Plant Cell Physiol. 45(4): 407–415 (2004)
JSPP © 2004
Thioredoxin Reduction Alters the Solubility of Proteins of Wheat Starchy
Endosperm: An Early Event in Cereal Germination
Joshua H. Wong 1, Nick Cai 1, Charlene K. Tanaka 2, William H. Vensel 2, William J. Hurkman 2 and Bob B.
Buchanan 1, 3
1
2
Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720, U.S.A.
USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, U.S.A.
;
A KCl-soluble, albumin/globulin fraction of wheat
(Triticum aestivum L.) starchy endosperm was further separated into a methanol-insoluble fraction that contained
metabolic proteins and a methanol-soluble fraction that
contained “chloroform-methanol” or CM-like proteins.
Reduction of the disulfide bonds of the CM proteins with
thioredoxin or dithiothreitol altered their properties so
that, like the metabolic proteins, they were insoluble in
methanol. Glutathione had little effect, indicating dithiol
specificity. Proteomic analysis of the CM protein fraction
revealed the presence of isoforms of low molecular weight
disulfide proteins (α-amylase, α-amylase/trypsin and WCI
proteinase inhibitors, lipid transfer proteins, γ-thionins),
stress enzymes (Cu-Zn superoxide dismutase and peroxidase), storage proteins (α-, γ- and ω-gliadins, low molecular weight glutenin subunits and globulins of the avenin N9
type), and a component of protein degradation (polyubiquitin). These findings support the view that, in addition to
modifying activity and increasing protease sensitivity,
reduction by thioredoxin alters protein solubility, thereby
promoting processes of the grain starchy endosperm, notably the mobilization of reserves during germination and
seedling development.
endosperm (henceforth endosperm), thereby (ii) increasing the
susceptibility of storage proteins to proteolysis and (iii) changing the activity of enzymes either directly by reduction or indirectly by inactivating specific inhibitor proteins (Besse et al.
1997, Jiao et al. 1992, Jiao et al. 1993, Kobrehel et al. 1992,
Lozano et al. 1996, Marx et al. 2003, Montrichard et al. 2003,
Serrato et al. 2001, Wong et al. 1995, Wong et al. 2003, Yano et
al. 2001). More recent work with germinating grain from transgenic barley that overexpresses thioredoxin in the endosperm
has broadened the role of thioredoxin to include, among other
effects, increasing protein solubility in aqueous solution (Wong
et al. 2002). While the results with transgenic barley are consistent with such a role, complementary in vitro evidence is
lacking.
The present study was undertaken to provide evidence for
the role of thioredoxin in modifying protein solubility using
methanol-soluble and -insoluble protein fractions of wheat
endosperm as a model system. We now report that reduction by
thioredoxin or dithiothreitol (DTT) alters the proteins in the
methanol-soluble fraction so that they become less soluble in
methanol and, in this respect, partition with enzymes and other
metabolic proteins of the seed that are insoluble in methanol.
The disulfide proteins undergoing this transition have been
identified as a mixture of chloroform-methanol (CM) and lipid
transfer proteins, thionins, stress enzymes and storage proteins. In the discussion below, this group is collectively referred
to as the “CM protein fraction.”
Keywords: Germination — Protein solubility — Redox regulation — Thioredoxin.
Abbreviations: CM, chloroform-methanol; 2-DE, two-dimensional electrophoresis; DTT, dithiothreitol; MS, mass spectroscopy;
mBBr, monobromobimane.
This paper is dedicated to Professor Keishiro Wada to commemorate his retirement from the University of Kanazawa. Work with Professor Wada on purothionin provided the first evidence for a role for
thioredoxin in seeds (Wada and Buchanan 1981, Johnson et al. 1987).
