- Email: [email protected]
[24] Thioredoxin and seed proteins
228
[241
THIOREDOXIN AND GLUTAREDOXIN
Determination o f Eh. The absolute values determined for the midpoint potential of the regulatory sulfhydryls on FBPase in Fig. 1 are dependent on the Eh values assigned to various ratios of DTTrea/DTT°x at pH 7.9. The initial value for the midpoint potential of DTT at pH 7 reported by Cleland 1° of -332 mV is well supported by measurements employing diverse techniques. 17,18Because the redox reaction of DTT involves protons, the Em of the redox buffer will have a substantial pH dependence which must be taken into account in Eh calculations. The reduced form of DTT is a very weak acid, with the pKa values of the thiols reported to be 9.2 for the first ionization and 10.1 for the secondJ 9 Because DTT °x is a very strong acid, the redox chemistry of DTT will involve one proton per electron at pH values below approximately 8.2 (at higher pH values ionization of the first thiol o n D T T red will lower the proton to electron ratio below 1). Thus for pH values below 8.2, the Em of DTT can be calculated according to Em~ = Em~7 - 0.059 (pH x - pH 7)
We have used the value of Lees and Whitesides is for the Era, 7 for DTI" of -327 mV at 25 °, giving a value for Era,7. 9 of --380 mV. It should be noted that Cleland's original value for Em,8.1 of -366 mV 1° (implying a - 3 0 mV per unit pH dependence) has not been substantiated by subsequent measurements and should not be used for calculations of Eh.
17 D. M. R o t h w a r f and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 89, 7944 (1992). is W. J. Lees and G. M. Whitesides, J. Org. Chem. 58, 642 (1993). 19 G. M. Whitesides, J. E. Liburn, and R. P. Szajewski, J. Org. Chem. 42, 332 (1977).
[24] By
Thioredoxin
and Seed Proteins
JOSHUA H. WONG, KAROLY KOBREHEL,
and BOB B.
BUCHANAN
Introduction Storage proteins, which are formed and stored within seeds, serve as the main source of nitrogen and as a primary source of carbon for the germination and growth of seedlings. Storage proteins are synthesized after pollination and accumulate during seed maturation. The proteins are mobilized, proteolytically degraded, and utilized when conditions are favorable METHODS1NENZYMOLOGY,VOL. 2.52
Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[24]
THIOREDOXIN AND SEED PROTEINS
229
for germinationJ '2 In the case of cereals, the bulk of the storage proteins are water insoluble after the grains mature. In most other types of seeds, the proteins remain soluble and enclosed within a membrane, thereby creating a structure known as a protein body. 3 In some cereals, the protein body m e m b r a n e is disrupted during maturation and drying of the grain, thereby exposing the storage proteins to endogenous proteases of the endosperm. 3,n However, in typically nonglutenous cereals such as corn, rice, sorghum, and millet, storage proteins remain in protein bodies in the mature grains (see references in Ref. 5). Disulfide bonds are one of the main stabilizing forces in storage proteins. The formation of disulfide bonds appears to function in cereals to provide increased structural stability on the one hand and decreased solubility on the other. Both features provide protection against proteolysis. The group includes typically large, insoluble storage proteins (60-100 kDa) as well as generally less abundant low molecular weight soluble proteins (5-30 kDa),4 rich in disulfide bonds, with four or more cystines per molecule. Examples include a wide variety of exogenous a-amylase and protease inhibitors. Except for the ot-amylase/subtilisin inhibitor of barley and other cereals, which inhibits endogenous a-amylase, 6,7 the physiological function of these small disulfide proteins within the seed remains a mystery. It has long been considered that proteins of this type play a bioprotection role by blocking digestive enzymes of invading pests. 8 Several investigators have shown that small disulfide proteins of seeds, like the larger insoluble storage proteins, are degraded during germination? a° Our laboratory has uncovered evidence that both protein groups undergo reduction in conjunction with the degradationJ ~ The NADP+/ thioredoxin system (NTS) appears to play a role in the reduction of critical disulfide groups of seed proteins (S-S ~ 2SH) and, thereby, as seen below, trigger germination. The thioredoxin is reduced by N A D P H via NADP+/ J A. M. Mayer and A. Poljakoff-Mayber, "The Germination of Seeds." 4th Ed,, p. 160. Pergamon, New York, 1989. 2 F. M. Ashton, Annu. Rev. Plant Physiol. 27, 95 (1976). 3 E. Weber and D. Neumann, Biochem. Physiol. Pflanz. 175, 279 (1980). 4 p. R. Shewry and B. J. Miflin, Adv. Cereal Sci. Technol. 7, 1 (1985). s D. B. Bechtel, R. L. Gaines, and Y. Pomeranz, Cereal Chem. 59, 336 (1982). 6j. Hejgaard, I. B. Svendsen, and J. Mundy, Carlsberg Res. Commun. 48, 91 (1983). 7 R. J. Weselake, A. W. MacGregor, R. D. Hill, and H. W. Duckworth, Plant Physiol. 73, 1008 (1983). s C. A. Ryan, Biochem. Plants 6, 351 (1981). 9 p. Hwang and W. Bushuk, Cereal Chem. 50, 147 (1973). 10j. E. Kruger and B. A. Marchylo, Cereal Chem. 62, 1 (1985). ~K. Kobrehel, J. H. Wong, A. Balogh, F. Kiss, B. C. Yee, and B. B. Buchanan, Plant Physiol. 99, 919 (1992).
230
THIOREDOXINAND GLUTAREDOXIN
[241
thioredoxin reductase (NTR), a flavoprotein [Eq. (1)]. The reduced thioredoxin, in turn, reduces disulfide groups on target proteins [Eq. (2)]. The evidence suggests that thioredoxin preferentially reduces intramolecular disulfide bonds relative to intermolecular counterpartsJ 2 Thioredoxin hox + N A D P H + H + -NTR thioredoxin hred + N A D P + (1) (--S--S--) (--SH HS--) Thioredoxin hred + proteinox --~ thioredoxin hox + proteinr~d (--SH HS--) (--S--S--) (--S--S--) (--SH HS--)
(2)
Thioredoxin h, a 12-kDa protein with a catalytically active disulfide group, seems to be ubiquitous in plant cells and to be localized in the cytosol, endoplasmic reticulum, and mitochondriaJ 3-15 At the onset of germination, thioredoxin h appears to be reduced by N A D P H generated by the oxidative pentose phosphate pathway of the cereal endospermJ 1 Once reduced, the thioredoxin, in turn, reduces the large storage proteins (leading to their solubilization), as well as the small disulfide inhibitor proteins (leading to loss of their ability to inhibit target enzymes). On reduction, both the large storage proteins 11A6,17 and the small disulfide enzyme inhibitor proteins ~8,19 show increased susceptibility to proteolysis. There is some evidence t h a t glutathione maintained in reduced form (GSH) by N A D P H and glutathione reductase may also function in the reduction of these proteins [Eqs. (3) and (4)], particularly after thioredoxin has initiated the reduction process. 11 GSSG + N A D P H + H +~ 2GSH + NADP ÷ (--S--S--) (--SH)
(3)
2 G S H + proteinox --~ GSSG + proteinred (--SH) (--S--S--) (--S--S--) (--SHHS--)
(4)
Much of the evidence for the role of thiols in seed germination has come from studies in our laboratory. For these studies, we have adapted a relatively simple, yet specific method for measurement of the thiol redox 12S. Shin, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Planta 189, 557 (1993). 13T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987). 14j. Bodenstein-Lang, A. Buch, and H. Follmann, FEBS Lett. 258, 22 (1989). 15F. Marcus, S. H. Chamberlain, C. Chu, F. R. Masiarz, S. Shin, B. C. Yee, and B. B. Buchanan, Arch. Biochem. Biophys. 287, 195 (1991). 16j. H. Wong, K. Kobrehel, C. Nimbona, B. C. Yee, A. Balogh, F. Kiss, and B. B. Buchanan, Cereal Chem. 70, 113 (1993). 17I. Besse, unpublished findings from our laboratory, 1995. ~8j. Jiao, B. C. Yee, K. Kobrehel, and B. B. Buchanan, J. Agric. Food Chem. 40, 2333 (1992). 19j. Jiao, B. C. Yee, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Plant Physiol. Biochem. 31, 799 (1993).
