Download Glycation by Ascorbic Acid Causes Loss of Activity of Ribulose

Plant Cell Physiol. 43(11): 1334–1341 (2002)
JSPP © 2002
Glycation by Ascorbic Acid Causes Loss of Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Its Increased Susceptibility to Proteases
Yasuo Yamauchi 1, Yukinori Ejiri and Kiyoshi Tanaka
Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori, 680-8553 Japan
;
Glycation is a process whereby sugar molecules form a
covalent adduct with protein amino groups. In this study,
we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein
in chloroplast tissues, and examined effects of glycation on
the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with
AsA and Rubisco in vitro at physiological conditions
(10 mM AsA, pH 7.5, 25°C in the presence of atmospheric
oxygen). At the early stage of glycation (phase 1), the
amount of AsA attaching to Rubisco increased at an almost
linear rate (0.5–0.7 mol AsA incorporated (mol Rubisco)–1
d–1). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N e(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation,
and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation
of high molecular weight aggregates cross-linked between
subunits. Glycation led to a decrease in Rubisco activity
(half-life about 7–8 d). Furthermore, glycated Rubisco of
phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of
phase 1 compared to that of native Rubisco. Results
obtained here suggest that AsA is possibly an important
factor in the loss of activity and turnover of Rubisco.
Keywords: Ascorbic acid — Cucumber (Cucumis sativus L.)
— Glycation — Protein modification — Rubisco — Senescence.
Abbreviations: AGE, advanced glycation end product(s); AsA,
ascorbic acid; CML, N e-(carboxymethyl)lysine; DHA, dehydroascorbic acid; HAP, highly aggregated product; Rubisco, ribulose-1,5bisphosphate carboxylase/oxygenase.
Introduction
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco,
EC 4.1.1.39) is responsible for the primary step of CO2 fixation in C3 plants and is speculated to be the most abundant protein on earth (Ellis 1979, Spreitzer 1993). The Rubisco content
1
in leaves is proportional to the rate of photosynthesis and therefore the turnover rate of Rubisco is an important factor in regulating the rate of leaf photosynthesis (Makino et al. 1984). It is
well known that Rubisco content changes drastically during
leaf development; for instance, it rapidly increases during leaf
expansion and then decreases during the initial stages of leaf
senescence in rice and cucumber (Makino et al. 1984,
Yamauchi et al. 2002). However, the degradation mechanism of
Rubisco protein is still a major mystery in plant physiology.
Many attempts to identify the proteases responsible for the
in vivo degradation of Rubisco have been made: however, the
potential candidates are few (Bushnell et al. 1993). On the
other hand, proteins are often conferred by various posttranslational modifications such as acetylation (Tsunasawa and
Sakiyama 1984), phosphorylation, N-terminal processing,
myristoylation (Farazi et al. 2001), deamidation, isomerization, racemization (Geiger and Clarke 1987), glutathiolation
(Cotgreave and Gerdes 1998), methylesterification (ZobelThropp et al. 2000) and glycation, and some of them are known
to change the stability of proteins. In the case of Rubisco, posttranslational acetylation of the N-terminus and trimethylation
of Lys-14 of the large subunit are known (Houtz et al. 1992),
and the trimethylation protects against tryptic proteolytic inactivation (Houtz et al. 1989). In addition, oxidative treatments
increase susceptibility to proteases (Peñarrubia and Moreno
1990, Garcìa-Ferris and Moreno 1993, Moreno and Spreitzer
1999). Therefore, chemical modifications of Rubisco protein
may influence protein turnover, in addition to protease degradation of the native protein.
Glycation is a process whereby carbonyl groups of sugar
molecules form a covalent adduct with protein amino groups
by the Maillard reaction (Maillard 1912). Maillard products
have been observed in vivo in association with several longlived proteins, e.g. lens proteins and collagen. In addition, the
in vivo presence of early stage glycation products such as
Schiff base adducts and Amadori rearrangement products has
been demonstrated for hemoglobin, erythrocyte membrane proteins, lens crystallin, collagen, albumin, and other serum proteins. Several sugars can participate in glycation, especially the
autoxidation products of L-ascorbic acid (AsA), which is
known to rapidly glycate and crosslink protein (Ortwerth and
Olesen 1988b). For example, bovine lens proteins are glycated
6.5-fold higher by AsA than by glucose (Ortwerth et al. 1992).