Results
The fractionation procedure summarized in Fig. 1 separates the KCl-soluble proteins (albumins and globulins) of
wheat endosperm (either the isolated endosperm itself or
derived flour) into two fractions: (1) a hydrophilic (methanolinsoluble) fraction containing primarily metabolic proteins—
enzymes and associated structural proteins, and (2) a hydrophobic (methanol-soluble) fraction consisting of CM and associated proteins discussed below (Hurkman and Tanaka, in preparation). In the present experiments, approximately 65% and
35% of the total protein extracted with KCl were recovered,
respectively, in the metabolic and CM protein fractions. The
KCl-soluble protein fraction was used in the experiments below
Introduction
Research accomplished during the past decade supports
the view that thioredoxin functions in the germination of cereals (wheat and barley) by (i) reducing the intramolecular
disulfide bonds of storage and other proteins of the starchy
3
Corresponding author: E-mail, [email protected]; Fax, +1-510-642-7356.
407
408
Thioredoxin and protein solubility
Fig. 1 Separation of metabolic (KCl-soluble/methanolinsoluble) and CM (KCl-soluble/methanol-soluble) proteins from wheat endosperm.
in which solubility was monitored as a function of reduction.
Proteins were identified in the methanol-soluble fraction in
order to observe their behavior in the different reduction treatments.
Two-dimensional electrophoresis (2-DE) showed that
major components of the CM protein fraction migrated to
acidic and neutral pI regions and had an Mr below 20 kDa
(Fig. 2). Proteomic analysis revealed six types of proteins: storage, enzyme inhibitor, protein degradation, non-specific lipid
transfer proteins, thionins and stress enzymes. The list
included: α-amylase, α-amylase/trypsin and WCI proteinase
inhibitors, several lipid transfer proteins and γ-thionins, polyubiquitin, Cu-Zn superoxide dismutase, peroxidase, α-, γ- and
ω-gliadins, low molecular weight glutenin subunits and globulins (Table 1). The proteins forming the bulk of this group, the
enzyme inhibitors, have historically been designated CM pro-
teins (Garcia-Olmedo et al. 1987). Except for the ω-gliadins
and polyubiquitin, all of the proteins identified contain conserved cysteines, and, with the three new additions given
below, all appear to be targets of thioredoxin (Kobrehel et al.
1991, Maeda et al. 2004, Marx et al. 2003, Wong et al. 2003).
The results in Figs. 3 and 4 (see below) show that two of these
additions, lipid transfer protein and WCI proteinase inhibitor,
are thioredoxin targets. It seems likely that the third addition,
Cu-Zn superoxide dismutase, is also a target, but low abundance prevented its identification under the conditions used for
the present gel analyses.
As seen in Table 1, the proteins identified as thioredoxin
targets fulfill functions in protein degradation and storage,
enzyme inhibition and stress response. Interestingly, two of the
disulfide storage proteins, the γ-gliadins and globulins (avenin
N9) are wheat allergens for baker’s asthma (Amano et al. 1998,
Fig. 2 2-DE map of the CM proteins of wheat
endosperm. Protein identifications are located in
Table 1.
Thioredoxin and protein solubility
Table 1
409
Identification of CM proteins of wheat endosperm
Spot No.
I. Storage Proteins
1
2
3
11
22
26
29
30
33
38
51
54
55
79a
81
125
126
149
II. Enzyme Inhibitors
42
66
78
81
83
85
86
87
89
90
91
92
94
95
96
97
99
100
101
102
104
105
106
107
108
109
110
140
Protein
Cys*
Swiss Prot No.