[24]
THIOREDOXIN AND SEED PROTEINS
231
status of proteins. The technique is a modification of an earlier procedure developed for analysis of chloroplast enzymes regulated by thioredoxin. 2° In that study, the chloroplast proteins of interest, because of their low abundance, were immunoprecipitated to permit quantitation of the reduction of the disulfide groups by light. An uncharged probe, m o n o b r o m o b i m a n e (mBBr), was used to bind sulfhydryl groups specifically and covalently, thereby rendering the p r o t e i n m B B r derivative fluorescent. 2°'21 The fluorescent m B B r - p r o t e i n products can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) and then visualized and quantified. The method can be applied equally well in vitro (both with purified proteins ls'19`2L22 and with extracts composed of many proteins 11,16)or in vivo (with intact seedsU). In either case, the redox status of the seed proteins can be assessed. A description of the procedure is presented below, using wheat as an example. Also, as seen below, the procedure can be applied equally well to disulfide proteins purified from a variety of sources.
S o u r c e s of Materials a n d C h e m i c a l s Materials. Either seeds or semolina of durum wheat (Triticum durum) or seeds or flour of bread wheat (Triticum aestivum) may be used. In our case, the respective cultivars are Desf. cv. M o n r o e or cv. Scout 66. Escherichia coli thioredoxin and N T R are either purchased from American Diagnostica (Greenwich, CT) or isolated from cells overexpressing each protein. 23'24 Wheat thioredoxin h and N T R are isolated from germ, following the procedures developed for spinach leaves. 25 Glutaredoxin (a gift of Prof. A. Holmgren, Karolinska Institutet, Stockholm) is isolated from E. coli cells genetically modified to overexpress the protein. 26 Chemicals. Reagents for S D S - P A G E are purchased from Bio-Rad Laboratories (Richmond, CA). M o n o b r o m o b i m a n e (mBBr) or Thiolyte is obtained from Calbiochem (San Diego, CA). Aluminum lactate and methyl green are products of Fluka Chemicals (Buchs, Switzerland).
20N. A. Crawford, M. Droux, N. S. Kosower, and B. B. Buchanan, Arch. Biochem. Biophys. 271, 223 (1989). 21N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 76. 22K. Kobrehel, B. C. Yee, and B. B. Buchanan, J. Biol. Chem. 266, 16135 (1991). ~3F. de la Motte-Guery, M. Mijiulac-Maslow, P. Decottiguies, M. Stein, P. Minard, and J. P. Jacquot, Eur. J. Biochem. 196, 287 (1991). 24M. Russel and P. Model, J. Biol. Chem. 263, 9015 (1988). 25F. J. Florencio, B. C. Yee, T. C. Johnson, and B. B. Buchanan, Arch. Biochem. Biophys. 266, 496 (1988). z6A. Holmgren, J. Biol. Chem. 264, 13963 (1989).
232
THIOREDOXINAND GLUTAREDOXIN
[241
Isolation of Protein Fractions Chemicals and Materials
Durum wheat semolina or bread wheat flour, 5 g Tris-HC1 buffer, pH 7.5, 50 mM Ethanol, 70% (v/v) Acetic acid, 0.1 M Procedure. For isolation of the soluble and insoluble proteins, semolina or flour (0.2 g) is extracted sequentially with 1 ml of the following solutions for the indicated times at 25°: (1) 50 mM Tris-HC1 buffer, pH 7.5 (20 min), for the albumins and globulins; (2) 70% ethanol (2 hr) for the gliadins; and (3) 0.1 M acetic acid (2 hr) for the glutenins. 1° Alternatively, the glutenins can be isolated by extraction with sodium myristate. In that case, 80 mg myristate is added to 1 g of flour together with 8 ml distilled water. 27 Similar conditions can be used to extract total proteins in one step. 28During extraction, samples are placed on an electrical rotator and, in addition, occasionally agitated with a vortex mixer. After extraction with each solvent, samples are centrifuged (12,000 rpm for 5 min at 4°) in an Eppendorf microcentrifuge, and supernatant fractions are saved for analysis. In between each extraction, pellets are washed with 1 ml of water and collected by centrifugation as before; the supernatant wash fractions are discarded. By convention, z9 the fractions are designated as follows: (1) albumin/globulin; (2) gliadin; and (3) glutenin. If desired, the albumins and globulins can be separated by dialysis against distilled water; the albumins remain soluble and the globulins precipitate. Following centrifugation (10 min, 10,000 g at 4°), the supernatant fraction containing the albumins is decanted and the globulins are redissolved in 50 mM Tris-HCl buffer, pH 7.5.
Reduction and in Vitro Labeling of Proteins In Vitro Labeling o f Proteins with Monobromobimane Reagents
Tris-HC1 buffer, pH 7.9 at 20 °, 100 mM Thioredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 NTR, E. coli, 0.1 mg/ml in 30 mM Tris-HCl, pH 7.9 NADPH, 20 mM in 30 mM Tris-HCl, pH 7.9 27K. Kobrehel and R. Alary, J. ScL Food Agric. 48, 441 (1989). 28K. Kobrehel and B. Matignon, Cereal Chem. 57, 73 (1980). 29T. B. Osborne and C. G. Voorhees,Am. Chem. J. 15, 392 (1893).
[241
THIOREDOXIN AND SEED PROTEINS
233
Dithiothreitol (DTT), 100 mM Glutathione, reduced, 20 mM in 30 mM Tris-HC1, pH 7.9 Glutathione reductase (purified from spinach leaves25), 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Glutaredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Monobromobimane (mBBr) or Thiolyte, 20 mM dissolved in acetonitrile and stored protected from light SDS, 10% (w/v) 2-Mercaptoethanol (2-ME), 100 mM Target proteins, 5-30/zg, in extraction solvent; alternatively, as seen below, 5-10 tzg pure disulfide proteins may be used Procedure. Both steps of the reaction are carried out at room temperature. As indicated, 7 txl NTR (0.7/~g) and 10/~1 thioredoxin (1 tzg) (both from E. coli unless specified otherwise) along with 10 t~l N A D P H are added to 70 tzl of 100 mM Tris-HC1 buffer, pH 7.9, and 30 txg of target protein. When thioredoxin is reduced by DTT, the N A D P H and NTR are omitted and 5 txl DTT is added to a final concentration of 1 mM. Reduction by reduced glutathione is performed similarly, but at a final concentration of 2 mM. After incubation for 20 min, 5/zl of mBBr (80 nmol) is added, and the reaction is continued for another 15 min. To stop the reaction and derivatize excess mBBr, 10/zl of 10% SDS and 10 tzl of 100 mM 2-ME are added, and the samples are then applied to the gels. For reduction by glutaredoxin, the thioredoxin and NTR are replaced by 10 Ixl E. coli glutaredoxin (1/xg), 10/xl glutathione reductase (1.4/xg), and 10 txl NADPH.
