 ml buffer A, and dialyzed for 24 h against 6 1 of buffer A. (ii) DE52 chromatography. The dialyzed 0-60% ammoniumsulfate fraction was applied to a DE52 column (32 m m diameter, 320 m m long) pre-equilibrated in buffer A. The column was eluted with 3.9 volumes of buffer A, then with 7 volumes of a linear salt gradient from 0 to 500 m M NaC1 in buffer A. Fractions of 13 ml were collected. NADP-thioredoxin reductase and thioredoxin h activities eluted together in the salt gradient. The active fractions were combined and concentrated by ultrafiltration. (iii) Sephadex G-75 superfine filtration. The concentrated fraction from the DE52 column was divided in two parts and applied separately to a Sephadex G-75 superfine column (26 m m diameter, 850 m m long) that was equilibrated and developed in buffer A containing 200 m M NaC1. The N T R and thioredoxin h activities were separated at this step, N T R eluting near the void volume and thioredoxin h in the included volume. Active fractions were pooled separately, concentrated by dialysis against solid sucrose, and then dialyzed for 24 h against 3 1 of buffer A.
Thioredoxin h Purification procedures All preparative procedures were carried out at 4 ~ C unless otherwise stated. Fast protein liquid chromatography (FPLC) on M o n o Q and Mono S columns was carried out at 20 ~ C using a Pharmacia system. Spectropore dialysis membrane (VWR Scientific, San Francisco, Cal., USA) with a 6-8 kilodalton (kDa) cut-off was used for dialysis throughout. Unless indicated otherwise, ultrafiltration was accomplished with a Ym-5 membrane (Amicon Corp., Danvers, Mass.). When indicated, protein fractions were filtered with a 0.22-gm nylon syringe filter (Rainin Instrument, Woburn, Mass.). All buffers were adjusted to the indicated pH at 20 ~ C.
Thioredoxin h and NTR from carrot tissue culture (i) Preparation of cell-free extract. Frozen carrot cells ( - 8 0 ~ C, 430 g) were thawed and added to 660 g of freshly harvested cells in order to bring the total lot to about 1 kg. The combined material was suspended in two volumes (w/v) of a breaking buffer containing 30 m M Tris-HCl buffer (pH 7.9), 0.5 m M phenylmethylsulfonyl fluoride (PMSF), 0.2 m M Na-EDTA, and 1.0% (w/v) polyvinyl poly-pyrrolidone, insoluble form. The
(iv) Fast protein liquid chromatography with Mono Q HR 5/5. The pooled thioredoxin-h fraction from step (iii) above was applied separately in equal parts to an FPLC M o n o Q H R 5/5 column (5 m m diameter, 50 m m long) equilibrated in buffer A. Protein was eluted with two volumes of buffer A, then with 27 volumes of a linear salt gradient 0-500 m M NaCI in buffer A, and lastly with three volumes of a linear salt gradient 50(~1000 m M NaC1 in buffer A (flow rate 1 m l . m i n i). Fractions of 1 ml were collected. A single peak of thioredoxin-h activity was recovered at 150 m M NaC1. The fractions showing thioredoxin-h activity were combined, and concentrated to 2 ml by ultrafiltration. (v) Sephadex G-50 superfine filtration. The concentrated M o n o Q H R 5/5 fraction was applied to a Sephadex G-50 superfine column (16 m m diameter, 900 m m long) equilibrated and developed in buffer A containing 200 m M NaC1. Fractions of 1.8 ml were collected and those showing thioredoxin-h activity were combined, and concentrated to 2 ml by ultrafiltration. The concentrated fraction was applied a second time to the Sephadex G-50 superfine column, and fractions containing thioredoxin,h activity were combined and concentrated as before. The result-
324 ing sample was judged to be homogeneous by sodiumdodecylsulfate-polyacrylamide gel electrophoresis.