Knowledge of glycation in plant tissues is limited: however, it
Corresponding author: E-mail, [email protected]; Fax, +81-857-31-6702.
1334
Glycation of Rubisco by ascorbic acid
1335
Fig. 1 Quantitative analysis of the incorporation of [14C]glycation agents into Rubisco. (A) Effect of temperature on the incorporation of
[14C]AsA (left) and [14C]glucose (Glc, right) into Rubisco. Number represented upside of column shows incubation temperature. (B) Temporal
changes in incorporated [14C]AsA into Rubisco at 4°C (open square) and 25°C (closed square).
is natural to speculate that chloroplast proteins are glycated by
AsA because AsA is present at high concentrations (12–
25 mM) within that organelle (Foyer et al. 1983). The aims of
this study are to investigate whether Rubisco is post-translationally modified by AsA glycation and if this process subsequently affects the activity or protease susceptibility of the
protein.
Results
Quantitative analysis of incorporation of [14C]AsA to Rubisco
during glycation
To prepare glycated Rubisco, glycation was performed by
a method conventionally adopted for the animal system. AsA
may be a potent glycation-inducing agent of Rubisco because
chloroplasts contain high physiological concentrations of AsA
(Foyer et al. 1983). In the presence of atmospheric oxygen, a
progressive yellow discoloration of an initially colorless sterile
10 mM AsA solution in phosphate buffer (pH 7.5), occurred
over the experimental period, which is an indicator of glycation. At physiological temperatures, the browning increased
during the first 10 d of incubation and slowed thereafter. After
30 d, extensive Maillard browning was observed.
Quantitative analysis using [14C]AsA showed that the rate
of AsA incorporation was dependant upon temperature (Fig.
1A). It was low at 4°C (0.1 mol (mol holoenzyme)–1 d–1) and
increased 5- to 7-fold at 25°C (0.5–0.7 mol (mol holoenzyme)–1
d–1) and 10-fold at 37°C (1 mol (mol holoenzyme)–1 d–1). These
rates were evidently higher than those of glucose, another
major candidate of glycation. The incorporation of [14C]AsA at
25°C, which is physiological temperature, was almost linear for
6 d and then nearly reached saturation, while the incorporation
of [14C]AsA at 4°C was linear throughout the experimental
period (Fig. 1B).
Hydroxyl radicals, caused by metal ion contamination,
were not involved in this process because after the addition of
EDTA to reaction mixture no difference in the results was
observed (data not shown), and the cleavage products of the
Rubisco large subunit by hydroxyl radicals (Ishida et al. 1999)
were not present in our analysis.
Table 1 Basic amino acid composition of glycated Rubisco
incubated with AsA a
Fig. 2 Absorption spectra of glycated Rubisco. After incubation of
purified Rubisco (80 mg ml–1) with 10 mM AsA at 25°C, the solution
was dialyzed extensively against distilled water and used for analysis.
Spectra of samples after 15 d incubation are not shown because the
pattern is almost same to that of 10 d.
Lys
Arg
His
a
0d
3d
10 d
30 d
30
33
17
29
33
16
28
33
17
25
33
17
Number of residues per large and small subunits (mol. wt 67,000).
1336
Glycation of Rubisco by ascorbic acid
Fig. 3 SDS-PAGE profile of glycated Rubisco. Purified Rubisco was incubated with 10 mM AsA or [14C]AsA at 25°C for indicated days.
Native Rubisco is represented as 0 day Rubisco. After the solution was dialyzed extensively against distilled water, an equal volume of SDSPAGE sample buffer was added and the sample denatured. After SDS-PAGE on 12% polyacrylamide gel, Rubisco was detected by Western blotting using anti-Rubisco antibody (A) and autoradiography (B). Each lane includes 1 mg (A) or 20 mg (B) of Rubisco protein. HAPs, L and S represent highly aggregated products (>150 kDa), large subunit and small subunit, respectively. Arrowhead indicates boundary between stacking and
separating gels.