Germination and in Vivo Labeling of Proteins Germination of Wheat Seeds Materials
Seeds of durum wheat Plastic petri dishes Whatman (Clifton, N J) No. 1 filter paper Growth chamber Procedure. Duplicate sets of 20 to 30 seeds are placed in a plastic petri dish on three layers of Whatman No. 1 filter paper moistened with 5 ml of deionized water. Petri dishes are then transferred to a darkened growth chamber. Germination is carried out for up to 4 days at room temperature, with 5 ml of deionized water being added daily to each petri dish. On the fifth day, the germinated seedlings are harvested.
234
THIOREDOXIN AND GLUTAREDOXIN
[24]
In Vivo Labeling of Proteins with Monobromobimane Chemicals and Materials Endosperm of germinated seedlings Liquid N2 mBBr, 2.0 mM, freshly diluted in 100 mM Tris-HC1 buffer, pH 7.9 Procedure. At the indicated times, the dry seeds or germinated seedlings (selected on the basis of similar radicle or shoot length) are taken from the petri dish and the embryos or germinated axes are removed with a razor blade. Five endosperm from each lot are weighed and then ground in liquid N2 with a mortar and pestle. One milliliter of 2.0 mM mBBr in 100 mM Tris-HCl buffer, pH 7.9, is added just as the last trace of liquid N2 disappears. The thawed mixture is ground for another minute and transferred to a microcentrifuge tube of 1.5 ml capacity. The volume of the suspension is adjusted to 1 ml with the appropriate mBBr or buffer solution. Protein fractions of albumin/globulin, gliadin, and glutenin from endosperm of germinated seedlings are extracted as described above for semolina and flour. The extracted protein fractions are stored at - 2 0 ° until use for electrophoresis. A buffer control (minus mBBr) is included for each time point. Analysis of Labeled Samples by Polyacrylamide Gel Electrophoresis
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Monobromobimane-Labeled Samples Analysis by S D S - P A G E of the mBBr-derivatized samples of each protein fraction is performed in 15% polyacrylamide gels at pH 8.5 as described by Laemmli. 3° Before sample application, an aliquot (20/xl) of 50% (v/v) glycerol containing 0.01% (w/v) bromphenol blue is added to each mBBr-labeled sample. Gels of 1.5 mm thickness are developed for 16 hr at a constant current of 9 mA.
Native Gel Electrophoresis for Separating Different Gliadins Reagents and Equipment A gel solution in 100 ml final volume contains the following: 6.0 g acrylamide, 0.3 g bisacrylamide, 0.024 g ascorbic acid, 0.2 mg ferrous sulfate heptahydrate, 0.25 g aluminum lactate, and 85% lactic acid (certified ACS); the gel solution is adjusted to pH 3.1 with lactic acid 30 U. K. Laemrnli, Nature (London) 227, 680 (1970).
[24]
THIOREDOXINAND SEEDPROTEINS
235
Catalyst solution, hydrogen peroxide, 3% Tank buffer, aluminum lactate buffer, 0.5 g/liter, is adjusted to pH 3.1 with lactic acid Tracking dye, methyl green, 0.6% (w/v) in 80% (w/v) sucrose in tank buffer Electrophoresis equipment, Hoefer Model SE600 (Hoefer Scientific Instrument, San Francisco, CA) Procedure. To resolve the different types of gliadins, native polyacrylamide gel electrophoresis is performed in 6% gels of 3 mm thickness (a procedure designed to separate gliadins into the four types, a,/3, y, and ~0) as described by Bushuk and ZiUman 31 and modified for vertical slab gels by Sapirstein and Bushuk. 32 A gel stock solution can be prepared beforehand by mixing acrylamide, bisacrylamide, and aluminum lactate, adjusting to pH 3.1 with lactic acid, and then storing at 4 °. Before use the ascorbic acid (0.024 g) and ferrous sulfate (0.2 rag) are added to 100 ml of the gel stock solution, which is then degassed for 2 hr at 4 °. Because polymerization occurs essentially instantaneously after adding the hydrogen peroxide catalyst, it is best to set up and precool the gel plate (16 × 18 cm) assembly, with the 3-mm spacers, in a cold room. It is also best to pour the gel solution as quickly as possible through a funnel, mounted on a stand, that fits into the space between the plates with the comb already in place. Before sample application an aliquot (20 /xl) of the tracking dye is added to each sample. The duration of electrophoresis is approximately 4 hr, with a constant current of 50 mA. The temperature of the tank buffer ( - 2 - 3 liters) is controlled by circulating water at 21 ° during electrophoresis. (Caution: As gliadins are positively charged at this pH, it is imperative to reverse the polarity of the two electrodes, i.e., by attaching the negative electrode of the gel box to the positive outlet of the power supply and vice versa for the positive electrode.) Electrophoresis is terminated when the lower mobility band of the methyl green tracking dye migrates to about ! cm from the end of the gel. Monobromobimane Removal and Fluorescence Photography Reagents and Equipment
12% (w/v) Trichloroacetic acid 40% Methanol/10% acetic acid (v/v)
31W. Bushuk and R. R. Zillman, Can. J. Plant. Sci. 58, 505 (1978). 32H. D. Sapirstein and W. Bushuk, Cereal Chem. 62, 372 (1985).
236
THIOREDOXIN AND GLUTAREDOXIN
[24]
Spectroline transilluminator, (Spectronics Corp., Westbury, NY), Model TS-365, 365 nm ultraviolet Polaroid Land camera Polaroid Positive/Negative Landfilm, type 55 Wratten gelatin filter No. 8 (cutoff of 460 nm) Procedure. Following electrophoresis, gels are placed in 200 ml trichloroacetic acid and soaked for 4 to 6 hr with one change of solution to fix the proteins; gels are then transferred to 200 ml of 40% methanol/10% acetic acid for 8 to 10 hr (with two to three changes of solution) to remove excess mBBr. The fluorescence of mBBr, both free and protein bound, is visualized by placing gels on a light box fitted with an ultraviolet light source (365 nm). Gels are photographed with Polaroid Positive/Negative Landfilm, type 55, through a yellow Wratten gelatin filter No. 8 (cutoff of 460 nm) (exposure time ranges from 25 to 60 sec at f4.5). The negatives are used for densitometric scanning after being processed and dried (see below).
