Fe2+-catalyzed Site-specific Cleavage of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase Close to the Active Site
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111072200
ISSN1083-351X
AutoresShen Luo, H. Ishida, Amane Makino, Tadahiko Mae,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoPrevious work has demonstrated that the large subunit (rbcL) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from wheat is cleaved at Gly-329 by the Fe2+/ascorbate/H2O2 system (Ishida, H., Makino, A., and Mae, T. (1999) J. Biol. Chem. 274, 5222–5226). In this study, we found that the rbcL could also be cleaved into several other fragments by increasing the incubation time or the Fe2+ concentration. By combining immunoblotting with N-terminal amino acid sequencing, cleavage sites were identified at Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Conformational analysis demonstrated that five of them are located in the α/β-barrel, whereas Gly-122 is in the N-terminal domain but near the bound metal in the adjacent rbcL. All of these residues are at or very close to the active site and are just around the metal-binding site within a radius of 12 Å. Furthermore, their CαH groups are completely or partially exposed to the bound metal. A radical scavenger, activation of RuBisCo, or binding of a reaction-intermediate analogue to the activated RuBisCo, inhibited the fragmentation. These results strongly suggest that the rbcL is cleaved by reactive oxygen species generated at the metal-binding site and that proximity and favorable orientation are probably the most important parameters in determining the cleavage sites. Previous work has demonstrated that the large subunit (rbcL) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from wheat is cleaved at Gly-329 by the Fe2+/ascorbate/H2O2 system (Ishida, H., Makino, A., and Mae, T. (1999) J. Biol. Chem. 274, 5222–5226). In this study, we found that the rbcL could also be cleaved into several other fragments by increasing the incubation time or the Fe2+ concentration. By combining immunoblotting with N-terminal amino acid sequencing, cleavage sites were identified at Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Conformational analysis demonstrated that five of them are located in the α/β-barrel, whereas Gly-122 is in the N-terminal domain but near the bound metal in the adjacent rbcL. All of these residues are at or very close to the active site and are just around the metal-binding site within a radius of 12 Å. Furthermore, their CαH groups are completely or partially exposed to the bound metal. A radical scavenger, activation of RuBisCo, or binding of a reaction-intermediate analogue to the activated RuBisCo, inhibited the fragmentation. These results strongly suggest that the rbcL is cleaved by reactive oxygen species generated at the metal-binding site and that proximity and favorable orientation are probably the most important parameters in determining the cleavage sites. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo, 1The abbreviations used are: RuBisCoribulose 1,5-bisphosphate carboxylase/oxygenaserbcLlarge subunit of RuBisCorbcSsmall subunit of RuBisCoROSreactive oxygen speciesanti-rbcL-Nsite-specific antibody against the N-terminal portion of wheat rbcLanti-rbcL-Csite-specific antibody against the C-terminal portion of wheat rbcLTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCABP2-carboxyarabinitol 1,5-bisphosphateCaps3-(cyclohexylamino)propanesulfonic acid 1The abbreviations used are: RuBisCoribulose 1,5-bisphosphate carboxylase/oxygenaserbcLlarge subunit of RuBisCorbcSsmall subunit of RuBisCoROSreactive oxygen speciesanti-rbcL-Nsite-specific antibody against the N-terminal portion of wheat rbcLanti-rbcL-Csite-specific antibody against the C-terminal portion of wheat rbcLTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCABP2-carboxyarabinitol 1,5-bisphosphateCaps3-(cyclohexylamino)propanesulfonic acidEC 4.1.1.39) is a bifunctional enzyme that catalyzes both photosynthetic CO2 assimilation and photorespiratory carbon oxidation in the stroma of the chloroplasts. It is the most abundant leaf protein, which accounts for 40–50% soluble protein and 20–30% total leaf nitrogen in the mature leaves of C3 plants (1.Makino A. Mae T. Ohira K. Plant Cell Physiol. 