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells Table 1. Purification of thioredoxin h from cultured carrot cells Step
Total activity a
Cell-free extract Acetone Ammonium-sulfate concentration (0-60%) DE52 chromatography Sephadex G-75 superfine filtration Mono Q chromatography Sephadex G-50 superfine filtration
62.1 n.d. ~ 40.1
12.0 n.d. 15.0
100 65
12.6 6.3
17.9 850
20 10
NADP-thioredoxin reductase Numerous attempts made to purify carrot NTR, in general, were unsuccessful because of low recovery of the enzyme during fractionation. The NTR was freed of interfering activities and purified about fourfold over the initial activity by subjecting the Sephadex G-75 superfine fraction from above (step iii) to three column steps: DEAE blue 2 Agarose, Mouo Q, and 2'-5' ADP-Sepharose affinity chromatography. The partially purified enzyme was stable when stored at - 2 0 ~ C, but was unstable when subjected to purification. A major reason for the loss of NTR activity during fractionation was traced to use of ultrafiltration as the method of concentration. Purified preparations of NTR had a high affinity for ultrafiltration membranes and could not be recovered following concentration. However, even after realizing this problem, it was not possible to purify NTR to homogeneity from cultured carrot cells despite repeated attempts. Thus only partially purified preparations could be used in the experiments below. Thioredoxin m from carrot leaves: Preparation of cell-free extract Thioredoxin m was partially purified from carrot leaves by following the DE52 chromatography and Sephadex G-75 superfine filtration as described in Crawford et al. (1986) except that MgClz, 2-mercaptoethanol and benzamidine-HC1 were omitted from the blending solution and the liquid-nitrogen treatment was eliminated. The thioredoxin-m fraction was free of interfering activities and could be stored at - 2 0 ~ C for several months without loss of activity. Preparation of antisera against NTR Antibodies to E. coli NTR were raised in a white female New Zealand rabbit injected with 0.15 mg enzyme that was previously emulsified in a 1 : 1 volume of Freund's complete adjuvant. Injections were subcutaneous and adjacent to the lymph nodes behind each leg. A 0.10-rag booster injection prepared in a similar manner with Freund's incomplete adjuvant was given after four weeks. Following one additional week, the rabbit was bled at weekly intervals for one month at the main ear artery after dilation by injection of droperidol-tentanyl (Tillman and Norman 1983). Approximately 30 ml of blood were collected at each bleeding; the serum was separated and stored at - 20 ~ C.
Results and discussion P u r i f i c a t i o n o f carrot t h i o r e d o x i n s T h i o r e d o x i n h. C a r r o t t h i o r e d o x i n h w a s p u r i f i e d to apparent homogeneity by the procedure outl i n e d in T a b l e I ( a c e t o n e p r e c i p i t a t i o n , a m m o n i um-sulfate fractionation, DE52 chromatography, S e p h a d e x G - 7 5 gel s u p e r f i n e f i l t r a t i o n , M o n o Q c h r o m a t o g r a p h y , a n d S e p h a d e x G - 5 0 s u p e r f i n e gel f i l t r a t i o n ) . A s d e t e r m i n e d in t h e c h l o r o p l a s t NADP-MDH assay, the procedure gave a 1 0 0 0 - f o l d p u r i f i c a t i o n a n d a y i e l d o f 0.46 m g t h i o r e d o x i n h f r o m 1090 g o f cells. T h i s y i e l d is l o w