Protein Staining/Destaining/Photography Reagents Coomassie Brilliant Blue R-250, 0.25% (w/v) 40% methanol/10% acetic acid (v/v) Coomassie Brilliant Blue R-250 (0.1 g dissolved in 10 ml of 95%, v/v, ethanol) Trichloroacetic acid, 12% Procedure. The SDS gels are stained with 100 ml of 0.25% Coomassie Brilliant Blue R-250 in 40% methanol/10% acetic acid for 1 to 2 hr and destained overnight with several changes (200 ml) of the above solvent without the dye. 2° Aluminum lacate native gels are stained overnight in a filtered solution containing 0.1 g Coomassie Brilliant Blue R-250 in 240 ml of 12% trichloroacetic acid. Gels are destained overnight in 200 ml of 12% trichloroacetic acid. 1l Protein-stained gels are photographed with Polaroid type 55 film to produce prints and negatives. Prints are used to determine band migration distances and loading efficiency as well as to quantitate protein SH. Quantitation of Fluorescence [Reduction)
Scanning of Gels The Polaroid negatives of fluorescent gels and prints of wet proteinstained gels are scanned with a laser densitometer (Pharmacia-LKB, Piscataway, N J, UltroScan XL). Intensities of fluorescent bands are quantified by evaluating peak areas after integration with GelScan XL software. The
[24]
237
THIOREDOXIN AND SEED PROTEINS Proteln
Fluorescence 2
3
I 5
4
~ 1
I 5
Protein
Fluorescence I 6
7
8
9
10
I
I
6
10
kDa 97.4 66.2 w
45.0
31.0
21.5
14.4
Gliadins:
Glutenins:
1. 2. 3. 4. 5,
6. Control 7. GSH 8. GSH/GR/NADPH 9. NGS 10. NTS
Control GSH GSH/GR/NADPH NGS NTS
FIG. 1. Relative effectiveness of the NADP+/thioredoxin (NTS), NADP+/glutaredoxin (NGS), and the glutathione/glutathione reductase system (GSH/GR/NADPH) in the reduction of seed proteins (SDS, polyacrylamide gel electrophoresis).
intensity of fluorescent bands, when totally reduced by 2 mM DTT at 100° for 3 min and derivatized with mBBr, is proportional to the amount of added protein (up to 30/zg). It is assumed that the intensity of fluorescence of the proteins under analysis is also a direct measure of percent reduction of sulfhydryl groups. Specific Applications The techniques described above have been used successfully in determining the reduction of seed storage proteins and purified disulfide proteins from seeds as well as other sources. The following examples serve to illustrate use of the technique.
238
[241
THIOREDOXIN AND GLUTAREDOXIN
Effectiveness of Thiols in Reduction of Seed Proteins. By employing the in vitro labeling technique described above, our laboratory has obtained evidence that the NADP+/thioredoxin system reduces wheat storage proteins--gliadins and glutenins--much more effectively than the glutaredoxin or glutathione systems (Fig. 1). T M Similar results have been obtained in an unpublished survey of extracted prolamines and glutelins from several nonglutenous cereals. The system works equally well for pure disulfide proteins from seeds (Fig. 2) as well as other s o u r c e s . 12'19,2°,22 Change in Redox Status of Proteins during Germination. Using the in vivo labeling technique described above, our laboratory has obtained evidence for a change in redox state of wheat proteins during germination.11 We observed a progressive increase of 2- to 3-fold in reduction of the albumin/globulin, gliadin, and glutenin fractions up to the second day of germination; thereafter, reduction declined (Fig. 3). This pattern of redox change was coincident with the proteolytic degradation (or mobilization) of storage proteins. A similar series of events has been observed with barley DSG-1
BBTI
•
,g"
DTT GSH
~ + , ÷1i t
DTT ÷
_
GSH II +
_
C ~l +
DTT II
.
GSH ,
+
C ,
+
I
+ and - refer to thioredoxin (E.coli) C = Control (no reductant) •~ = Thioredoxin
Fie. 2. Dithiothreitol-reduced thioredoxin as a reductant for seed a-amylase and trypsin inhibitor proteins. DSG-1, Wheat a-amylase inhibitor; BBTI, soybean Bowman-Birk trypsin inhibitor (SDS, polyacrylamide gel electrophoresis).
[241
THIOREDOXIN AND SEED PROTEINS
5
I
I
,
o
.~a
i
a
•
,
,
I
239 I
t
o,/
i
i
~ ~ 3
~
o
0
0
1
2
3
4
Day
FIG. 3. In vivo reduction status of protein fractions during germination. [Data obtained by analysis of SDS-polyacrylamide gels.]
(C. Marx, J. H. Wong, and B. B. Buchanan, unpublished results); also in unpublished experiments, the reduction of disulfide bonds of gliadins and glutenins by the NTS was found to facilitate their degradation by proteases (I. Besse, and B. B. Buchanan, unpublished findings). Change in Redox Status of Thiol-Containing Proteins during Seed Maturation. Samples (wheat ears) are harvested at different stages of seed maturation up to complete maturity. Immediately after harvest, samples are put into liquid N2, and grains are dehuUed manually in a mortar in the presence of liquid N2. In vivo labeling with mBBr is performed under the conditions described for the in vivo labeling of germinated seeds. Results to date provide evidence that the storage proteins are synthesized in the reduced form and become oxidized during maturation and drying. Redox Changes during Technological Transformations. An extension of these experiments has revealed that the NADP+/thioredoxin system reduces selected flour proteins and strengthens the dough network formed
240
[251
THIOREDOXIN AND GLUTAREDOXIN
with flour of poor cooking quality, as measured by farinograph 16and baking tests. 33 These results suggest that addition of the NADP+/thioredoxin system improves products prepared from wheat and other cereals. Monobromobimane is currently being used as a probe to identify the major sulfhydryl/disulfide redox changes taking place in proteins during dough strengthening. Samplings are taken at different stages of the dough making process. Dough samples are put into liquid N2, and protein is fractionated and labeled with mBBr as described above.
33 K. Kobrehel, C. Nimbona, B. B. Buchanan, D. Bergmann, J. H. Wong, and B. C. Yee, "Gluten Proteins 1993," p. 381. Assoc. of Cereal Research, Detmold, Germany.
[251 A n a l y s i s a n d M a n i p u l a t i o n o f T a r g e t E n z y m e s Thioredoxin Control
By J E A N - P I E R R E
for
J A C Q U O T , E M M A N U E L L E ISSAKIDIS,
PAULETTE DECOTTIGNIES, MARTINE LEMAIRE,
and
MYROSLAWA MIGINIAC-MAsLOW
Introduction Several systems are able to control the activity, stability, and correct folding of enzymes through dithiol/disulfide isomerization reactions including the enzyme protein disulfide-isomerase, the glutathione-dependent glutaredoxin system, and the thioredoxin systems. 1-3 Plants contain two different thioredoxin systems, one extrachloroplastic in which thioredoxin is reduced through NADPH and the flavoenzyme NADPH-thioredoxin reductase (EC 1.6.4.5), and the other chloroplastic in which the reducing system is photoreduced ferredoxin and the iron-sulfur enzyme ferredoxinthioredoxin reductase. 4'5 To study thioredoxins and their reducing systems, it is necessary to use a target enzyme, the properties of which can be modified after reaction with reduced thioredoxin. Several proteins with this property have been isolated (including fructose-l,6-bisphosphatase, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, glu1 R. B. Freedman, FEBS Lett. 97, 201 (1979). z A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). 3 H. Eklund, F. K. Gleason, and A. Holmgren, Proteins: Struct. Funct, Genet. 11, 13 (1991). 4 B. B. Buchanan, Annu. Rev. Plant Physiol. 31, 341 (1980). 5 T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987).