ω-Gliadin, 1B
ω-Gliadin, 1B
ω-Gliadin, 1B
LMW glutenin subunit
γ-Gliadin
γ-Gliadin
γ-Gliadin
γ-Gliadin
γ-Gliadin
α-Gliadin
Globulin (Avenin N9)
γ-Gliadin
Globulin (Avenin N9)
Globulin (Avenin N9)
Grain softness protein 1b
LMW glutenin subunit
LMW glutenin subunit
α-Gliadin
0
0
0
6
6
7
7
7
7
6
nd
4
nd
nd
10
6
6
6
a
Q8W3W4
Q94G96
Q94G95
Q94G97
Q94G97
Q94G95
Q9M4M0
P80356
P06659
P80356
P80356
Q43659 b
Q8GU18
Q8GU18
Q9M4L6
α-Amylase inhibitor 0.19
α-Amylase inhibitor/trypsin inhibitor CM3
α-Amylase/subtilisin inhibitor (WASI)
α-Amylase inhibitor/trypsin inhibitor CM3
α-Amylase inhibitor/trypsin inhibitor CM3
α-Amylase inhibitor/trypsin inhibitor CM3
α-Amylase inhibitor/trypsin inhibitor CM3
α-Amylase inhibitor 0.28
α-Amylase inhibitor 0.53
α-Amylase inhibitor 0.19
α-Amylase inhibitor/trypsin inhibitor CM17
α-Amylase inhibitor/trypsin inhibitor CM16
WCI proteinase inhibitor
α-Amylase inhibitor 0.19
α-Amylase inhibitor 0.19
α-Amylase inhibitor 0.19
α-Amylase inhibitor 0.19
α-Amylase inhibitor 0.19
α-Amylase inhibitor 0.53
α-Amylase inhibitor 0.53
α-Amylase inhibitor 0.19
α-Amylase inhibitor/trypsin inhibitor CM16
α-Amylase inhibitor/trypsin inhibitor CM17
α-Amylase inhibitor Ima1
α-Amylase inhibitor Ima1
α-Amylase inhibitor Ima1
α-Amylase inhibitor Ima1
α-Amylase inhibitor/trypsin inhibitor CM17
10
7
3
7
7
7
7
10
9
10
7
7
6
10
10
10
10
10
9
9
10
7
7
7
7
7
7
7
P01085
P17314
P16347
P17314 b
P17314
P17314
P17314
P01083
P01084
P01085
Q41540
P16159
P83207
P01085
P01085
P01085
P01085
P01085
P01084
P01084
P01085
P16159
Q41540
Q49956
Q49956
Q49956
Q49956
Q41540
a
a
410
Thioredoxin and protein solubility
Table 1
Spot No.
Cont.
Protein
Cys*
III. Thionins
69
γ-Thionin
70
γ-Thionin
72
γ-Thionin
IV. Stress-related Enzymes
87
Superoxide dismutase [Cu-Zn]
93
Superoxide dismutase [Cu-Zn]
130
Peroxidase
131
Peroxidase
V. Lipid Transfer Proteins
111
Nonspecific lipid-transfer protein
112
Nonspecific lipid-transfer protein
119
Lipid transfer protein
VI. Protein Degradation
149
Polyubiquitin
Swiss Prot No.
8
8
8
Q39999
Q39999
Q39999
2
2
8
8
P23345
Q96123
Q8LK23
Q8LK23
8
8
7
P24296
P24296
Q9FEK9
0
Q9ZRI6
* Number of conserved cysteines.
a
Proteins identified by DuPont et al. (2000).
b
Spot number 81 contained two components: grain softness protein 1b and α-amylase inhibitor/trypsin
inhibitor CM3.
Buchanan et al. 1997). The role of others, lipid transfer proteins and thionins, is unknown. The three isoforms of ω-gliadin
were the only proteins identified that lack disulfide groups
(Shewry and Miflin 1985).
In support of earlier findings (Kobrehel et al. 1991,
Kobrehel et al. 1992, Jiao et al. 1993, Johnson et al. 1987,
Marx et al. 2003, Wada and Buchanan 1981, Wong et al. 2003),
we observed in experiments with the fluorescent probe, monobromobimane (mBBr), that components of the CM protein
fraction were effectively reduced by thioredoxin or DTT but
not by GSH (Fig. 3). When examining their properties further,
we made the unexpected observation that, on reduction, properties of the CM and associated proteins changed such that they
became insoluble in methanol as determined by protein staining of 2-DE gels (Fig. 4) and chemical measurements (Fig. 5).
