* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Glycation by Ascorbic Acid Causes Loss of Activity of Ribulose
Expression vector wikipedia , lookup
Magnesium transporter wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Point mutation wikipedia , lookup
Interactome wikipedia , lookup
Genetic code wikipedia , lookup
Catalytic triad wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Specialized pro-resolving mediators wikipedia , lookup
Protein purification wikipedia , lookup
Biosynthesis wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Metalloprotein wikipedia , lookup
Photosynthesis wikipedia , lookup
Biochemistry wikipedia , lookup
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. References Andersson, I., Knight, S., Schneider G, Lindqvist, Y., Lundqvist, T., Brändén, C.I. and Lorimer, G.H. (1989) Crystal structure of the active site of ribulosebisphosphate carboxylase. Nature 377: 229–234. Bartoli, C.G., Pastoli, G.M. and Foyer, C. (2000) Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol. 123: 335–343. Brownlee, M., Vlassara, H., Kooney, A., Ulrich, P. and Cerami, A. (1986) Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232: 1629–1632. Bushnell, T.P., Bushnell, D. and Jagendorf, A.T. (1993) A purified zinc protease of pea chloroplast, EP1, degrades the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Physiol. 103: 585–591. Cotgreave, I.A. and Gerdes, R.G. (1998) Recent trends in glutathione biochemistry-glutathione-protein interactions. Biochem. Biophys. Res. Commun. 242: 1–9. Drickamer, K. (1996) Breaking the curse of the AGEs. Nature 382: 211–212. Dunn, J.A., Ahmed, M.U., Murtiashaw, M.H., Richardson, J.M., Walla, M.D., Thorpe, S.R. and Baynes, J.W. (1990) Reaction of ascorbate with lysine and protein under autoxdizing conditions: Formation of N e-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate. Biochemistry 29: 10964–10970. Ellis, R.J. (1979) The most abundant protein in the world. Trends Biochem. Sci. 4: 241–244. Farazi, T.A., Waksman, G. and Gordon, J.I. (2001) The biology and enzymology of protein N-myristoylation. J. Biol. Chem. 276: 39501–39504. Foyer, C., Rowell, J. and Walker, D. (1983) Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157: 239–244. Garcìa-Ferris, C. and Moreno, J. (1993) Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis. Photosynth. Res. 35: 55–66. Geiger, T. and Clarke, S. (1987) Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. J. Biol. Chem. 262: 785–794. Glomb, M.A. and Pfahler, C. (2001) Amides are novel protein modifications formed by physiological sugars. J. Biol. Chem. 276: 41638–41647. Hartman, F.C. and Lee, E.H. (1989) Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction. J. Biol. Chem. 246: 11784–11789. Hartman, F.C., Soper, T.S., Niyogi, S.K., Mural, R.J., Foote, R.S., Mitra, S., Lee, E.H., Machanoff, R. and Larimer, F.W. (1987) Function of Lys-166 of Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis. J. Biol. Chem. 262: 3496–3501. Herrmann, J. and Andrae, W. (1963) Oxydative abbauprodukte der L-ascorbinsäure. Nährung 7: 243–260. Horiuchi, S., Araki, N. and Morino, Y. (1991) Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. J. Biol. Chem. 266: 7329–7332. Houtz, R.L., Poneleit, L., Jones, S.B., Royer, M. and Stults, J.T. (1992) Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species. Plant Physiol. 98: 1170–1174. Glycation of Rubisco by ascorbic acid Houtz, R.L., Stults, J.T., Mulligan, R.M. and Tolbert, N.E. (1989) Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. USA 86: 1855–1859. Ishida, H., Makino, A. and Mae, T. (1999) Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329. J. Biol. Chem. 274: 5222–5226. Itzhaki, H., Naveh, L., Lindahl, M., Cook, M. and Adam, Z. (1998) Identification and characterization of DegP, a serine protease associated with the luminal side of the thylakoid membrane. J. Biol. Chem. 273: 7094–7098. Kawano, N., Ella, E., Ito, O., Yamauchi, Y. and Tanaka, K. (2002) Comparison of adaptability to flash flood between different flash flood tolerant rice cultivars. Soil Sci. Plant Nutr. 48: 659–665. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. Lindahl, M., Tabak, S., Cseke, L., Pichersky, E., Andersson, B. and Adam, Z. (1996) Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants. J. Biol. Chem. 271: 29329–29334. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275. Mae, T., Kamei, C., Funaki, K., Miyadai, K., Makino, A., Ohira, K. and Ojima, K. (1989) Degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase in the lysates of the chloroplasts isolated mechanically from wheat (Triticum aestivum L.) leaves. Plant Cell Physiol. 