METHODSIN ENZYMOLOGY,VOL.252
Copyright © 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[241
THIOREDOXIN AND GLUTAREDOXIN
Determination o f Eh. The absolute values determined for the midpoint potential of the regulatory sulfhydryls on FBPase in Fig. 1 are dependent on the Eh values assigned to various ratios of DTTrea/DTT°x at pH 7.9. The initial value for the midpoint potential of DTT at pH 7 reported by Cleland 1° of -332 mV is well supported by measurements employing diverse techniques. 17,18Because the redox reaction of DTT involves protons, the Em of the redox buffer will have a substantial pH dependence which must be taken into account in Eh calculations. The reduced form of DTT is a very weak acid, with the pKa values of the thiols reported to be 9.2 for the first ionization and 10.1 for the secondJ 9 Because DTT °x is a very strong acid, the redox chemistry of DTT will involve one proton per electron at pH values below approximately 8.2 (at higher pH values ionization of the first thiol o n D T T red will lower the proton to electron ratio below 1). Thus for pH values below 8.2, the Em of DTT can be calculated according to Em~ = Em~7 - 0.059 (pH x - pH 7)
We have used the value of Lees and Whitesides is for the Era, 7 for DTI" of -327 mV at 25 °, giving a value for Era,7. 9 of --380 mV. It should be noted that Cleland's original value for Em,8.1 of -366 mV 1° (implying a - 3 0 mV per unit pH dependence) has not been substantiated by subsequent measurements and should not be used for calculations of Eh.
17 D. M. R o t h w a r f and H. A. Scheraga, Proc. Natl. Acad. Sci. U.S.A. 89, 7944 (1992). is W. J. Lees and G. M. Whitesides, J. Org. Chem. 58, 642 (1993). 19 G. M. Whitesides, J. E. Liburn, and R. P. Szajewski, J. Org. Chem. 42, 332 (1977).
[24] By
Thioredoxin
and Seed Proteins
JOSHUA H. WONG, KAROLY KOBREHEL,
and BOB B.
BUCHANAN
Introduction Storage proteins, which are formed and stored within seeds, serve as the main source of nitrogen and as a primary source of carbon for the germination and growth of seedlings. Storage proteins are synthesized after pollination and accumulate during seed maturation. The proteins are mobilized, proteolytically degraded, and utilized when conditions are favorable METHODS1NENZYMOLOGY,VOL. 2.52
Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[24]
THIOREDOXIN AND SEED PROTEINS
229
for germinationJ '2 In the case of cereals, the bulk of the storage proteins are water insoluble after the grains mature. In most other types of seeds, the proteins remain soluble and enclosed within a membrane, thereby creating a structure known as a protein body. 3 In some cereals, the protein body m e m b r a n e is disrupted during maturation and drying of the grain, thereby exposing the storage proteins to endogenous proteases of the endosperm. 3,n However, in typically nonglutenous cereals such as corn, rice, sorghum, and millet, storage proteins remain in protein bodies in the mature grains (see references in Ref. 5). Disulfide bonds are one of the main stabilizing forces in storage proteins. The formation of disulfide bonds appears to function in cereals to provide increased structural stability on the one hand and decreased solubility on the other. Both features provide protection against proteolysis. The group includes typically large, insoluble storage proteins (60-100 kDa) as well as generally less abundant low molecular weight soluble proteins (5-30 kDa),4 rich in disulfide bonds, with four or more cystines per molecule. Examples include a wide variety of exogenous a-amylase and protease inhibitors. Except for the ot-amylase/subtilisin inhibitor of barley and other cereals, which inhibits endogenous a-amylase, 6,7 the physiological function of these small disulfide proteins within the seed remains a mystery. It has long been considered that proteins of this type play a bioprotection role by blocking digestive enzymes of invading pests. 8 Several investigators have shown that small disulfide proteins of seeds, like the larger insoluble storage proteins, are degraded during germination? a° Our laboratory has uncovered evidence that both protein groups undergo reduction in conjunction with the degradationJ ~ The NADP+/ thioredoxin system (NTS) appears to play a role in the reduction of critical disulfide groups of seed proteins (S-S ~ 2SH) and, thereby, as seen below, trigger germination. The thioredoxin is reduced by N A D P H via NADP+/ J A. M. Mayer and A. Poljakoff-Mayber, "The Germination of Seeds." 4th Ed,, p. 160. Pergamon, New York, 1989. 2 F. M. Ashton, Annu. Rev. Plant Physiol. 27, 95 (1976). 3 E. Weber and D. Neumann, Biochem. Physiol. Pflanz. 175, 279 (1980). 4 p. R. Shewry and B. J. Miflin, Adv. Cereal Sci. Technol. 7, 1 (1985). s D. B. Bechtel, R. L. Gaines, and Y. Pomeranz, Cereal Chem. 59, 336 (1982). 6j. Hejgaard, I. B. Svendsen, and J. Mundy, Carlsberg Res. Commun. 48, 91 (1983). 7 R. J. Weselake, A. W. MacGregor, R. D. Hill, and H. W. Duckworth, Plant Physiol. 73, 1008 (1983). s C. A. Ryan, Biochem. Plants 6, 351 (1981). 9 p. Hwang and W. Bushuk, Cereal Chem. 50, 147 (1973). 10j. E. Kruger and B. A. Marchylo, Cereal Chem. 62, 1 (1985). ~K. Kobrehel, J. H. Wong, A. Balogh, F. Kiss, B. C. Yee, and B. B. Buchanan, Plant Physiol. 99, 919 (1992).
230
THIOREDOXINAND GLUTAREDOXIN
[241
thioredoxin reductase (NTR), a flavoprotein [Eq. (1)]. The reduced thioredoxin, in turn, reduces disulfide groups on target proteins [Eq. (2)]. The evidence suggests that thioredoxin preferentially reduces intramolecular disulfide bonds relative to intermolecular counterpartsJ 2 Thioredoxin hox + N A D P H + H + -NTR thioredoxin hred + N A D P + (1) (--S--S--) (--SH HS--) Thioredoxin hred + proteinox --~ thioredoxin hox + proteinr~d (--SH HS--) (--S--S--) (--S--S--) (--SH HS--)
(2)
Thioredoxin h, a 12-kDa protein with a catalytically active disulfide group, seems to be ubiquitous in plant cells and to be localized in the cytosol, endoplasmic reticulum, and mitochondriaJ 3-15 At the onset of germination, thioredoxin h appears to be reduced by N A D P H generated by the oxidative pentose phosphate pathway of the cereal endospermJ 1 Once reduced, the thioredoxin, in turn, reduces the large storage proteins (leading to their solubilization), as well as the small disulfide inhibitor proteins (leading to loss of their ability to inhibit target enzymes). On reduction, both the large storage proteins 11A6,17 and the small disulfide enzyme inhibitor proteins ~8,19 show increased susceptibility to proteolysis. There is some evidence t h a t glutathione maintained in reduced form (GSH) by N A D P H and glutathione reductase may also function in the reduction of these proteins [Eqs. (3) and (4)], particularly after thioredoxin has initiated the reduction process. 11 GSSG + N A D P H + H +~ 2GSH + NADP ÷ (--S--S--) (--SH)
(3)
2 G S H + proteinox --~ GSSG + proteinred (--SH) (--S--S--) (--S--S--) (--SHHS--)
(4)
Much of the evidence for the role of thiols in seed germination has come from studies in our laboratory. For these studies, we have adapted a relatively simple, yet specific method for measurement of the thiol redox 12S. Shin, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Planta 189, 557 (1993). 13T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987). 14j. Bodenstein-Lang, A. Buch, and H. Follmann, FEBS Lett. 258, 22 (1989). 15F. Marcus, S. H. Chamberlain, C. Chu, F. R. Masiarz, S. Shin, B. C. Yee, and B. B. Buchanan, Arch. Biochem. Biophys. 287, 195 (1991). 16j. H. Wong, K. Kobrehel, C. Nimbona, B. C. Yee, A. Balogh, F. Kiss, and B. B. Buchanan, Cereal Chem. 70, 113 (1993). 17I. Besse, unpublished findings from our laboratory, 1995. ~8j. Jiao, B. C. Yee, K. Kobrehel, and B. B. Buchanan, J. Agric. Food Chem. 40, 2333 (1992). 19j. Jiao, B. C. Yee, J. H. Wong, K. Kobrehel, and B. B. Buchanan, Plant Physiol. Biochem. 31, 799 (1993).