Changes in spectra profile and basic amino acid composition
By UV absorption analysis of glycated Rubisco after dialysis against distilled water, typical changes of the spectrum of
glycated protein were also observed (Fig. 2). As glycation proceeded, an increase in UV absorption was observed. Tailing
above 300 nm is responsible for the brown color of the material (Pongor et al. 1984). The increase in UV absorption around
280 nm is due to aromatic glycation adducts of Rubisco lysyl
residues, Pongor et al. (1984) showed that extensive increases
of UV absorption below 300 nm was due to glycated poly
(L-lysine). This is also supported by data shown in Table 1, i.e.
as glycation proceeded, intact Lys residues decreased. Other
basic amino acids apparently did not decrease through the
glycation process. This rate of loss of Lys residues was higher
than that of incorporation of AsA, e.g. for 3 d incubation,
eight Lys residues per holoenzyme were lost (Table 1) while
1.5–2 mole of AsA was attached to one holoenzyme (Fig. 1).
This is probably due to linkage by one AsA molecule between
two Lys residues and production of many by-products that
also cause glycation reaction during AsA glycation process
(Herrmann and Andrae 1963, Nakamura et al. 1992, Obayashi
et al. 1996, Glomb and Pfahler 2001).
Aggregation of Rubisco subunits and incorporation of AsA
during glycation
The alteration of the molecular mass of Rubisco during
glycation was determined by SDS-PAGE. As glycation progressed, many bands of high molecular masses were observed
in the presence of AsA (Fig. 3A), in contrast to little change in
the absence of AsA (data not shown). These bands reacted with
anti-Rubisco antiserum, thus they were derived from Rubisco
subunits. Judging from their molecular masses they are homologous and heterogeneous aggregates between large and small
subunits. These aggregations are due to cross-linked modifica-
tion of subunits of Rubisco between basic amino acid residues and AsA, for instance, the formation of pentosidine
and crossline (Sell and Monnier 1989, Nakamura et al. 1992,
Obayashi et al. 1996). Cross-linking via disulfide bonds did not
contribute to this aggregation because the treatment of glycated Rubisco samples used for SDS-PAGE with the reductant
2-mercaptoethanol did not remove the presence of high molecular mass aggregated Rubisco bands. In contrast, aggregated
Rubisco bands formed during oxidization stress disappeared in
reductive SDS-PAGE conditions (Peñarrubia and Moreno
1990, Mehta et al. 1992). Attachment of [14C]AsA to Rubisco
protein was also confirmed by autoradiography (Fig. 3B). In
this experiment, radioactivity was detected both in monomeric
and aggregated subunits after 6 d incubation (Fig. 3B). From
the results of incorporation of [14C]AsA and changes in spectra
curve (Fig. 1B, 2), the incorporation of AsA to Rubisco was
almost completed within 10 d, and thereafter cross-linking
between glycation adducts of subunits is a major event.
From results obtained in the above experiments, the
process of glycation can be divided into two phases and we used
these terms in the following experiments; i.e. phase 1 (until 10 d
of incubation) where AsA is incorporated linearly and Rubisco
seldom aggregates, and phase 2 (after 10 d) where incorporation of Rubisco has saturated and aggregation proceeds.
Immunochemical analysis of glycation adduct formation
Monoclonal antibodies against glycation products were
developed by Horiuchi et al. (1991) in order to facilitate physiological and biochemical analysis of glycation products. In this
study, we examined the formation of glycation adducts in glycated Rubisco using these antibodies. Anti-advanced glycation
end product(s) (AGE) antibody is raised against glycated BSA,
and it reacts with glycated BSA but not native BSA, therefore
it recognizes chemical adducts formed by glycation. Also in
Glycation of Rubisco by ascorbic acid
1337
Fig. 4 Immunoblot analyses of glycated Rubisco using monoclonal antibody against glycation-derived epitopes, anti-AGE antibody (A), antiCML antibody (B), and anti-pentosidine antibody (C). Each lane contains 1 mg of protein. Legends and abbreviations are the same as Fig. 3.
our experiment, it reacted with glycated Rubisco of phases1
and 2 but not native Rubisco (Fig. 4A). Anti N e-(carboxymethyl)lysine (CML) antibody also reacted with glycated Rubisco
in a similar manner to anti-AGE antibody (Fig. 4B). Moreover,
a monoclonal antibody raised against a glycation adduct, pentosidine that forms intra- and intermolecular cross-links
between Lys and Arg residues, also reacted mainly with glycated Rubisco of phase 2 (Fig. 4C).
