Biochimica et Biophysica Acta 873 (1986) 415-418 415 Elsevier

BBA30156 BBA Report

Characterizat ion of three di f ferent th ioredox ins in wheat

K l a u s V o g t a n d H a r t m u t F o l l m a n n

Fachbereich Chemie der Philipps-Universitlit, Biochemie, Hans-Meerwein-Strasse, D-3550 Marburg (F..R.G.)

(Received 1 July 1986)

Key words: Thioredoxin; Isoprotein; Amino acid composition; Enzyme activation; (Wheat seed)

Heat-stable protein extracts from diploid, tetraploid and hexaploid wheat seeds (Triticum monococcum, T. durum, T. aestivum) all contain three different thioredoxins of molecular weight 12000-12500. This distribution is comparable to the thioredoxin patterns found in soybean seeds and in chloroplast-free green algae. Non-photosynthetic plant cells thus appear to have a much more uniform thioredoxin content than green plants. The newly characterized wheat seed protein, thioredoxin III, is a cysteine-rich polypeptide of unusual composition and apparently unelated to isothioredoxins I and II, but is still capable of stimulating thioredoxin-activated enzymes and crossreacting with antibodies against E. coil thioredoxin.

The distribution of thioredoxins in plant cells is very complex. Several such dicysteine polypep- tides are located in the chloroplast and others appear to be of cytoplasmic origin [1-6]. The total number is hard to establish because thioredoxins lack endogeneous activity, necessitating the use of various indicator enzymes, and because some iso- thioredoxins in the 11-12 kDa category are dif- ficult to separate from each other. It is also un- known whether all plants have the same thio- redoxi n pattern. This situation is unsatisfactory in view of the physiological significance of thioredo- xins, which participate, inter alia, in de- oxyribonucleotide biosynthesis, light regulation of chloroplast enzymes, and assimilatory sulfate re- duction [7].

To simplify the approach to plant thioredoxins we have focussed our attention on non-photosyn- thetic tissues and have recently characterized three different thioredoxins of M r 12000 (designated I, II, III) in soybean (Glycine max) seeds and roots

Correspondence address: Dr. H. Follmann, Fachbereich Chemie der Philipps-Universit~it, Biochemie, Hans-Meerwein- Strasse, D-3550 Marburg, F.R.G.

[8] and in a chloroplast-free mutant of the unicell- ular green algae, Scenedesmus obliquus [4]; in con- trast, only two such polypeptides could be dif- ferentiated in wheat (Triticum aestioum) [8]. To verify whether this is a speciality of the mono- cotyledon, or of the allohexaploid cultivated plant, we have reinvestigated the thioredoxin content of wheat, including the diploid and tetraploid species. Herein we demonstrate that they all contain three thioredoxins, one of which had previously escaped detection.

Seeds of the diploid (T. monococcum, 'Einkorn'), tetraploid (T. durum, durum wheat) and hexaploid (T. aestivum, common bread wheat) species were kindly supplied by Institut fiir Pflanzenbau, Freis- ing-Weihenstephan, and Bundesanstalt fiir Ge- treideverarbeitung, Detmold, F.R.G. The test en- zymes, NADP-malate dehydrogenase from spinach leaves, fructose-bisphosphatase from wheat, and ribonucleotide reductase of E. coli, were purified and assayed by published procedures [9-11]. The thioredoxin reductase assay with 5,5'-dithiobis(2- nitrobenzoate) has been described [12]. Analytical and preparative electrophoresis on SDS-contain- ing 15% polyacrylamide gels were also performed

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

416

as previously [8]. Amino acid analyses were done on a Biotronik LC 7000 system after performic acid oxidation of the cysteine residues; E. coli thioredoxin served as reference. Antibodies against E. coli thioredoxin coupled to Sepharose [13] and a sample of glutaredoxin were generous gifts from Dr. A. Holmgren, Karolinska Institutet, Stock- holm.