[24]
THIOREDOXIN AND SEED PROTEINS
231
status of proteins. The technique is a modification of an earlier procedure developed for analysis of chloroplast enzymes regulated by thioredoxin. 2° In that study, the chloroplast proteins of interest, because of their low abundance, were immunoprecipitated to permit quantitation of the reduction of the disulfide groups by light. An uncharged probe, m o n o b r o m o b i m a n e (mBBr), was used to bind sulfhydryl groups specifically and covalently, thereby rendering the p r o t e i n m B B r derivative fluorescent. 2°'21 The fluorescent m B B r - p r o t e i n products can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) and then visualized and quantified. The method can be applied equally well in vitro (both with purified proteins ls'19`2L22 and with extracts composed of many proteins 11,16)or in vivo (with intact seedsU). In either case, the redox status of the seed proteins can be assessed. A description of the procedure is presented below, using wheat as an example. Also, as seen below, the procedure can be applied equally well to disulfide proteins purified from a variety of sources.
S o u r c e s of Materials a n d C h e m i c a l s Materials. Either seeds or semolina of durum wheat (Triticum durum) or seeds or flour of bread wheat (Triticum aestivum) may be used. In our case, the respective cultivars are Desf. cv. M o n r o e or cv. Scout 66. Escherichia coli thioredoxin and N T R are either purchased from American Diagnostica (Greenwich, CT) or isolated from cells overexpressing each protein. 23'24 Wheat thioredoxin h and N T R are isolated from germ, following the procedures developed for spinach leaves. 25 Glutaredoxin (a gift of Prof. A. Holmgren, Karolinska Institutet, Stockholm) is isolated from E. coli cells genetically modified to overexpress the protein. 26 Chemicals. Reagents for S D S - P A G E are purchased from Bio-Rad Laboratories (Richmond, CA). M o n o b r o m o b i m a n e (mBBr) or Thiolyte is obtained from Calbiochem (San Diego, CA). Aluminum lactate and methyl green are products of Fluka Chemicals (Buchs, Switzerland).
20N. A. Crawford, M. Droux, N. S. Kosower, and B. B. Buchanan, Arch. Biochem. Biophys. 271, 223 (1989). 21N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 76. 22K. Kobrehel, B. C. Yee, and B. B. Buchanan, J. Biol. Chem. 266, 16135 (1991). ~3F. de la Motte-Guery, M. Mijiulac-Maslow, P. Decottiguies, M. Stein, P. Minard, and J. P. Jacquot, Eur. J. Biochem. 196, 287 (1991). 24M. Russel and P. Model, J. Biol. Chem. 263, 9015 (1988). 25F. J. Florencio, B. C. Yee, T. C. Johnson, and B. B. Buchanan, Arch. Biochem. Biophys. 266, 496 (1988). z6A. Holmgren, J. Biol. Chem. 264, 13963 (1989).
232
THIOREDOXINAND GLUTAREDOXIN
[241
Isolation of Protein Fractions Chemicals and Materials
Durum wheat semolina or bread wheat flour, 5 g Tris-HC1 buffer, pH 7.5, 50 mM Ethanol, 70% (v/v) Acetic acid, 0.1 M Procedure. For isolation of the soluble and insoluble proteins, semolina or flour (0.2 g) is extracted sequentially with 1 ml of the following solutions for the indicated times at 25°: (1) 50 mM Tris-HC1 buffer, pH 7.5 (20 min), for the albumins and globulins; (2) 70% ethanol (2 hr) for the gliadins; and (3) 0.1 M acetic acid (2 hr) for the glutenins. 1° Alternatively, the glutenins can be isolated by extraction with sodium myristate. In that case, 80 mg myristate is added to 1 g of flour together with 8 ml distilled water. 27 Similar conditions can be used to extract total proteins in one step. 28During extraction, samples are placed on an electrical rotator and, in addition, occasionally agitated with a vortex mixer. After extraction with each solvent, samples are centrifuged (12,000 rpm for 5 min at 4°) in an Eppendorf microcentrifuge, and supernatant fractions are saved for analysis. In between each extraction, pellets are washed with 1 ml of water and collected by centrifugation as before; the supernatant wash fractions are discarded. By convention, z9 the fractions are designated as follows: (1) albumin/globulin; (2) gliadin; and (3) glutenin. If desired, the albumins and globulins can be separated by dialysis against distilled water; the albumins remain soluble and the globulins precipitate. Following centrifugation (10 min, 10,000 g at 4°), the supernatant fraction containing the albumins is decanted and the globulins are redissolved in 50 mM Tris-HCl buffer, pH 7.5.
Reduction and in Vitro Labeling of Proteins In Vitro Labeling o f Proteins with Monobromobimane Reagents
Tris-HC1 buffer, pH 7.9 at 20 °, 100 mM Thioredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 NTR, E. coli, 0.1 mg/ml in 30 mM Tris-HCl, pH 7.9 NADPH, 20 mM in 30 mM Tris-HCl, pH 7.9 27K. Kobrehel and R. Alary, J. ScL Food Agric. 48, 441 (1989). 28K. Kobrehel and B. Matignon, Cereal Chem. 57, 73 (1980). 29T. B. Osborne and C. G. Voorhees,Am. Chem. J. 15, 392 (1893).
[241
THIOREDOXIN AND SEED PROTEINS
233
Dithiothreitol (DTT), 100 mM Glutathione, reduced, 20 mM in 30 mM Tris-HC1, pH 7.9 Glutathione reductase (purified from spinach leaves25), 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Glutaredoxin, E. coli, 0.1 mg/ml in 30 mM Tris-HC1, pH 7.9 Monobromobimane (mBBr) or Thiolyte, 20 mM dissolved in acetonitrile and stored protected from light SDS, 10% (w/v) 2-Mercaptoethanol (2-ME), 100 mM Target proteins, 5-30/zg, in extraction solvent; alternatively, as seen below, 5-10 tzg pure disulfide proteins may be used Procedure. Both steps of the reaction are carried out at room temperature. As indicated, 7 txl NTR (0.7/~g) and 10/~1 thioredoxin (1 tzg) (both from E. coli unless specified otherwise) along with 10 t~l N A D P H are added to 70 tzl of 100 mM Tris-HC1 buffer, pH 7.9, and 30 txg of target protein. When thioredoxin is reduced by DTT, the N A D P H and NTR are omitted and 5 txl DTT is added to a final concentration of 1 mM. Reduction by reduced glutathione is performed similarly, but at a final concentration of 2 mM. After incubation for 20 min, 5/zl of mBBr (80 nmol) is added, and the reaction is continued for another 15 min. To stop the reaction and derivatize excess mBBr, 10/zl of 10% SDS and 10 tzl of 100 mM 2-ME are added, and the samples are then applied to the gels. For reduction by glutaredoxin, the thioredoxin and NTR are replaced by 10 Ixl E. coli glutaredoxin (1/xg), 10/xl glutathione reductase (1.4/xg), and 10 txl NADPH.