That is, they partitioned like metabolic proteins after reduction. The extent of change in solubility followed the relative
effectiveness of the individual reductants in reducing the relevant disulfide groups: compared with total reduction by DTT,
thioredoxin was effective in bringing about the solubility
change, whereas GSH was not (Fig. 5). Moreover, this shift in
solubility was not specific to E. coli Trx. Fig. 6 shows that
reduction by Trx and NTR from barley catalyzed a similar
change. Again, GSH was ineffective and reduction by DTT and
heat completely altered the solubility of components except for
residual levels of several proteins below 20 kDa. That reduction
Fig. 3 Reduction of metabolic and CM protein fractions of wheat endosperm. Proteins in the KCl-soluble fraction were either not treated (control) or reduced using the NADP/glutathione system (GSH), NADP/thioredoxin system (NTS), or with DTT. Following reduction, proteins were
derivatized with mBBr, separated into metabolic and CM protein fractions and analyzed by 2-DE.
Thioredoxin and protein solubility
411
Fig. 4 Distribution of proteins between the metabolic and CM protein fractions following reduction. Gels as in Fig. 3 except stained with
Coomassie Blue for protein.
was responsible for the change in properties was supported by
the finding that loss of methanol solubility was dependent on
the concentration of added reductant (DTT in Fig. 7). Moreover, reduction of the isolated CM protein fraction with DTT
changed the constituent proteins so that their solubility in aqueous buffer increased from 3% to 80%, presumably as a result of
Fig. 5 Distribution of proteins between the metabolic and CM protein fractions following reduction. Fractions were assayed directly for
protein.
conversion from disulfide (S-S) to sulfhydryl (-SH) form (data
not shown). It remains to be seen whether the change in solubility of the CM (α-amylase inhibitor) proteins is involved in
the accelerated onset of activity of α-amylase observed in
transgenic barley overexpressing thioredoxin (Wong et al.
2002). It should be noted that investigators have long used DTT
to enhance the solubility of cereal seed proteins, such as gliadins and glutenins of wheat (Kim and Bushuk 1995), but more
soluble components such as those in the CM protein fraction
appear not to have been examined in this respect.
The results suggest that thioredoxin alters the structure of
components of the CM protein fraction so that they not only
lose biochemical activity (if present) and resistance to proteolysis (Jiao et al. 1992, Jiao et al. 1993), but also change in solu-
Fig. 6 The change in solubility of the CM protein fraction is also
effected by the barley NADP/thioredoxin system. Protein reduction
was as described in Fig. 3 except that barley thioredoxin and NADPthioredoxin reductase were included in one treatment and the resulting
fractions were analyzed by 1-D SDS/PAGE. CM, CM protein fraction;
M, metabolic protein fraction.
412
Thioredoxin and protein solubility
Fig. 9 Role of thioredoxin in solubilizing proteins of endosperm during germination.
Fig. 7 The change in solubility of the CM protein fraction depends
on extent of reduction. Proteins in the KCl-soluble fraction were
reduced with increasing concentrations of DTT. Following reduction,
proteins were derivatized with mBBr, separated into metabolic (M)
and CM (CM) protein fractions, and analyzed by 1-DE. The SDSPAGE gel shown was stained for protein.
Fig. 8 Reduction changes gliadin solubility. Immunoblots of KClsoluble proteins were reacted with gliadin anti-serum. The gliadin
standard used as a positive control was extracted from flour with 70%
ethanol (top panel) and 50% propanol (bottom panel). Reduction was
carried out as in Fig. 3. Based on molecular weight, the cross-reactive
bands were α- and γ-type gliadins.
bility. To this end, they become more hydrophilic and fractionate like classical enzymes. This change in solubility on
reduction of the methanol-soluble proteins was assessed independently in immunoblot experiments with anti-gliadin serum
(Fig. 8). The proteins of this family consist of the ω-gliadins,
acidic to neutral pI, 55–70 kDa proteins, and the α- and γgliadins, neutral pI, 36–55 kDa proteins (Fig. 2). The antigliadin serum reacted with the α- and γ-gliadins, but we did not
detect the higher molecular weight ω-gliadins (Fig. 8). The
immunoblots and 2-D gels of the same samples confirmed that
reduction changes protein solubility: the α- and γ-gliadins were
less soluble in methanol following reduction (data not shown).
In contrast, the distribution of ω-gliadins, as detected in 2-D
gels, did not change dramatically in the presence of reductant—a finding in keeping with the lack of a solubility change
as would be expected from the absence of disulfide bonds.