Inhibition of glycation by GSH, Lys, and aminoguanidine
We examined the inhibitory effects of several amino acids,
GSH, urea and aminoguanidine (Fig. 5). The most effective
agent was GSH, a major intracellular antioxidant, which inhibited incorporation of AsA to Rubisco at its physiological concentration. Lys and aminoguanidine, an analogue of the side
chain of Arg, were also effective. Arg and Gly showed a slight
effect and other agents had no effect upon glycation.
Loss of Rubisco activity by glycation
Amino groups of proteins are known to participate in the
Fig. 5 Inhibition of the incorporation of [14C]AsA into Rubisco by
several agents. Purified Rubisco was incubated with [14C]AsA in the
absence (Cont) or presence of each agent at 5 mM for 72 h. In control
sample, 1.5 mol AsA was incorporated into 1 mol Rubisco and it was
defined as 100%. Abbreviations are citrulline (Cit) and aminoguanidine (AG).
Fig. 6 Loss of the activity of Rubisco by glycation. Rubisco was incubated at 25°C (A) or 4°C (B) in the absence (open square) or presence of
10 mM AsA (closed square) or 1 mM DHA (closed circle). Rubisco activity of control on 0 day (1.1 mmol min–1 mg–1) was defined as 100%.
1338
Glycation of Rubisco by ascorbic acid
Fig. 7 Susceptibility of glycated Rubisco to commercial proteases. Rubisco (600 ng) glycated with 10 mM AsA for days indicated over the panels was incubated without (A) or with 0.5 ng of trypsin (B), chymotrypsin (C) and V8 protease (D) for 15 min at 37°C. Native Rubisco is represented as 0 day Rubisco. After incubation, the reaction was stopped by mixing with an equal volume of SDS-sample buffer and boiling, and
Rubisco protein was detected by Western blotting using anti-Rubisco antiserum. Legends and abbreviations are the same as Fig. 3.
binding of sugars, in particular the e-amino groups of lysine
side chains are the most reactive sites (Ortwerth et al. 1992).
Lys residues of Rubisco are implicated in Rubisco catalysis
(Hartman et al. 1987, Hartman and Lee 1989). Therefore, we
examined whether glycation by AsA contributed towards the
loss of Rubisco activity. In our results, glycated Rubisco of
phase 1 decreased in activity linearly, and then the activity of
Rubisco of phase 2 slowly decreased further (Fig. 6A). At
physiological conditions, the half time of loss of Rubisco activity was about 7–8 d. The oxidative form of AsA, dehydroascorbic acid (DHA) is a more effective inactivator, i.e. physiological concentration of DHA (1 mM) inactivated 80% of activity
only 1 d after incubation and it reacted with anti-AGE and antiCML monoclonal antibodies (data not shown). Loss of activity
by AsA is also dependent on temperature corresponding to
incorporation of AsA, i.e. it was low at 4°C (Fig. 6B). Rates of
loss of activity correlate to AsA incorporation at each temperature (Fig. 1B, 6).
Increased susceptibility of glycated Rubisco to proteases
It is hypothesized that glycated Rubisco has an increased
susceptibility to proteases due to the disturbance of the native
conformation by chemical modifications. To explore this
hypothesis further, glycated and native Rubisco were treated
with proteases and their degradation was analyzed. In these
experiments, we used three commercial proteases of different
substrate specificities, trypsin (basic amino acid-specific), chy-
motrypsin (preferentially hydrophobic amino acids), and V8
protease (acidic amino acids-specific). In our results, glycation
changed susceptibility of Rubisco to all proteases (Fig. 7). Glycated Rubisco in early period of phase 1 (within 3 d of incubation) did not show any change of susceptibility to native
Rubisco. However, glycated Rubisco of phase 2 showed drastically increased susceptibility to commercial proteases, especially to chymotrypsin (Fig. 7C). Highly aggregated products
(HAPs) and small subunits showed some limited resistance to
proteases.