Germination and in Vivo Labeling of Proteins Germination of Wheat Seeds Materials
Seeds of durum wheat Plastic petri dishes Whatman (Clifton, N J) No. 1 filter paper Growth chamber Procedure. Duplicate sets of 20 to 30 seeds are placed in a plastic petri dish on three layers of Whatman No. 1 filter paper moistened with 5 ml of deionized water. Petri dishes are then transferred to a darkened growth chamber. Germination is carried out for up to 4 days at room temperature, with 5 ml of deionized water being added daily to each petri dish. On the fifth day, the germinated seedlings are harvested.
234
THIOREDOXIN AND GLUTAREDOXIN
[24]
In Vivo Labeling of Proteins with Monobromobimane Chemicals and Materials Endosperm of germinated seedlings Liquid N2 mBBr, 2.0 mM, freshly diluted in 100 mM Tris-HC1 buffer, pH 7.9 Procedure. At the indicated times, the dry seeds or germinated seedlings (selected on the basis of similar radicle or shoot length) are taken from the petri dish and the embryos or germinated axes are removed with a razor blade. Five endosperm from each lot are weighed and then ground in liquid N2 with a mortar and pestle. One milliliter of 2.0 mM mBBr in 100 mM Tris-HCl buffer, pH 7.9, is added just as the last trace of liquid N2 disappears. The thawed mixture is ground for another minute and transferred to a microcentrifuge tube of 1.5 ml capacity. The volume of the suspension is adjusted to 1 ml with the appropriate mBBr or buffer solution. Protein fractions of albumin/globulin, gliadin, and glutenin from endosperm of germinated seedlings are extracted as described above for semolina and flour. The extracted protein fractions are stored at - 2 0 ° until use for electrophoresis. A buffer control (minus mBBr) is included for each time point. Analysis of Labeled Samples by Polyacrylamide Gel Electrophoresis
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis of Monobromobimane-Labeled Samples Analysis by S D S - P A G E of the mBBr-derivatized samples of each protein fraction is performed in 15% polyacrylamide gels at pH 8.5 as described by Laemmli. 3° Before sample application, an aliquot (20/xl) of 50% (v/v) glycerol containing 0.01% (w/v) bromphenol blue is added to each mBBr-labeled sample. Gels of 1.5 mm thickness are developed for 16 hr at a constant current of 9 mA.
Native Gel Electrophoresis for Separating Different Gliadins Reagents and Equipment A gel solution in 100 ml final volume contains the following: 6.0 g acrylamide, 0.3 g bisacrylamide, 0.024 g ascorbic acid, 0.2 mg ferrous sulfate heptahydrate, 0.25 g aluminum lactate, and 85% lactic acid (certified ACS); the gel solution is adjusted to pH 3.1 with lactic acid 30 U. K. Laemrnli, Nature (London) 227, 680 (1970).
[24]
THIOREDOXINAND SEEDPROTEINS
235
Catalyst solution, hydrogen peroxide, 3% Tank buffer, aluminum lactate buffer, 0.5 g/liter, is adjusted to pH 3.1 with lactic acid Tracking dye, methyl green, 0.6% (w/v) in 80% (w/v) sucrose in tank buffer Electrophoresis equipment, Hoefer Model SE600 (Hoefer Scientific Instrument, San Francisco, CA) Procedure. To resolve the different types of gliadins, native polyacrylamide gel electrophoresis is performed in 6% gels of 3 mm thickness (a procedure designed to separate gliadins into the four types, a,/3, y, and ~0) as described by Bushuk and ZiUman 31 and modified for vertical slab gels by Sapirstein and Bushuk. 32 A gel stock solution can be prepared beforehand by mixing acrylamide, bisacrylamide, and aluminum lactate, adjusting to pH 3.1 with lactic acid, and then storing at 4 °. Before use the ascorbic acid (0.024 g) and ferrous sulfate (0.2 rag) are added to 100 ml of the gel stock solution, which is then degassed for 2 hr at 4 °. Because polymerization occurs essentially instantaneously after adding the hydrogen peroxide catalyst, it is best to set up and precool the gel plate (16 × 18 cm) assembly, with the 3-mm spacers, in a cold room. It is also best to pour the gel solution as quickly as possible through a funnel, mounted on a stand, that fits into the space between the plates with the comb already in place. Before sample application an aliquot (20 /xl) of the tracking dye is added to each sample. The duration of electrophoresis is approximately 4 hr, with a constant current of 50 mA. The temperature of the tank buffer ( - 2 - 3 liters) is controlled by circulating water at 21 ° during electrophoresis. (Caution: As gliadins are positively charged at this pH, it is imperative to reverse the polarity of the two electrodes, i.e., by attaching the negative electrode of the gel box to the positive outlet of the power supply and vice versa for the positive electrode.) Electrophoresis is terminated when the lower mobility band of the methyl green tracking dye migrates to about ! cm from the end of the gel. Monobromobimane Removal and Fluorescence Photography Reagents and Equipment
12% (w/v) Trichloroacetic acid 40% Methanol/10% acetic acid (v/v)
31W. Bushuk and R. R. Zillman, Can. J. Plant. Sci. 58, 505 (1978). 32H. D. Sapirstein and W. Bushuk, Cereal Chem. 62, 372 (1985).
236
THIOREDOXIN AND GLUTAREDOXIN
[24]
Spectroline transilluminator, (Spectronics Corp., Westbury, NY), Model TS-365, 365 nm ultraviolet Polaroid Land camera Polaroid Positive/Negative Landfilm, type 55 Wratten gelatin filter No. 8 (cutoff of 460 nm) Procedure. Following electrophoresis, gels are placed in 200 ml trichloroacetic acid and soaked for 4 to 6 hr with one change of solution to fix the proteins; gels are then transferred to 200 ml of 40% methanol/10% acetic acid for 8 to 10 hr (with two to three changes of solution) to remove excess mBBr. The fluorescence of mBBr, both free and protein bound, is visualized by placing gels on a light box fitted with an ultraviolet light source (365 nm). Gels are photographed with Polaroid Positive/Negative Landfilm, type 55, through a yellow Wratten gelatin filter No. 8 (cutoff of 460 nm) (exposure time ranges from 25 to 60 sec at f4.5). The negatives are used for densitometric scanning after being processed and dried (see below).