Discussion
A growing body of evidence has shown that thioredoxin
acts as an early signal in the germination of cereal (see above)
and possibly dicot seeds (Montrichard et al. 2003) by effecting, along with other changes, the reduction of proteins, including the CM proteins examined in this study (Kobrehel et al.
1992, Marx et al. 2003). The results described in this paper
demonstrate that reduction is accompanied by a change in protein solubility, thus confirming recent findings with transgenic
grain (barley) overexpressing thioredoxin (Wong et al. 2002). It
is noted that other effects of thioredoxin were observed in the
transgenic studies, notably an acceleration of germination and
α-amylase synthesis and an increase in pullulanase activity
(Cho et al. 1999, Kim et al. 2003, Wong et al. 2002).
In the present experiments, reduction by thioredoxin was
found to change the properties of proteins in the albumin/globulin fraction—low molecular weight disulfide proteins, storage
proteins and associated enzymes—so that they became more
hydrophilic and fractionated like metabolic proteins. The
results thus show that, in reducing proteins of the endosperm,
thioredoxin not only activates enzymes and accelerates proteolysis, but also enhances solubilization (Fig. 9), thereby collectively promoting the mobilization of storage reserves—a prerequisite to germination and seedling development (Bewley and
Black 1994). It remains to be seen whether an increase in solubility influences the function of other proteins identified in this
study—e.g., the Cu-Zn superoxide dismutase and peroxidase
stress enzymes. It is also of interest to know whether solubility
changes of this nature occur during grain development—a
process in which redox regulation appears to play a central role
(Gobin et al. 1996, Rhazi et al. 2003, De Gara et al. 2003).
Finally, it is noted that the solubility changes effected by
thioredoxin extend to proteins from other sources. Rancourt et
al. (2004) recently reported a dramatic increase in the solubility of proteins of sputum of patients suffering from cystic fibrosis following reduction by thioredoxin.
Thioredoxin and protein solubility
Materials and Methods
Plant material
Wheat plants (Triticum aestivum L., cv. Butte 86) were grown in
a climate controlled greenhouse with an average maximum daytime
temperature of 25°C and nighttime temperature of 17°C (Altenbach et
al. 2003). Starchy endosperm was harvested from grain 40 d postanthesis (dpa), frozen in liquid nitrogen and stored at –80°C. Mature
grain was tempered and milled to flour with a Brabender Quadramat
Junior (South Hakensack, NJ, U.S.A.) using standard procedures at the
Western Wheat Quality Laboratory (U.S. Department of Agriculture,
Agricultural Research Service, Pullman, WA, U.S.A.).
Protein isolation
Proteins from endosperm or flour were separated into three fractions based on solubility in KCl and methanol (Vensel et al. 2002).
Endosperm was ground and flour was suspended in cold (4°C) KCl
buffer (50 mM Tris-HCl, 100 mM KCl, 5 mM EDTA, pH 7.8) using
200 µl of buffer per 50 mg endosperm or flour. For endosperm fractionation, protease inhibitors (Mini Complete Protease Inhibitor Cocktail, Roche Applied Science, Indianapolis, IN, U.S.A.) were added to
the KCl buffer (1 tablet/10 ml). Samples were incubated on ice for
5 min with intermittent mixing (Vortex Genie 2, Scientific Industries,
Inc., Bohemia, NY, U.S.A.) and centrifuged at 4°C for 15 min at
14,000 rpm (Tomy MRX-151, Peninsula Laboratories, Inc., Belmont,
CA, U.S.A.). The pellet, containing the gliadins and glutenins, was not
further analyzed. The proteins of the supernatant fraction, designated
the KCl-soluble fraction, were subjected to reduction and fractionated
as described below. The isolation procedure is summarized in Fig. 1.
In vitro protein reduction
The disulfide proteins of the KCl-soluble fraction from
endosperm or flour were identified after reduction using a fluorescent
thiol-specific probe, mBBr, coupled with proteomics (Marx et al.