All preparative work was carried out at 0-5 ° C. Thioredoxins were extracted from 300 g batches of finely ground wheat flour in I 1 of 50 mM Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA. In mod- ification of the published procedure [8] the protein extract was first subjected to (NH4)2SO4 precipi- tation at 50-90% saturation, and the sample was redissolved and subjected to heat treatment (5 min at 80°C). The supernatant was fractionated on CM cellulose in 0.02 M sodium acetate buffer, pH 4.60, and thioredoxins were eluted with 0.05-0.25 M NaC1 gradient (Fig. 1). Introduction of this initial chromatography step at acidic pH greatly

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0 I I I I [ I I I I

0 IO 20 30 40 f r a c t i o n

Fig. 1. Chromatography of heat-stable protein extracts from wheat flour on columns of CM-cellulose (Whatman CM-52; 2 .8×6.5 cm; flow rate, 50 m l . h -1) equilibrated in 20 mM sodium acetate buffer, pH 4.60. The arrow indicates the start of a 50 to 250 mM NaCl gradient. Closed lines and symbols: common bread wheat (T. aestivum); dashed lines and oF.=n symbols: one-grained wheat (T. monococcum). The slight dis- placement of activity profiles is within the reproducibility limits of chromatography runs. T. durum extracts show the same chromatographic behaviour. Thioredoxin activity (e, O) was measured as stimulation of spinach NADP-mala te dehy- drogenase and is expressed as the factor of rate increase over thioredoxin-free enzyme assays.

improved the isolation of thioredoxins from the highly viscous wheat extracts. Proteins I and II cochromatographed under these conditions and could then be purified further by chromatography on DEAE-cellulose, CM-cellulose and Sephadex G-75 as previously described [8]. An additional fraction of enzyme-stimulatory activity eluted from the first CM-cellulose column at higher ionic strength (fraction III) and by the criteria discussed below this new heat-stable protein was also desig- nated a thioredoxin. Thioredoxin III was purified separately by rechromatography on CM-cellulose, gel filtration on Sephadex G-75, and by chro- matography on hydroxyapatite in a 50 to 500 mM Tris-HC1 buffer gradient, pH 7.80. Hydroxy- apatite did not bind thioredoxins I and II. The latter step provided apparently homogeneous pro- tein III which showed only one band on SDS- polyacrylamide electrophoresis gels (Fig. 2). The yield was 0.7 mg /kg dry wheat. The three wheat thioredoxins were also chromatographed on other media such as Blue Sepharose and alkyl agaroses but in no case could the activity peaks be resolved any further.

Thioredoxin III shares the following properties with all other regular thioredoxins of bacterial and eucaryotic origin: a molecular weight of 12000- 12500, as established in Fig. 2; strong binding occurred on a column of immobilized antibodies against E. coli thioredoxin (not shown); the pro- tein served as substrate for the endogenous NADPH-dependent thioredoxin reductase from wheat; and, in the reduced form, it was capable of stimulating three different thioredoxin-activatable enzymes (Table I). Protein III is not a glutare- doxin [16] because it failed to function in com- bination with E. coli ribonucleotide reductase, glutathione reductase, NADPH, and glutathione in place of dithiothreitol. It is not one of the more heterogeneous group of f-type thioredoxins [1-4] because leaf fructose-bisphosphatase is not specif- ically stimulated. The figures in Table I do not permit assignation of a biochemical function be- cause the few test enzymes cannot represent all conceivable thioredoxin-dependent processes; considering the lower activity of thioredoxin III towards these typical enzymes, its physiological target in seed metabolism or germination may rather be altogether unknown.

U QIimmm

D

Q

Q

1 2 3 4 5 6 Fig. 2. SDS-polyacrylamide gel electrophoresis of wheat (7". aestivum) thioredoxin preparations. Lane 1, fractions 17-23; lane 2, fractions 24-34 from CM-cellulose chromatography (Fig. 1); lanes 3 and 4, thioredoxins I and 11, separated and further purified by column chromatography and preparative electrophoresis as described in Ref. 8; lane 5, thioredoxin III, purified by chromatography on hydroxyapatite as described above. Lane 6, marker proteins. From top to bottom: ovalbu- min ( + impurity), M r = 45 000; chymotrypsinogen, M r = 25000; cytochrome c, Mr =12500; and aprotinin, Mr =6500.

We deemed it poss ib le that the t r ip l ic i ty of th ioredoxins in c o m m o n wheat was a consequence of its hexaplo idy . Such a cor re la t ion has been es tab l i shed in the case of the three wheat puro th ion ins , i.e., toxic po lypep t ides of M r = 5000