1984; 25: 429-437Google Scholar, 2.Makino A. Mae T. Ohira K. Plant Physiol. (Bethesda). 1985; 79: 57-61Crossref PubMed Google Scholar, 3.Evans J.R. Seemann J.R. Briggs W. Photosynthesis. Alan R. Liss, Inc., New York1989: 183-205Google Scholar). During senescence, RuBisCo is rapidly degraded, and its nitrogen is remobilized and translocated into growing organs, thus affecting photosynthesis and nitrogen economy in plants (4.Mae T. Makino A. Ohira K. Thomson W.W. Nothnagel E.A. Huffaker R. Plant Senescence: Its Biochemistry and Physiology. The American Society of Plant Physiologists, Rockville, MD1987: 123-131Google Scholar). Recent studies (5.Desimone M. Henke A. Wagner E. Plant Physiol. (Bethesda). 1996; 111: 789-796Crossref PubMed Scopus (157) Google Scholar, 6.Desimone Wagner E. Johanningmeier U. Planta. 1998; 205: 459-466Crossref Scopus (57) Google Scholar, 7.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar, 8.Ishida H. Shimizu S. Makino A. Mae T. Planta. 1998; 204: 305-309Crossref PubMed Scopus (89) Google Scholar, 9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) have suggested that reactive oxygen species (ROS) may trigger the degradation of RuBisCo in plants. Our previous works (7.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar, 8.Ishida H. Shimizu S. Makino A. Mae T. Planta. 1998; 204: 305-309Crossref PubMed Scopus (89) Google Scholar) indicated that the large subunit of RuBisCo (rbcL) is directly cleaved into 37- and 16-kDa fragments by ROS in both chloroplast lysates and intact chloroplasts under illumination. A similar fragmentation was also observed when the purified RuBisCo was incubated with the Fe2+/ascorbate/H2O2 system (7.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar). Furthermore, the cleavage site has been identified at glycine 329, close to the active site of RuBisCo, and it has been suggested that ROS generated at the catalytic site of the rbcL by Fenton reaction may lead to this site-specific fragmentation (9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Therefore, the mechanism for the Fe2+-catalyzed oxidative cleavage of RuBisCo needs to be confirmed.It has been well documented that various biomolecules are susceptible to damage by ROS generated by the presence of reduced transition metals such as Fe2+ and Cu1+ (10.Halliwell B. Gutteridge G.M.C. Methods Enzymol. 1984; 105: 47-56Crossref PubMed Scopus (163) Google Scholar, 11.Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1254) Google Scholar). When these transition metals specifically bind to a protein, the polypeptide backbone can be cleaved by ROS in the presence of H2O2 and/or O2 and a reducing agent such as ascorbate or dithiothreitol (12.Kim K. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1985; 260: 15394-15397Abstract Full Text PDF PubMed Google Scholar, 13.Soundar S. Colman R.F. J. Biol. Chem. 1993; 268: 5264-5271Abstract Full Text PDF PubMed Google Scholar, 14.Chou W-Y. Tsai W-P. Lin C-C. Chang G-G. J. Biol. Chem. 1995; 270: 25935-25941Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15.Zhang Z. Barlow J.N. Baldwin J.E. Schofield C.J. Biochemistry. 1997; 36: 15999-16007Crossref PubMed Scopus (85) Google Scholar, 16.Cao W. Barany F. J. Biol. Chem. 1998; 273: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17.Shimon M.B. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1998; 273: 34190-34195Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18.Hlavaty J.J. Benner J.S. Hornstra L.J. Schildkraut I. Biochemistry. 2000; 39: 3097-3105Crossref PubMed Scopus (42) Google Scholar, 19.Ishida, H., Anzawa, D., Kokubun, N., Makino, A., and Mae, T. (2002) Plant Cell Environ., in pressGoogle Scholar). Protein fragmentation by ROS produced by bound metal ions through Fenton reaction is not a random process. Kim et al. (12.Kim K. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1985; 260: 15394-15397Abstract Full Text PDF PubMed Google Scholar) has suggested that ROS are generated locally around irons bound at specific sites on enzymes, and these ROS are responsible for the specific cleavage of proteins such as glutamine synthetase, adenylyltransferase, and pyruvate kinase. Wei et al. (20.Wei C-H. Chou W-Y. Huang S-M. Lin C-C. Chang G-G. Biochemistry. 1994; 33: 7931-7936Crossref PubMed Scopus (53) Google Scholar, 21.Wei C-H. Chou W-Y. Chang G-G. Biochemistry. 1995; 34: 7949-7954Crossref PubMed Scopus (40) Google Scholar) demonstrated that malic enzyme is specifically cleaved at the metal-binding site by the Fe2+/ascorbate system. Similarly, this affinity cleavage has been used to identify the metal-binding sites in several other enzymes including isocitrate dehydrogenase (13.Soundar S. Colman R.F. J. Biol. Chem. 1993; 268: 5264-5271Abstract Full Text PDF PubMed Google Scholar), 1-aminocyclopropane-1-carboxylate oxidase (15.Zhang Z. Barlow J.N. Baldwin J.E. Schofield C.J. Biochemistry. 1997; 36: 15999-16007Crossref PubMed Scopus (85) Google Scholar), phosphoenolpyruvate carboxykinase (22.Hlavaty J.J. Nowak T. Biochemistry. 1997; 36: 15514-15525Crossref PubMed Scopus (22) Google Scholar), and restriction endonucleases (18.Hlavaty J.J. Benner J.S. Hornstra L.J. Schildkraut I. Biochemistry. 2000; 39: 3097-3105Crossref PubMed Scopus (42) Google Scholar). Moreover, metal-catalyzed oxidative cleavage of proteins has also been used to identify TaqI endonuclease active site residues (16.Cao W. Barany F. J. Biol. Chem. 1998; 273: 33002-33010Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and to provide information on spatial organization of Na and K-ATPase (17.Shimon M.B. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1998; 273: 34190-34195Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,23.Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar), because the bound transition metals catalyze the site-specific cleavages of peptide bonds close to the bound metals.On the other hand, metal-catalyzed site-specific cleavage of proteins could be achieved by introducing a specific metal-binding site on a protein. Some "bifunctional chelating agents," which incorporate a strong metal-chelating group and a functional group capable of reacting with a specific group on the protein (24.Rana T.M. Adv. Inorg. Biochem. 1994; 10: 177-200PubMed Google Scholar), have been synthesized and used for this approach. Rana and Meares (25.Rana T.M. Meares C.F. J. Am. Chem. Soc. 1990; 112: 2457-2458Crossref Scopus (186) Google Scholar, 26.Rana T.M. Meares C.F. J. Am. Chem. Soc. 1991; 113: 1859-1861Crossref Scopus (115) Google Scholar) reported a cysteine-specific Fe-EDTA derivative that could be covalently attached to a protein via an α-bromoketone. Ermácora et al.(27.Ermácora M.R. Dellfino J.M. Cuenoud B. Schepartz A. Fox R.O. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6383-6387Crossref PubMed Scopus (147) Google Scholar, 28.Ermácora M.R. Ledman D.W. Hellinga H.W. Hsu G.W. Fox R.O. Biochemistry. 1994; 33: 13625-13641Crossref PubMed Scopus (42) Google Scholar) developed another Fe-EDTA derivative, which is attached through a mixed disulfide to a cysteine residue of a protein. When Fe-EDTA is attached to a protein, site-specific cleavage occurs close to the attachment site in the presence of H2O2and/or O2 and ascorbate.In this study, the site-specific cleavage of purified wheat RuBisCo by the Fe2+/ascorbate/H2O2 system was investigated in detail. We found that several other new fragments besides 37- and 16-kDa appeared when the incubation time was prolonged or the Fe2+ concentration was raised. These fragments were identified by their cross-reactivity with the site-specific antibodies against the N-terminal portion and the C-terminal portion of the rbcL, and the N-terminal amino acid sequences of the fragments containing the C-terminal portion of the rbcL were analyzed. The results demonstrated that all the C-terminal fragments analyzed had free N termini making it possible