a b c d
1.3 5.6
Specific activity (103) b
10060 12200
Recovery (%)
2 9d
gmol NADPH oxidized-min 1 lamol NADPH oxidized, min- 1. (mg protein) - 1 n.d.=not determined Represents 0.46 mg from 1090 g of cultured carrot cells
when compared with thioredoxins from bacterial s o u r c e s , s u c h as C h r o m a t i u m v i n o s u m , w h e r e 20-fold higher yields are common. A reason for t h e l o w y i e l d o f t h i o r e d o x i n h lies p e r h a p s in p a r t in t h e n a t u r e o f t h e p a r e n t tissue, w h i c h is e x t e n sively v a c u o l a t e d a n d r i c h in i n t e r f e r i n g l i p i d - l i k e materials. The latter consistently caused problems (i.e. f l o a t i n g p e l l e t s ) in i n i t i a l l y s e p a r a t i n g c e l l u l a r debris from soluble proteins by differential centrifu g a t i o n u n t i l t h e a c e t o n e - p r e c i p i t a t i o n s t e p w a s inc l u d e d (see a b o v e ) . A s seen p r e v i o u s l y w i t h C h r o m a t i u m v i n o s u m ( J o h n s o n et al. 1984), c a r r o t N T R a n d t h i o r e d o x i n copurified on DE52, but were easily separated on gel s i z i n g c o l u m n s . W i t h S e p h a d e x G - 7 5 s u p e r f i n e , NTR eluted with the bulk of the protein near the excluded volume whereas thioredoxin h eluted l a t e r , w e l l w i t h i n t h e i n c l u d e d v o l u m e ( F i g . 1). C a r rot thioredoxin h was further purified effectively b y c h r o m a t o g r a p h y o n a M o n o Q H R 5/5 c o l u m n (anion-exchange column) developed with a fast p r o t e i n l i q u i d c h r o m a t o g r a p h y s y s t e m a n d w a s fin a l l y p u r i f i e d to a p p a r e n t h o m o g e n e i t y (see b e l o w ) by serial filtration with a Sephadex G-50 superfine column. T h i o r e d o x i n m. C e l l - f r e e e x t r a c t s o f c a r r o t l e a v e s were found to require both the addition of polyvinyl poly-pyrrolidone and rapid processing to yield active thioredoxin-m preparations. When screened with the NADP-MDH assay, a single peak of thioredoxin activity that eluted with 125 m M N a C 1 w a s r e c o v e r e d f r o m t h e i n i t i a l D E 5 2 c o l u m n . T h e f r a c t i o n s c o n t a i n i n g this t h i o r e d o x i n rn a c t i v i t y w e r e a p p l i e d t o a S e p h a d e x G - 7 5 s u p e r -
T.C. Johnson et al. : NADP/thioredoxinsystemin carrot cells
12.o
I"
I
0.25 E o_ 8.0
I
325
I
I
Protein .~
I
/ ]
NTR
O'~-
z
! jl
~ 0.20
th,oredox,nh0.04
,
=-
= o -
o.la 0.02 I'-- .":--
0
~
0
10
3o
i
50
7o
9o
0
Froction Number Fig. 1. Separation of thioredoxin h and NTR from cultured carrot cells by Sephadex G-75 superfine filtration. Aliquots of the indicated fractions, 0.12 ml, were assayed for thioredoxin activity by measuring their ability to activate corn chloroplast NADPMDH. The NTR activitywas determined by followingthe NADPH- and thioredoxin-dependentreduction of DTNB at 412 nm. The assay contained 20 gg of E. coli thioredoxin and a 0.02-ml aliquot of the indicated fractions. Details in Materials and methods fine column which proved to be effective in separating it from the bulk of the protein and from interfering activities.
thioredoxin, which is itself low in carrot cells when compared with bacteria (Holmgren 1977; Pigiet and Conley 1977).