Discussion
In chloroplasts, the concentration of AsA and DHA
together has been estimated to be 12–25 mM (Foyer et al.
1983), while glucose in chloroplasts is likely to be found in
much lower concentrations (Wagner 1979). In addition, AsA
binds to Rubisco much faster than glucose (Fig. 1), therefore
AsA is the most likely glycation agent in chloroplasts. Glycation by AsA is principally caused by the oxidative products of
AsA, such as DHA (Slight et al. 1992), xylosone (Nagaraj et al.
1991) and threose (Dunn et al. 1990), rather than AsA itself. In
our experiment, 1 mM DHA decreased Rubisco activity much
faster than AsA (Fig. 6A). Furthermore, half of AsA was
degraded after 3 d incubation (data not shown), and GSH partially inhibited the glycation (Fig. 5). Therefore, oxidative
products containing carbonyl groups derived from AsA con-
Glycation of Rubisco by ascorbic acid
tributed to the glycation of Rubisco, similar to the case of lens
proteins (Slight et al. 1992). Atmospheric and photosynthesisderived oxygen in chloroplasts is an important factor for glycation process by AsA in vivo. Usually, plants are exposed under
intensive light conditions, and photosynthesis generates many
reactive oxygen species. CML levels of the sun-exposed skin
area of individuals was significantly higher than that of the
sun-unexposed area of the same individuals (Mizutari et al.
1997). This active contribution of reactive oxygen species is
involved in the formation of AGEs, which are preferentially
sequestered to UV-exposed and photo-aged skin. Therefore
glycation of plant proteins might proceed at a higher rate under
sunlight.
Amino acid residues of Rubisco to which AsA bound
were thought to be Lys and Arg because AsA bound to only
Lys and Arg residues in lens proteins (Ortwerth and Olesen
1988b). From our results, the markedly reduced activity and
number of Lys residues of glycated Rubisco (Fig. 6, Table 1)
suggest that it is highly possible that modification by glycation
occurs at catalytic Lys residues. For example, Lys-166, Lys191 and Lys-329 of the Rubisco large subunit of Rhodospirillum rubrum are catalytic residues and potential candidates
(Hartman et al. 1987, Hartman and Lee 1989) and they are conserved among various plant species (Andersson et al. 1989).
Aggregation of Rubisco subunits is probably due to
intermolecular cross-linking glycation adducts such as pentosidine (linkage between Lys and Arg residues, Sell and
Monnier 1989), crossline (linkage between Lys and Lys residues, Nakamura et al. 1992, Obayashi et al. 1996), 1,3-bis(5amino-5-carboxypentyl)imidazolium and N-{2-[(5-amino-5carboxypentyl)amino]-2-oxoethyl}lysine (linkage between Lys
and Lys residues, Glomb and Pfahler 2001). Aggregated
Rubisco after 10 d incubation reacted with anti-pentosidine antibody (Fig. 4C), suggesting at least pentosidine was produced
via the pathway proposed by Nagaraj et al. (1991). However,
the contribution of pentosidine might be a small because intact
Arg residues apparently showed no decrease during glycation
(Table 1). Therefore, linkage between Lys and Lys would
mainly contribute towards cross-linking between subunits.
Spontaneous glycation by AsA will occur under normal
physiological conditions in vivo: however, its rate is thought to
be lesser than that obtained in our in vitro experiments, because
GSH and several amino acids inhibited glycation at their physiological concentrations (Fig 5). Inhibition by GSH is likely to
prevent the oxidation of AsA (Ortwerth and Olesen 1988a),
and inhibition by amino acids and their analogs is due to trap
glycation adducts, similar to the case of glycation by glucose
(Brownlee et al. 1986, Drickamer 1996). However, DHA usually exists as about 10% of total ascorbate, and the ratio of
AsA/DHA decreases due to senescence (Bartoli et al. 2000),
environmental stresses such as high light (Yoshimura et al.