417

which show weak th ioredoxin act ivi ty [14]; one or two puro th ion ins are found in d ip lo id and te t ra- p lo id wheat, respect ively [15]. Hea t - s t ab le extracts f rom d ip lo id one-gra ined wheat (T. monococcurn), t e t rap lo id d u r u m wheat (T. durum) and c o m m o n b read wheat (T. aestivum) were therefore analyzed in para l le l for th ioredoxin content . Closely com- pa rab le amoun t s of th ioredoxins I, II, and I I I were found in all the three species (Fig. 1). In fact one-gra ined wheat would be par t i cu la r ly good source for the p r epa ra t i on of wheat th ioredoxins because it lacks m a n y o ther p ro te ins of the M r = 1 0 0 0 0 - 1 5 0 0 0 size class which compl ica te the pur i f ica t ion f rom bread wheat flour. Conse- quent ly , the genomic origin of wheat th ioredoxins has no th ing in c o m m o n with that of the puro- thionins. The presence of three M r - - 1 2 0 0 0 th ioredoxins in non-pho tosyn the t i c cells of very diverse p lants ( Scenedesmus obliquus; Triticum sp.; Glycine max) ra ther suggests that the t r iplet repre- sents a bas ic set of p lan t th ioredoxins in general .

The amino acid compos i t ion of homogenous wheat th ioredoxin samples has been de te rmined and is l isted in Table II together with that of E. coli th ioredoxin. The number of 110-118 amino acid residues and the ca lcula ted molecu la r weights are in good agreement with those of th ioredoxins f rom other sources, and es tabl i shed by SDS-poly- ac ry lamide gel e lect rophores is (Fig. 2). The figures also demons t r a t e that th ioredoxins I and II, which resemble each other in act ivi ty (Table I), are in- deed closely re la ted and be long to the regular

TABLE I

SUBSTRATE OR ENZYME-STIMULATING ACTIVITY OF WHEAT SEED THIOREDOXINS

Relative figures are given because the various enzymes differ greatly in absolute activities. The E. coli protein is included for its high activity in heterologous systems.

Enzyme Relative activity (%) of thioredoxin

none wheat I wheat II wheat III E. coil

Wheat seed NADPH-thioredoxin reductase a 0 90 Wheat leaf fructose-bisphosphatase 15 80 Spinach leaf NADP-malate dehydrogenase 10 50 E. coli ribonucleotide reductase 20 35

100 50 5 100 b 40 90 55 25 100 ¢ 40 30 100 d

a Equal amounts of oxidized thioredoxins as substrate; 100% corresponds to a rate of 24 nmol SH formation, min- 1. Specific thioredoxin activities tb-d~ were obtained after subtraction of the unstimulated ( - thioredoxin) enzyme activity:

b 0 . 2 # mol phosphate liberation, min - 1. rag- 1; c 10 # mol NADPH oxidized, min - 1. mg- i; d 1.2 #tool CDP reduction.h-l.mg -1.

418

TABLE II

AMINO ACID COMPOSITION OF WHEAT (7", AESTI- VUM) THIOREDOXINS

Amino Wheat thioredoxin E. coli

acid I II III thioredoxin

Ala 18 14 6 12 Arg 5 5 6 1 Asx 8 9 7 15 Cys 2 2 10 2 Glx 17 15 13 8 Gly 12 13 10 9 His 2 3 0 1 lie 3 3 3 9 Leu 5 5 9 13 Lys 8 9 9 10 Met 3 2 3 1 Phe 4 3 2 4 Pro 3 2 13 5 Ser 7 9 5 3 Thr 8 7 9 6 Trp a (1-2) (1-2) (1-2) 2 Tyr 2 2 3 2 Val 7 6 8 5

Total 115-116 110-111 117-118 108

Calculated M r 12255 11752 12770 11657

+_ 280 _+ 280 + 280

a Tryptophan was not determined. The close similarities be- tween the ultraviolet absorption and fluorescence spectra of wheat and other thioredoxins indicate the presence of 1-2 tryptophan residues.

dicysteine polypeptide family where the occur- rence of pairs of isoproteins is not uncommon [2,4,6,17]. In contrast, thioredoxin III is a differ- ent, cysteine- and proline-rich, histidine-free pro- tein. Thioredoxins of such composition have not as yet been isolated from bacterial or mammalian sources nor from spinach chloroplasts but we have found another cysteine-rich representative among soybean seed thioredoxins (Ref. 18 and unpub- lished data). While their enzyme-activating capac- ity alone would probably not suffice for classifi-

cation as thioredoxins [14,19] we feel that the observed crossreactivity with antibodies against E. coli thioredoxin and the other properties together leave no other choice, and that the thioredoxin family may be of greater structural diversity than anticipated.

This work has been supported by Deutsche Forschungsgemeinschaft, Sonderforschungs- bereich 305 (Okophysiologie). We thank Mrs. Gabriele Schimpff-Weiland for her assistance with the ribonucleotide reductase assays.

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