to identify six cleavage sites including Gly-329. Using the high resolution crystal structures of RuBisCo as models, the sites of cleavage were mapped. The results indicated that all these sites were around the metal-binding site in the rbcL, strongly suggesting that the backbone of the rbcL was cleaved by ROS generated at the metal-binding site. The structural basis of the cleavage reaction was analyzed in detail, and the mechanisms of Fe2+-catalyzed cleavage of RuBisCo were examined.EXPERIMENTAL PROCEDURESPlant Material and RuBisCo PurificationWheat (Triticum aestivum L. cv. Aoba) seeds were planted on a plastic net floating on tap water in a pot and grown in a phytotron with a day and night temperature of 20 and 18 °C, respectively, and 70% relative humidity. The photoperiod was 12 h with a quantum flux density of 300 μmol of quanta m−2 s−1at plant height. RuBisCo was purified from the primary and secondary leaves of 9-day-old seedlings according to a previously described procedure (7.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar).Cleavage of RuBisCo by the Fe2+/Ascorbate/H2O2SystemPurified RuBisCo was first passed through an Econo-Pac 10DG (Bio-Rad) column previously equilibrated with 100 mmHepes-NaOH buffer, pH 7.5, for a change of buffer. In a typical cleavage experiment, 0.5 μl of 0.02–20 mmFeSO4, 0.5 μl of 200 mm sodium ascorbate, and 1 μl of 10 mm H2O2 were added to 8 μl of RuBisCo solution (the rbcL protomer concentration, 18.5 μm) so that the final concentrations in this 10-μl mixture would be 14.8 μm rbcL, 1–1000 μm FeSO4, 10 mm sodium ascorbate, and 1 mm H2O2. The cleavage reaction was held at 4 °C for the given time, and then 10 μl of 2 × SDS sample buffer (200 mm Tris-HCl, pH 8.5, 2% (w/v) SDS, 20% (v/v) glycerol, and 5% (v/v) 2-mercaptoethanol) was added followed by heating of the mixture at 100 °C for 3 min. Boiled samples were analyzed by gel electrophoresis followed by immunoblotting or stored at −40 °C until use. Further experimental details are given in the figure legends.Gel Electrophoresis and ImmunoblottingSDS-PAGE (12.5%) was performed according to a reported procedure (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar) with a modification in the SDS sample buffer as described above. Two-dimensional polyacrylamide gel electrophoresis was carried out by the method of O'Farrell (30.O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) with the exception that 2% (v/v) ampholine (pH range 3.5–10, Amersham Biosciences) was used for isoelectric focusing. Tricine-SDS-PAGE was performed according to the method of Schägger and von Jagow (31.Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10439) Google Scholar) using a 16.5% T (total percentage concentration of acrylamide-bisacrylamide mixture), 6% C (percentage concentration of the cross-linker relative to the total concentration T) separating gel. The electrophoresis run started at 30 V, and then the voltage was raised to 100 V after the sample had completely moved into the separating gel. Immunoblot analysis was carried out with site-specific anti-rbcL-N and anti-LSU-C antibodies against the N-terminal portion corresponding to residues 3–17 and against the C-terminal portion corresponding to residues 463–477 of the wheat rbcL, respectively (7.Ishida H. Nishimori Y. Sugisawa M. Makino A. Mae T. Plant Cell Physiol. 1997; 38: 471-479Crossref PubMed Scopus (132) Google Scholar).Isolation of the rbcL FragmentsFor sequence analysis, the 40-, 30.5-, and 20-kDa fragments were separated by SDS-PAGE, and the 16-, 10.5-, and 8-kDa fragments were separated by Tricine-SDS-PAGE. Gel electrophoresis was performed as described above with the following modifications. First, the gel was cast overnight prior to use to allow the decomposition of ammonium peroxodisulfate. Second, the samples were treated with SDS sample buffer at 25 °C (not at 100 °C) for more than 1 h prior to loading the gel to avoid possible modification of the fragments. After gel electrophoresis, the separated fragments were transferred to a polyvinylidene difluoride