2000) and submergence (Kawano et al. 2002). Thus the rate of
glycation might increase and glycated proteins might accumulate in such conditions.
1339
In phase 1, AsA attached to Rubisco (Fig. 1), however the
native conformation of Rubisco of phase 1 was likely not to be
seriously disturbed considering the unchanged susceptibility to
proteases compared to native Rubisco (Fig. 7). However, the
structure of Rubisco of phase 2 was disturbed by saturated AsA
incorporation because Rubisco showed drastically increased
susceptibility to proteases (Fig. 7). CML formed on side chains
of Lys residue eliminates the positive charge of Lys residues
and raises the negative charge, resulting in possible conformational changes and increasing the susceptibility to protease degradation. Taking the drastically increased susceptibility of
Rubisco of phase 2 to proteases into consideration, Rubisco of
phase 2 is likely to be rapidly degraded by endogenous chloroplast proteases in vivo. A study using isolated wheat chloroplasts showed that proteolytic activity against SDS-denatured
Rubisco was observed but not against native Rubisco (Mae et
al. 1989). Glycated Rubisco of phase 2 has increased susceptibility to commercial proteases (Fig. 7) as seen in Rubisco denatured by SDS, therefore it might be degraded by various chloroplast proteases. Recent genetic analysis revealed the
existence of various proteases similar to proteases of other
organisms in chloroplasts, for example, ClpP, PrcA (Ostersetzer et al. 1996), FtsH (Lindahl et al. 1996), and DegP
(Itzhaki et al. 1998).
Results in this study suggest the possibility that AsA is
an important factor for loss of Rubisco activity and turnover
of Rubisco. Glycation is a non-specific and non-enzymatic
reaction proceeding spontaneously under physiological conditions, thus it is natural to speculate that glycation occurs in
whole plant tissues containing reducing sugars and AsA, and
might be implicated in protein turnover. To clear this hypothesis, further evidence that existence of glycation process in vivo
is necessary.
Materials and Methods
Materials
Anti-AGE, anti-CML, and anti-pentosidine monoclonal antibodies were purchased from Dojindo Laboratories (Kumamoto, Japan).
14
14
14
L-[carboxyl- C] Ascorbic acid, D-[U- C]glucose, and NaH CO3 were
purchased from Amersham Pharmacia Biotech. (Buckinghamshire,
U.K.). All other reagents were purchased from Wako Pure Chemical
Industries (Osaka, Japan). Cucumber (C. sativus L. suyo) purchased
from Yamato Nohen (Nara, Japan) was grown in the field, and healthy
leaves were harvested, frozen in liquid N2 and stored at –80°C until use.
Preparation of Rubisco
Cucumber leaves frozen by liquid nitrogen were homogenized
with five volumes of 100 mM Tris-HCl, pH 8.0 containing 5 mM
dithiothreitol and 0.1 mM EDTA. After centrifugation at 20,000´g for
20 min, proteins in the supernatant were fractionated by ammonium
sulfate precipitation between 33% and 45% saturation. The fractionated proteins were dissolved in a minimum volume of 100 mM TrisHCl, pH 8.0 containing 5 mM dithiothreitol and 0.1 mM EDTA, and
applied to DEAE-Toyopearl (Tosoh, Japan) equilibrated with the same
buffer. Proteins were eluted by a linear gradient of 0–0.5 M NaCl. The
fractions containing the majority of the protein, monitored by absorb-
1340
Glycation of Rubisco by ascorbic acid
ance at 280 nm, were combined and proteins were precipitated by 45%
saturated ammonium sulfate. The precipitate was dissolved in 100 mM
Tris-HCl, pH 8.0 containing 5 mM dithiothreitol, 0.1 mM EDTA and
0.2 M NaCl, and then applied to a gel filtration column (HW-55F,
Tosoh, Japan) equilibrated with same buffer. After the addition of
glycerol to a final concentration of 40%, the purified Rubisco was
stored at –20°C, or 4°C with 0.05% NaN3 until use. Only purified
Rubisco showing both electrophoretic homogeneity and greater than
1.0 mmol min–1 mg–1 specific activity, was used for analysis. Protein
was measured by the method of Lowry et al. (1951).