30: 193–200. Maillard, L.C. (1912) Action des acides amines sur les sucres: formation des melanoidines par voie methodique. C. R. Acad. Sci. (Paris) 154: 66–68. Makino, A., Mae, T. and Ohira, K. (1984) Relation between nitrogen and ribulose-1,5-bisphosphate carboxylase in rice leaves from emergence through senescence. Plant Cell Physiol. 25: 429–437. Mehta, R.A., Fawcett, T.W., Porath, D. and Mattoo, K. (1992) Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5bisphosphate carboxylase/oxygenase. J. Biol. Chem. 267: 2810–2816. Mizutari, K., Ono, T., Ikeda, K., Kayashima, K.-I. and Horiuchi, S. (1997) Photo-enhanced modification of human skin elastin in actinic elastosis by Ne(carboxymethyl)lysine, one of the glycoxidation products of the Maillard reaction. J. Clin. Dermatol. 108: 797–802. Moreno, J. and Spreitzer, R.J. (1999) C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase. J. Biol. Chem. 274: 26789– 26793. Nagaraj, R.H., Sell, D.R., Prabhakaram, M., Ortwerth, B.J. and Monnier, V.M. (1991) High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis. Proc. Natl. Acad. Sci. USA 88: 10257–10261. Nakamura, K., Hasegawa, T., Fukunaga, Y. and Ienaga, K. (1992) Crosslines A and B as candidates for the fluorophores in age- and diabete-related crosslinked proteins, and their diacetates produced by Maillard reaction of a-Nacetyl-L-lysine with D-glucose. J. Chem. Soc. Chem. Commun. 992–994. Obayashi, H., Nakano, K., Shigeta, H., Yamaguchi, M., Yoshimori, K., Fukui, M., Fujii, M., Kitagawa, Y., Nakamura, N., Nakamura, K., Nakazawa, Y., 1341 Ienaga, K., Ohta, M., Nishimura, M., Fukui, I. and Kondo, M. (1996) Formation of crossline as a fluorescent advanced glycation end product in vitro and in vivo. Biochem. Biophys. Res. Commun. 226: 37–41. Ortwerth, B.J. and Olesen, P.R. (1988a) Glutathione inhibits the glycation and crosslinking of lens proteins by ascorbic acid. Exp. Eye Res. 47: 737–750. Ortwerth, B.J. and Olesen, P.R. (1988b) Ascorbic acid-induced crosslinking of lens proteins: evidence supporting a Maillard reaction. Biochim. Biophys. Acta 956: 10–22. Ortwerth, B.J., Slight, S.H., Prabhakaram, M., Sun, Y. and Smith, J.B. (1992) Site-specific glycation of lens crystallins by ascorbic acid. Biochim. Biophys. Acta 1117: 207–215. Ostersetzer, O., Tabak, S., Yarden, O., Shapira, R. and Adam, Z. (1996) Immunological detection of proteins similar to bacterial proteases in higher plant chloroplasts. Eur. J. Biochem. 236: 932–936. Peñarrubia, L. and Moreno, J. (1990) Increased susceptibility of ribulose-1,5bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments. Arch. Biochem. Biophys. 281: 319–323. Pongor, S., Ulrich, P.C., Bencsath, F.A. and Cerami, A. (1984) Aging of proteins: Isolation and identification of a fluorescent chromophore from the reaction of polypeptides with glucose. Proc. Natl. Acad. Sci. USA 81: 2684–2688. Sell, D.R. and Monnier, V.M. (1989) Structure elucidation of a senescence cross-link from human extracellular matrix. J. Biol. Chem. 264: 21597– 21602. Slight, S.M., Prabhakaram, M., Shin, D.B., Feather, M.S. and Ortwerth, B.J. (1992) The extent of N e-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid. Biochim. Biophys. Acta 1117: 199–206. Spreitzer, R.J. (1993) Genetic dissection of Rubisco structure and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 411–434. Takabe, T., Rai, A.K. and Akazawa, T. (1984) Interaction of constituent subunits in ribulose 1,5-bisphosphate carboxylase from Aphanothece halophytica. Arch. Biochem. Biophys. 229: 202–211. Takata, K., Horiuchi, S., Araki, N., Shiga, M., Saitoh, M. and Morino, Y. (1988) Endocytic uptake of nonenzymatically glycosylated proteins is mediated by a scavenger receptor for aldehyde-modified proteins. J. Biol. Chem. 263: 14819–14825. Tsunasawa, S. and Sakiyama, F. (1984) Amino-terminal acetylation of proteins: an overview. Methods Enzymol. 106: 165–170. Wagner, G.J. (1979) Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts. Plant Physiol. 64: 88–93. Yamauchi, Y., Sugimoto, T., Sueyoshi, K., Oji, Y. and Tanaka, K. (2002) Appearance of endopeptidases during the senescence of cucumber leaves. Plant Sci. 162: 615–619. Yoshimura, K., Yabuta, Y., Ishikawa, T. and Shigeoka, S. (2000) Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiol. 123: 223–233. Zobel-Thropp, P., Yang, M.C., Machado, L. and Clarke, S. (2000) A novel posttranslational modification of yeast elongation factor 1A. J. Biol. Chem. 275: 37150–37158. (Received June 12, 2002; Accepted September 7, 2002)