membrane (Bio-Rad) using a transfer buffer containing 10 mm Caps-NaOH, pH 11.0, and 20% (v/v) methanol. The membrane was then stained with 0.1% (w/v) Coomassie Blue R-250 in 50% (v/v) methanol for 5 min at room temperature followed by destaining in 50% (v/v) methanol for three times within 25 min. The membrane was allowed to air-dry overnight, and the bands of interest were cut out and stored at −20 °C until analysis.N-terminal Amino Acid Sequencing and Conformational AnalysesThe N-terminal amino acid sequence of the isolated fragments was automatically analyzed with an Applied Biosystems-pulsed liquid protein sequencer (Model 491), equipped with an on-line analyzer of the phenylthiohydantoin derivative of the amino acids. The transblot membranes containing the protein fragments were directly sequenced by Edman degradation for 10 cycles. Identified cleavage sites were mapped on the available structures of RuBisCo in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (32.Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26649) Google Scholar). Furthermore, conformational analyses were carried out using Insight II (Biosym Technologies, Inc.) and Weblab ViewerLite (Molecular Simulations, Inc.) programs.RESULTSTime and Fe2+ Concentration-dependent Cleavage of the rbcL by the Fe2+/Ascorbate/H2O2 SystemAs with all other higher plant RuBisCo (33.Hartman F.C. Harpel M.R. Annu. Rev. Biochem. 1994; 63: 197-234Crossref PubMed Google Scholar), RuBisCo from wheat is a hexadecamer of eight large subunits and eight small subunits (rbcS) with molecular masses of ∼53 (34.Terachi T. Ogihara Y. Tsunewaki K. Jpn. J. Genet. 1987; 62: 375-387Crossref Scopus (39) Google Scholar) and 15 kDa (35.Broglie R. Coruzzi G. Lamppa G. Keith B. Chua N.H. Bio/Technology. 1983; 1: 55-61Crossref Scopus (145) Google Scholar), respectively. We have previously demonstrated that the rbcL can be directly broken down into 37- and 16-kDa fragments when incubated with 1 mmH2O2, 10 μm FeSO2, and 1 mm ascorbic acid at 4 °C for 15 min in 50 mm Hepes-NaOH buffer containing 5% (v/v) glycerol and 1 mm dithiothreitol (9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). However, our recent work has indicated that the rbcL can be degraded into several other fragments as well as 37- and 16-kDa when the conditions of cleavage reaction are changed. It can be seen from Fig. 1A that several new fragments including four major fragments (43-, 40-, 33-, and 20-kDa) and two minor fragments (45- and 30.5-kDa) appeared gradually at 4 °C with an increase of incubation time up to 22 h, whereas the quantity of the rbcL decreased slowly. On the other hand, no changes in the rbcS can be detected. Increasing the Fe2+ concentration also led to similar changes in cleavage pattern after 1-h incubation at 4 °C (Fig. 1B). The 37-kDa fragment appeared first and was the most dominant one after 0.5-h incubation or in the presence of 1–10 μm Fe2+. However, unlike the others, no further increase in its quantity could be seen until 22 h of incubation or in the presence of 1 mm Fe2+. The 16-kDa fragment, which is complementary to the 37-kDa fragment (9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), could not be separated from the rbcS by SDS-PAGE because of their similar molecular masses, but it was identified using two-dimensional PAGE and Tricine-SDS-PAGE (see below). These results clearly demonstrate that the rbcL can be cleaved at not only Gly-329 (9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) but also at several other sites by the Fe2+/ascorbate/H2O2 system when the incubation time or the Fe2+ concentration is increased. However, the cleavage of Gly-329 seems to be easier. Note that glycerol was omitted from the Hepes-NaOH buffer in this study, because Hlavaty et al. (18.Hlavaty J.J. Benner J.S. Hornstra L.J. Schildkraut I. Biochemistry. 2000; 39: 3097-3105Crossref PubMed Scopus (42) Google Scholar, 22.Hlavaty J.J. Nowak T. Biochemistry. 