Preparation of anti-Rubisco antiserum
Two mg of purified Rubisco was dissolved in 100 ml of 10 mM
potassium phosphate buffer, pH 7.5 containing 137 mM NaCl and
3 mM KCl. After mixing with an equal volume of complete Freund
adjuvant, the emulsion was injected to a mouse (strain ICR). After a
week, the same amount of antigen was injected as booster.
In vitro glycation of Rubisco
Glycation of Rubisco was done by the method of Takata et al.
(1988) with slight modifications. Purified Rubisco (1 mg ml–1) was
incubated with AsA (10 mM) in 0.2 M sodium phosphate buffer, pH
7.5. Reagents were sterilized by filtration (0.42 mm). After incubation,
the solution was dialyzed extensively against distilled water or sodium
phosphate buffer, and used for analysis. Absorption spectra were taken
on a Shimadzu spectrophotometer (MPS-2000, Kyoto, Japan).
Incorporation of [14C]AsA and [14C]glucose into Rubisco
After purified Rubisco (200 mg) was incubated with either
10 mM [14C]AsA or [14C]glucose (18.5 kBq each) in a total volume of
100 ml (sodium phosphate buffer, pH 7.5), proteins were precipitated
by the addition of 100 ml of 10% (w/v) trichloroacetic acid and were
incubated for 10 min on ice. After centrifuging for 5 min, the precipitate was washed with 200 ml of 5% trichloroacetic acid, and was further washed with 100 ml of diethyleter. The resultant precipitate was
dissolved in 100 ml of 10% (w/v) SDS, 10 ml of Scintisol 500 (Dojindo Laboratories) was added and radioactivity was measured by scintillation counting (Walac, Perkin Elmer, MA, U.S.A.).
Amino acid composition analysis
After native or glycated Rubisco was dialyzed against water, it
was freeze-dried. Powder was resuspended in 266 ml of 6 M HCl containing 0.2% phenol, and then incubated at 110°C for 24 h in vacuo.
After drying under N2, residue was dissolved in 0.3 ml of 0.02 M HCl
and the insoluble materials were removed by filtration (0.45 mm). The
amino acid composition was analyzed by amino acid analyzer (L8800, Hitachi, Japan) using an ion exchange column (#2622, Hitachi).
Rubisco activity
Rubisco activity was measured according to the method of Takabe et al. (1984) with slight modifications. Briefly, Rubisco was fully
activated in reaction mixture (100 mM Tris-HCl, pH 7.8, 10 mM
MgCl2, and 10 mM NaH14CO3) at 25°C, then 12.5 ml of 20 mM ribulose-1,5-bisphosphate (Sigma, St. Louis, MO, U.S.A.) was added.
After incubation for 1 min, the reaction was stopped by the addition of
20 ml of acetic acid. Then incorporated 14C into acid stable fraction
was measured by scintillation counting.
Electrophoresis
SDS-PAGE was performed by the method of Laemmli (1970)
using 12% polyacrylamide gel under reducing conditions. Fluorograms of 14C-labeled samples were prepared from dried gels that had
been impregnated with EN3HANCE (New England Nuclear). Western
blotting was performed according to manufacturers’ instructions after
transblotting to polyvinyldifluoride membrane (Atto, Tokyo, Japan).
Monoclonal antibodies were diluted 1 : 3,000 prior to use, and alkaline phosphatase-conjugated anti-mouse IgG antibody (StressGen Biotechnologies, Canada) was used as a secondary antibody.
Degradation of native and glycated Rubisco by commercial proteases
Native or glycated Rubisco (each 600 ng) was incubated with
0.5 ng of trypsin, chymotrypsin, or V8 protease in total reaction volume of 20 ml containing 50 mM HEPES-KOH, pH 7.0, for 15 min at
37°C. Reaction was stopped by the addition of 20 ml of SDS-sample
buffer and boiling for 3 min. The degradation of Rubisco was detected
by SDS-PAGE and subsequent Western blotting using anti-Rubisco
antiserum.
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(Received June 12, 2002; Accepted September 7, 2002)