2003, Yano et al. 2001, Balmer et al. 2003, Wong et al. 2003, Vensel et
al. 2002). Reduction of protein disulfides was effected with: (i) the
NADP/thioredoxin system, consisting of 0.125 µmol NADPH, 0.7 µg
E. coli NTR, and 0.8 µg E. coli thioredoxin (Jiao et al. 1992), or (ii)
the NADP/glutathione system, composed of 0.125 µmol NADPH,
0.1 µmol reduced GSH, and 2.0 µg yeast GSH reductase (Sigma, St.
Louis, MO, U.S.A.). As indicated, the proteins of the E. coli thioredoxin system were replaced by their counterparts from barley. The
reductant was pre-incubated at 37°C for 15 min in 30 mM Tris-HCl
buffer, pH 7.5, in a volume of 40 µl, then 10 µl (18 µg protein) of the
KCl extract was added and incubated for another 20 min at 37°C. To
stop the enzymatic reaction and label the targets, 0.1 µmol mBBr in
5 µl acetonitrile was added to each sample, which was then incubated
for an additional 15 min at room temperature. The labeling reaction
was terminated by adding 1.0 µl of 100 mM 2-mercaptoethanol. Total
reduction of disulfide proteins was carried out by incubating an aliquot of the extract with 2.5 mM DTT at 90–95°C for 5 min and derivatizing with mBBr as described above.
Protein solubility in response to reduction by different DTT concentrations
To confirm that reduction of protein disulfide bonds was the main
basis for the shift in protein solubility in the different solvents, the
KCl-soluble fraction from flour was treated with increasing concentrations of DTT, 0, 1.0, 2.5, 5,0, 10.0, 20.0, 40.0 mM, as indicated, at
37°C for 20 min. Total reduction was achieved with 2.5 mM DTT at
90–95°C for 5 min. The reduced proteins were derivatized with mBBr
as described above and the resulting reaction mixture was fractionated
and analyzed by 1-D SDS/PAGE as described below.
413
Protein fractionation
After reduction, the KCl-soluble fraction was further separated
into methanol-insoluble and -soluble fractions. For this separation, 5
volumes of 0.1 M ammonium acetate in methanol were added to the
KCl-soluble fraction. Following incubation overnight at –20°C, methanol-insoluble proteins were collected by centrifugation at 14,000 rpm
for 15 min at 4°C. The supernatant or methanol-soluble fraction was
decanted and transferred to another set of tubes. The pellet was rinsed
with acetone, air-dried, and solubilized in either urea buffer (9 M urea,
4% NP-40, 1% DTT and 2% ampholytes) for 2-DE or Laemmli (1970)
sample buffer for 1-D SDS/PAGE. Analysis by mass spectroscopy
revealed that the major constituents of this fraction were metabolic
proteins. The proteins in the methanol-soluble fraction were recovered
by precipitation with four volumes of ice-cold acetone. The pellet was
rinsed with acetone, air dried, and solubilized in (a) the urea buffer for
2-DE, or (b) Laemmli (1970) sample buffer for 1-D SDS/PAGE.
Because the most abundant proteins in this fraction were identified as
CM-like proteins, it is referred to as the CM protein fraction.
One-dimensional SDS/PAGE analysis
mBBr-labeled proteins were separated in 10–20% Criterion TrisHCl gels (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.) run at
constant 150 V for 70 min at room temperature in Tris/glycine/SDS
buffer, pH 8.3 (Laemmli 1970). Fluorescence of the SH groups of protein bands was captured on a UV 365 nm light-box by Gel Doc-1000
with the Quantity One program, version 4.1 (BioRad Laboratories,
Inc., Hercules, CA, U.S.A.). Exposure (aperture and time) was determined under a non-saturated condition with the totally reduced DTT
sample. The same exposure setting was then used to capture fluorescent images of all other treatments. The gels were stained with
Coomassie Blue G-250 and protein patterns were captured similarly on
a white light light-box. Molecular weights were estimated on 1-D and
2-DE gels using Mark 12 unstained protein standards (Invitrogen,
Carlsbad, CA, U.S.A.).