1997; 36: 15514-15525Crossref PubMed Scopus (22) Google Scholar) had reported that glycerol could act as a radical scavenger, which protected the phosphoenolpyruvate carboxykinase and endonuclease against Fe2+/ascorbate-induced backbone cleavage.Estimation of Putative Cleavage Sites of the rbcLTo determine the cleavage sites of the primary sequence of the rbcL, the cross-reactivities of the fragments with two site-specific antibodies (anti-rbcL-N and anti-rbcL-C) were examined. As shown in Fig. 2, two-dimensional PAGE (30.O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) was used for separating the polypeptides having similar molecular masses but different isoelectric points (for example, 16-kDa fragment and rbcS). All the fragments found in Fig. 1, as well as the 16-kDa fragment, were detected by two-dimensional PAGE (Fig. 2A). In these fragments, the 45-, 37-, and 33-kDa fragments cross-reacted with the anti-rbcL-N but not with the anti-rbcL-C, indicating they are N-terminal fragments. In contrast, the 40-, 30.5-, 20-, and 16-kDa fragments cross-reacted with only the anti-rbcL-C, demonstrating that they are C-terminal fragments. The band of 43-kDa, which cross-reacted with both the anti-rbcL-N and anti-rbcL-C, seems to be two different fragments that contain the N and C terminus, respectively, of the rbcL. Further experiments on this band demonstrated that there were two fragments that could barely be separated by SDS-PAGE containing 10% (w/v) acrylamide (data not shown). Thus, it can be considered that these two 43-kDa fragments are the N- and C-terminal fragments, respectively, but that they have similar molecular masses and isoelectric points so that they cannot be separated by two-dimensional PAGE. It should be noticed in Fig. 2 that most of the spots were multiple spots with the same molecular weight. This finding could be explained as resulting from the charge heterogeneities of the polypeptides (30.O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar), which could be attributed to the ROS-generated oxidative modification of the rbcL fragments, such as formation of carbonyl derivatives on amino acid side chains (36.Eckardt N.A. Pell E.J. Plant Physiol. Biochem. 1995; 33: 273-282Google Scholar).Figure 2Cross-reactivity of the site-specific anti-rbcL-N or anti-rbcL-C antibody to the fragments of the rbcL separated by two-dimensional polyacrylamide gel electrophoresis. RuBisCo (the rbcL protomer concentration, 14.8 μm) was incubated at 4 °C for 3 h in 100 mm Hepes-NaOH buffer, pH 7.5, in the presence of 100 μmFeSO4, 10 mm sodium ascorbate, and 1 mm H2O2. After incubation, the reaction mixture was analyzed by two-dimensional polyacrylamide gel electrophoresis. Separated polypeptides were transferred to a polyvinylidene difluoride membrane followed by Coomassie Blue staining (A) or analyzed by immunoblotting with anti-rbcL-N antibody (B) or anti-rbcL-C antibody (C). Arrowheads in the panels indicate the positions of the rbcL, rbcS, and the fragments of the rbcL with their molecular masses (kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT)According to the molecular mass of the rbcL (53 kDa), the following two pairs of fragments seem to be complementary based on their apparent molecular sizes and those mentioned above, 37- and 16-kDa fragments (which were previously demonstrated) and 33- and 20-kDa fragments. However, the complementary fragment of the minor fragment (30.5-kDa), which may have a calculated mass of 22.5 kDa, cannot be seen in Figs. 1 and 2 and was barely visible when SDS-PAGE was applied to a >5-fold concentrated sample (data not shown), indicating that the cleavage at this site was minor in comparison with the others.On the other hand, several complements to the other fragments, which may have calculated masses smaller than 15 kDa, remain to be resolved. Here it can be seen from Fig. 1 that there are at least two weak bands under the rbcS (Fig. 1, A and B, rightmost lanes). To separate these small fragments more effectively, Tricine-SDS-PAGE (31.Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10439) Google Scholar) was used (Fig. 3). Based on the molecular mass markers (range of 2.5–17 kDa), three new fragments were found to have molecular masses of ∼10.5, 9, and 8 kDa, respectively (Fig. 3, lane 1). The cross-reactivities with the site-specific antibodies indicated that the 9-kDa fragment contained the N terminus of the rbcL, and that the 10.5- and 8-kDa fragments contained the C termini (Fig. 3, lanes 2 and 3). In addition, a new 13-kDa fragment cross-reacting with the anti-rbcL-N was detected in lane 2, but this band was very weak in Coomassie Blue-stained gel. On the other hand, it was found that this PAGE could also separate the C-terminal 16-kDa fragment. From these results and those shown in Fig. 2, the following complementary fragments can be considered as pairs: 45 and 8 kDa, 43 and 10.5 kDa, 13 and 40 kDa, and 9 and 43 kDa.Figure 3Cross-reactivity of the site-specific anti-rbcL-N or anti-rbcL-C antibody to the fragments of the rbcL separated by Tricine-SDS-PAGE. RuBisCo was cleaved by the Fe2+/ascorbate/H2O2 system under the same conditions as described in Fig. 2. After the cleavage reaction, sample was analyzed by Tricine-SDS-PAGE followed by Coomassie Blue staining (lane 1) or immunoblot analysis using anti-rbcL-N antibody (lane 2) or anti-rbcL-C antibody (lane 3). Molecular masses (kDa) of the fragments are indicated beside the lanes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of Cleavage SitesThe apparently complementary pairs of fragments (see above) are summarized in Table I. In the seven C-terminal fragments, six were isolated using SDS-PAGE or Tricine-SDS-PAGE followed by electroblotting onto polyvinylidene difluoride membranes as described under "Experimental Procedures." The remainder of the C-terminal 43-kDa fragment, which could not be isolated from the N-terminal 43-kDa fragment by the methods used for this study, was not investigated further. The six purified C-terminal fragments were subjected to N-terminal Edman degradation sequence analysis. As shown in Table I, all these fragments were sequenced successfully demonstrating that they had free N termini. The N-terminal sequences of 8-, 10.5-, 16-, 20-, 30.5-, and 40-kDa fragments correspond to residues 405–414, 381–390, 330–339, 297–306, 204–213, and 123–132 of wheat rbcL, respectively (34.Terachi T. Ogihara Y. Tsunewaki K. Jpn. J. Genet. 1987; 62: 375-387Crossref Scopus (39) Google Scholar). These results indicate that the rbcLs are cleaved by the Fe2+/ascorbate/H2O2system at the following sites: Gly-404, Gly-380, Gly-329, Ala-296, Asp-203, and Gly-122. Note that the Gly-329 agrees with the site found previously (9.Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The molecular masses of these C-terminal fragments calculated from the amino acid sequence are 7891, 10,500, 16,181, 19,872, 30,429, and 39,538 Da, respectively, corresponding to their masses measured by SDS-PAGE.Table IAnalysis of the fragments produced by Fe2+-catalyzed cleavage of the rbcLPairs of fragmentsN-terminal sequence of the C-terminal fragmentsrbcL sequencebActive-site residues identified by the three-dimensional structure (37) are underlined.Cleavage sitesN-terminal fragmentsaResults from Figs. 2 and 3.C-terminal fragmentsaResults from Figs. 2 and 3.kDakDa458GTLGH P XGNAFGG404GTLGH PWGNA414Gly-4044310.5GIHV X HMPALASG380GIHVW HMPAL390Gly-3803716TVVGK L XXXRHSG329TVVGKLEGER339Gly-3293320MHAVI DRQKNHRA296MHAVI DRQKN306Ala-296Not detected30.5ENVN X Q XXMRKDD203ENVNS QPFMR213Asp-2031340NVFGF KALRAIVG122NVFGF KALRA132Gly-122943Not sequencedCleavage experiments were performed as described under "Experimental Procedures." Produced fragments were separated by SDS-PAGE or Tricine-SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Isolated C-terminal fragments were subjected to sequential Edman degradation.a Results from Figs. 2 and 3.b Active-site residues identified by the three-dimensional structure (37.Knight S. Andersson I. Brändén C.-I. J. Mol. Biol. 1990; 215: 113-160Crossref PubMed Scopus (278) Google Scholar) are underlined. Open table in a new tab According to a crystallographic study on RuBisCo (37.Knight S. Andersson I. Brän
Referência(s)