Protein Staining/Destaining/Photography Reagents Coomassie Brilliant Blue R-250, 0.25% (w/v) 40% methanol/10% acetic acid (v/v) Coomassie Brilliant Blue R-250 (0.1 g dissolved in 10 ml of 95%, v/v, ethanol) Trichloroacetic acid, 12% Procedure. The SDS gels are stained with 100 ml of 0.25% Coomassie Brilliant Blue R-250 in 40% methanol/10% acetic acid for 1 to 2 hr and destained overnight with several changes (200 ml) of the above solvent without the dye. 2° Aluminum lacate native gels are stained overnight in a filtered solution containing 0.1 g Coomassie Brilliant Blue R-250 in 240 ml of 12% trichloroacetic acid. Gels are destained overnight in 200 ml of 12% trichloroacetic acid. 1l Protein-stained gels are photographed with Polaroid type 55 film to produce prints and negatives. Prints are used to determine band migration distances and loading efficiency as well as to quantitate protein SH. Quantitation of Fluorescence [Reduction)
Scanning of Gels The Polaroid negatives of fluorescent gels and prints of wet proteinstained gels are scanned with a laser densitometer (Pharmacia-LKB, Piscataway, N J, UltroScan XL). Intensities of fluorescent bands are quantified by evaluating peak areas after integration with GelScan XL software. The
[24]
237
THIOREDOXIN AND SEED PROTEINS Proteln
Fluorescence 2
3
I 5
4
~ 1
I 5
Protein
Fluorescence I 6
7
8
9
10
I
I
6
10
kDa 97.4 66.2 w
45.0
31.0
21.5
14.4
Gliadins:
Glutenins:
1. 2. 3. 4. 5,
6. Control 7. GSH 8. GSH/GR/NADPH 9. NGS 10. NTS
Control GSH GSH/GR/NADPH NGS NTS
FIG. 1. Relative effectiveness of the NADP+/thioredoxin (NTS), NADP+/glutaredoxin (NGS), and the glutathione/glutathione reductase system (GSH/GR/NADPH) in the reduction of seed proteins (SDS, polyacrylamide gel electrophoresis).
intensity of fluorescent bands, when totally reduced by 2 mM DTT at 100° for 3 min and derivatized with mBBr, is proportional to the amount of added protein (up to 30/zg). It is assumed that the intensity of fluorescence of the proteins under analysis is also a direct measure of percent reduction of sulfhydryl groups. Specific Applications The techniques described above have been used successfully in determining the reduction of seed storage proteins and purified disulfide proteins from seeds as well as other sources. The following examples serve to illustrate use of the technique.
238
[241
THIOREDOXIN AND GLUTAREDOXIN
Effectiveness of Thiols in Reduction of Seed Proteins. By employing the in vitro labeling technique described above, our laboratory has obtained evidence that the NADP+/thioredoxin system reduces wheat storage proteins--gliadins and glutenins--much more effectively than the glutaredoxin or glutathione systems (Fig. 1). T M Similar results have been obtained in an unpublished survey of extracted prolamines and glutelins from several nonglutenous cereals. The system works equally well for pure disulfide proteins from seeds (Fig. 2) as well as other s o u r c e s . 12'19,2°,22 Change in Redox Status of Proteins during Germination. Using the in vivo labeling technique described above, our laboratory has obtained evidence for a change in redox state of wheat proteins during germination.11 We observed a progressive increase of 2- to 3-fold in reduction of the albumin/globulin, gliadin, and glutenin fractions up to the second day of germination; thereafter, reduction declined (Fig. 3). This pattern of redox change was coincident with the proteolytic degradation (or mobilization) of storage proteins. A similar series of events has been observed with barley DSG-1
BBTI
•
,g"
DTT GSH
~ + , ÷1i t
DTT ÷
_
GSH II +
_
C ~l +
DTT II
.
GSH ,
+
C ,
+
I
+ and - refer to thioredoxin (E.coli) C = Control (no reductant) •~ = Thioredoxin
Fie. 2. Dithiothreitol-reduced thioredoxin as a reductant for seed a-amylase and trypsin inhibitor proteins. DSG-1, Wheat a-amylase inhibitor; BBTI, soybean Bowman-Birk trypsin inhibitor (SDS, polyacrylamide gel electrophoresis).
[241
THIOREDOXIN AND SEED PROTEINS
5
I
I
,
o
.~a
i
a
•
,
,
I
239 I
t
o,/
i
i
~ ~ 3
~
o
0
0
1
2
3
4
Day
FIG. 3. In vivo reduction status of protein fractions during germination. [Data obtained by analysis of SDS-polyacrylamide gels.]
(C. Marx, J. H. Wong, and B. B. Buchanan, unpublished results); also in unpublished experiments, the reduction of disulfide bonds of gliadins and glutenins by the NTS was found to facilitate their degradation by proteases (I. Besse, and B. B. Buchanan, unpublished findings). Change in Redox Status of Thiol-Containing Proteins during Seed Maturation. Samples (wheat ears) are harvested at different stages of seed maturation up to complete maturity. Immediately after harvest, samples are put into liquid N2, and grains are dehuUed manually in a mortar in the presence of liquid N2. In vivo labeling with mBBr is performed under the conditions described for the in vivo labeling of germinated seeds. Results to date provide evidence that the storage proteins are synthesized in the reduced form and become oxidized during maturation and drying. Redox Changes during Technological Transformations. An extension of these experiments has revealed that the NADP+/thioredoxin system reduces selected flour proteins and strengthens the dough network formed
240
[251
THIOREDOXIN AND GLUTAREDOXIN
with flour of poor cooking quality, as measured by farinograph 16and baking tests. 33 These results suggest that addition of the NADP+/thioredoxin system improves products prepared from wheat and other cereals. Monobromobimane is currently being used as a probe to identify the major sulfhydryl/disulfide redox changes taking place in proteins during dough strengthening. Samplings are taken at different stages of the dough making process. Dough samples are put into liquid N2, and protein is fractionated and labeled with mBBr as described above.
33 K. Kobrehel, C. Nimbona, B. B. Buchanan, D. Bergmann, J. H. Wong, and B. C. Yee, "Gluten Proteins 1993," p. 381. Assoc. of Cereal Research, Detmold, Germany.
[251 A n a l y s i s a n d M a n i p u l a t i o n o f T a r g e t E n z y m e s Thioredoxin Control
By J E A N - P I E R R E
for
J A C Q U O T , E M M A N U E L L E ISSAKIDIS,
PAULETTE DECOTTIGNIES, MARTINE LEMAIRE,
and
MYROSLAWA MIGINIAC-MAsLOW
Introduction Several systems are able to control the activity, stability, and correct folding of enzymes through dithiol/disulfide isomerization reactions including the enzyme protein disulfide-isomerase, the glutathione-dependent glutaredoxin system, and the thioredoxin systems. 1-3 Plants contain two different thioredoxin systems, one extrachloroplastic in which thioredoxin is reduced through NADPH and the flavoenzyme NADPH-thioredoxin reductase (EC 1.6.4.5), and the other chloroplastic in which the reducing system is photoreduced ferredoxin and the iron-sulfur enzyme ferredoxinthioredoxin reductase. 4'5 To study thioredoxins and their reducing systems, it is necessary to use a target enzyme, the properties of which can be modified after reaction with reduced thioredoxin. Several proteins with this property have been isolated (including fructose-l,6-bisphosphatase, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, glu1 R. B. Freedman, FEBS Lett. 97, 201 (1979). z A. Holmgren, Annu. Rev. Biochem. 54, 237 (1985). 3 H. Eklund, F. K. Gleason, and A. Holmgren, Proteins: Struct. Funct, Genet. 11, 13 (1991). 4 B. B. Buchanan, Annu. Rev. Plant Physiol. 31, 341 (1980). 5 T. C. Johnson, K. Wada, B. B. Buchanan, and A. Holmgren, Plant Physiol. 85, 446 (1987).
METHODSIN ENZYMOLOGY,VOL.252
Copyright © 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.