Purification of carrot N T R
Properties of thioredoxin h
Cell-free extracts of cultured carrot cells were found to contain high levels of NADPH-dependent DTNB reduction activity without the addition of exogenous thioredoxin h or oxidized glutathione. However, both of these hydrogen accepters markedly enhanced the reduction of DTNB, indicating the presence of both N T R and glutathione reductase. We, therefore, embarked on a study to purify and characterize this putative carrot N T R activity. While this activity was found to be caused by NTR, we observed in numerous purification attempts that the activity of the enzyme decayed in a manner approximately proportional to its extent of purification, resulting in low yields of enzyme. Partially purified preparations of carrot N T R were obtained by using acetone precipitation, ammonium-sulfate fractionation, DE52 chromatography, Sephadex G-75 superfine gel filtration, DEAE reactive blue 2 Agarose chromatography, Mono Q chromatography, and 2"-5" ADP-Sepharose affinity chromatography. The most effective step in the purification sequence was the 2'-5' ADP-Sepharose column in which N T R binds and is subsequently eluted with 5 mM NADP. As for other sources, carrot N T R was found to represent a much smaller fraction of the total protein of cultured cells than
Carrot thioredoxin h was homogeneous in gradient SDS (10 to 20%) slab polyacrylamide gel electrophoresis when purified as described above (Fig. 2). The final purification step, filtration on Sephadex G-50 superfine, removed contaminants that consistently were present earlier in the preparation. This step was also successfully used in the purification of thioredoxin from Clostridium pasteurianum (Hammel and Buchanan 1981). Filtration of the homogeneous carrot preparation on a calibrated Sephadex G-50 superfine column yielded an M, (relative molecular mass) of 10500 (Fig. 3) - a value in the range typical of thioredoxins such as those from Escherichia coli and spinach chloroplasts (Laurent et al. 1964; Schfirmann et al. 1981; Tsugita et al. 1983; Maeda et al. 1986). Gradient SDS polyacrylamide gel electrophoresis gave a similar molecular weight for carrot thioredoxin h (11000; Fig. 2) although this value must be accepted with reservation as gradient gels do not resolve low-molecular-weight proteins linearly by size (Anderson et al. 1983). Thioredoxin h from carrot was much more active in the chloroplast N A D P - M D H assay (originally devised for thioredoxin m) than in the chloroplast FBPase assay (originally devised for thiore-
326
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells Table 2. Relative effectivenessof carrot thioredoxin h in activating chloroplast FBPase, compared with other plant thioredoxins Thioredoxin b Carrot thioredoxin h Carrot thioredoxin m Spinach thioredoxin f Spinach thioredoxin m None
FBPase a 1.8 0.0 60.0 0.6 0.0
nmol Pi released.min 1 b In each case, an amount of thioredoxin showing an activity of 16 nmol NADPH oxidized-min- 1in the NADP-MDH assay was used doxin f). Based on equal activities (determined in the N A D P - M D H assay) thioredoxin f effectively activated F B P a s e whereas thioredoxin h, like thioredoxin m, did not (Table 2). Similar results were shown previously for thioredoxins from several bacteria ( J a c q u o t et al. 1979; H a m m e l and Buc h a n a n 1981) and f r o m wheat seeds (Berstermann et al. 1983).
Properties of N T R from carrot cells
Fig. 2. Photograph of gradient SDS-PAGE of thioredoxin h from cultured carrot cells. The standards (stds) in kDa were: bovine serum albumin (68), ovalbumin (43), chymotrypsinogen (25), cytochrome c (12.4), and bovine trypsin inhibitor (6.5). Carrot thioredoxin h (Thh) samples contained 5, 10, and 15 p.g of protein, from right to left
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Early in this study, N T R was identified as a constituent o f c a r r o t callus g r o w n u n d e r h e t e r o t r o p h i c (dark) conditions and a partially purified preparation was obtained by using m e t h o d s described above. H o w e v e r , glutathione reductase, which typically is easily and completely separated f r o m N T R (Pigiet and Conley 1977), could be only partially eliminated f r o m our p r e p a r a t i o n s because o f the instability o f N T R . Thus, even t h o u g h the enzymes were partially separated by Sephadex G-100 superfine gel filtration, gluthathione reductase progressively b e c a m e relatively m o r e a b u n d a n t during the later purification steps in consequence o f the steady loss o f N T R activity. This was observed b o t h for p r e p a r a t i o n s f r o m c a r r o t cells and, as shown in Fig. 4, f r o m wheat seeds. The residual glutathione reductase interfered in repeated attempts to determine whether N T R c o n t r i b u t e d to the flavin present in the preparations. In other organisms, glutathione reductase is similar to N T R in that it is an N A D P - s p e c i f i c flavoprotein dimer c o m p o s e d o f two identical subunits but its subunit molecular weight is higher t h a n that o f N T R , 52000 versus 35000 ( H o l m g r e n 1981; Meister and A n d e r s o n 1983). Like its c o u n t e r p a r t f r o m o t h e r sources ( H o l m gren 1981), c a r r o t N T R was arsenite-sensitive, activity in the D T N B assay being reduced 74% by 0.5 m M sodium arsenite. T h e partially purified en-
T.C. Johnson et al. : NADP/thioredoxin system in carrot cells
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