Immunoblotting with anti-gliadin
Proteins in the KCl/methanol-soluble and -insoluble fractions
were separated by 1-D SDS/PAGE and transferred to nitrocellulose
using a Criterion Gel/Plate Blot System (Bio-Rad) in Transfer Buffer
at pH 8.3 (25 mM Tris, 150 mM glycine, 20% (v/v) methanol and
0.1% (w/v) SDS) at 50 V for 60 min at 4°C. Blots were probed first
with rabbit anti-gliadin serum (Sigma, St. Louis, MO, U.S.A.) in a
1 : 1,000 dilution after blocking with 5% non-fat powder milk in TBS
buffer (20 mM Tris-HCl, pH 7.5 containing 0.15 M NaCl). After washing three times with TBS buffer containing 0.05% Nonidet P-40, the
blot was probed with goat anti-rabbit IgG conjugated to horseradish
peroxidase (Bio-Rad). Color development was carried out using a
TMB substrate kit for peroxidase (Vector Laboratories, Inc., Burlingame, CA, U.S.A.). Molecular weights were estimated on immunoblots using Plus 2 Prestained Standards (Invitrogen, Carlsbad, CA,
U.S.A.).
Protein determination
Protein in the KCl-soluble fraction and the methanol-soluble and
-insoluble fractions was determined by the Bio-Rad assay based on the
dye-binding procedure (Bradford 1976). Protein dissolved in the urea
rehydration buffer was determined by two methods: (1) the RC DC
procedure (Bio-Rad) based on the Lowry protocol (Lowry et al. 1951);
and (2) the Non-Interfering Protein Assay (Geno Technology, Inc., St
Louis, MO) based on the specific binding of copper ions to the peptide backbone of proteins after the interfering agents were removed
from the assay solutions by a Universal Protein Precipitating Agent.
414
Thioredoxin and protein solubility
2-DE and proteomic analysis
Proteins in the metabolic and CM protein fractions were separated by 2-DE (Vensel et al. 2002). Two-dimensional gels of the
mBBr-labeled proteins were stained with Coomassie Blue G-250 and
identified using a CM protein map. For the map, CM proteins were
isolated from Butte 86 flour, separated by 2-DE and identified by mass
spectrometry (MS) and tandem MS as described by Vensel et al.
(2002). In addition, electrospray ionization mass spectrometry was
performed using a QSTAR Pulsar i quadrupole time-of-flight mass
spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada)
equipped with a Proxeon Biosystems (Odense, Denmark) nanoelectrospray source. In-gel digestion of protein spots was as described
previously (Vensel et al. 2002), except that acetonitrile was omitted
from the final extraction step and 10% formic acid used instead. For
LC/MS/MS, 20 µl of the resulting extract was loaded onto a C-18 trap
cartridge and chromatographed on a reversed-phase column (Vydac
238EV5.07515, 75 µ × 150 mm, Hesperia, CA, U.S.A.) fitted at the
effluent end with a coated spray tip (FS360-50-5-CE, New Objective
Inc., Woburn, MA, U.S.A.). An LC Packings nano-flow LC system
(Dionex, Sunnyvale, CA, U.S.A.) with autosampler, column switching
device, loading pump, and nano-flow solvent delivery system was used
to elute the column. Elution solvents were: A (0.5% acetic acid) and B
(80% acetonitrile, 0.5% acetic acid). Samples were eluted at 220 nl
min–1 with the following profile: 8% B at 0 min to 80% B by 12 min
through 13 min to 8% B by 14 min continuing at 8%B to 28 min.
Spectra were transferred to a PC and proteins identified using a suite
of programs from Genomic Solutions (Madison, WI, U.S.A.); the
Knexus automation client was used for both peptide mass mapping and
MS/MS data analysis. Reported mass spectrometer identifications
from the NCBInr plant protein database search had expectation values
of 1×10–3 (one chance in 1,000 that the match was due to a random
event) or less.
Acknowledgments
This work was supported by funds from Syngenta, Inc. and the
Agricultural Experiment Station of the University of California. We
thank Dr. Myeong-Je Cho for a sample of recombinant barley thioredoxin and NADP-thioredoxin reductase.
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(Received October 31, 2003; Accepted January 13, 2004)