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The Thylakoid Lumen of Chloroplasts

1998; Elsevier BV; Volume: 273; Issue: 12 Linguagem: Inglês

10.1074/jbc.273.12.6710

1083-351X

Thomas Kieselbach, Åsa Hagman, Bertil Andersson, Wolfgang P. Schröder,

Antioxidant Activity and Oxidative Stress

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60–65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase. The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60–65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase. The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1Andersson B. Barber J. Adv. Mol. Cell Biol. 1994; 10: 1-53Crossref Scopus (23) Google Scholar, 2Hall D.O. Rao K.K. Photosynthesis. 5th Ed. Cambridge University Press, Cambridge, UK1994Google Scholar). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3Weibull C. Albertsson P.-Å. J. Ultrastruct. Mol. Struct. Res. 1988; 100: 55-59Crossref Scopus (5) Google Scholar). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment. By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4Andersson B. Åkerlund H.-E. Biochim. Biophys. Acta. 1978; 503: 462-472Crossref PubMed Scopus (110) Google Scholar). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5Åkerlund H.-E. Jansson C. FEBS Lett. 1981; 124: 229-232Crossref Scopus (128) Google Scholar, 6Åkerlund H.-E. Jansson C. Andersson B. Biochim. Biophys. Acta. 1982; 681: 1-10Crossref Scopus (207) Google Scholar) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7Murata N. Miyao M. Trends Biochem. Sci. 1985; 10: 122-124Abstract Full Text PDF Scopus (152) Google Scholar, 8Vermaas W.F.J. Styring S. Schröder W.P. Andersson B. Photosynth. Res. 1993; 38: 249-263Crossref PubMed Scopus (75) Google Scholar). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capable of assembling into photosystem II (10Hashimoto A. Yamamoto Y. Theg S.M. FEBS Lett. 1996; 391: 29-34Crossref PubMed Scopus (47) Google Scholar). Furthermore, it was found that during photoinhibitory conditions the extrinsic proteins were released from the membrane into the lumen (11Hundal T. Virgin I. Styring S. Andersson B. Biochim. Biophys. Acta. 1990; 1017: 235-241Crossref Scopus (83) Google Scholar,12Eisenberg-Domovich Y. Oelmüller R. Herrmann R.G. Ohad I. J. Biol. Chem. 1995; 270: 30181-30186Crossref PubMed Scopus (29) Google Scholar). Other important components of the thylakoid lumen are plastocyanin, the primary electron donor of photosystem I (13Haehnel W. Berzborn R.J. Andersson B. Biochim. Biophys. Acta. 1981; 637: 389-399Crossref Scopus (45) Google Scholar, 14Haehnel W. Annu. Rev. Plant. Physiol. 1984; 35: 659-693Crossref Google Scholar), and PsaN, a photosystem I subunit that is extrinsically bound to the lumenal side of the thylakoid membrane (15He W.-Z. Malkin R. FEBS Lett. 1992; 308: 298-300Crossref PubMed Scopus (20) Google Scholar). Recent investigations have revealed that polyphenol oxidases (16Sommer A. Ne'eman E. Steffens J.C. Mayer A.M. Harel E. Plant Physiol. 1994; 105: 1301-1311Crossref PubMed Scopus (110) Google Scholar, 17Sokolenko A. Fulgosi H. Gal A. Altschmied L. Ohad I. Herrmann R.G. FEBS Lett. 1995; 371: 176-180Crossref PubMed Scopus (39) Google Scholar) and violaxanthin de-epoxidase are also present in the thylakoid lumen (18Hager H. Holocher K. Planta. 1994; 192: 581-589Crossref Scopus (205) Google Scholar). Furthermore, the carboxyl-terminal processing protease for the D1 protein (19Inagaki N. Mori H. Fujita S. Yamamoto Y. Satoh K. Mathis P. Photosynthesis: From Light to Biosphere. 3. Kluwer Academic Publishers Group, Drodrecht, Netherlands1995: 783-786Google Scholar, 20Oelmüller R. Herrmann R.G. Pakrasi H.B. J. Biol. Chem. 1996; 271: 21848-21852Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and a processing protease for plastocyanin (21Kirwin P.M. Elderfield P.D. Williams R.S. Robinson C. J. Biol. Chem. 1988; 263: 18128-18132Abstract Full Text PDF PubMed Google Scholar) were found on the lumenal surface of the thylakoids, whereas chaperones may be located in the lumen (22Schlicher T. Soll J. FEBS Lett. 1996; 379: 302-304Crossref PubMed Scopus (38) Google Scholar). So far all lumenal proteins have been found to be nuclear-encoded and synthesized as precursors in the cytoplasm. These precursor proteins have characteristic amino-terminal bipartite transit peptides, which direct their import into the chloroplast stroma and across the thylakoid membrane into the lumen (23von Heijne G. Steppuhn J. Herrmann R.G. Eur. J. Biochem. 1989; 180: 535-545Crossref PubMed Scopus (910) Google Scholar, 24Robinson C. Klösgen R.B. Plant Mol. Biol. 1994; 26: 15-24Crossref PubMed Scopus (75) Google Scholar, 25Robinson C. Knott T.G. Andersson B. Salter A.H. Barber J. Molecular Genetics of Photosynthesis. IRL Press at Oxford University Press, Oxford1996: 145-159Google Scholar). On the basis of this property, bipartite transit peptides have become typical markers for lumenal proteins. However, not all chloroplast proteins encoded with such presequences are routed into the lumenal space. The PsbW protein and CFoII, for instance, are synthesized with bipartite transit peptides but have been shown to be integral proteins of the thylakoid membrane (26Michl D. Robinson C. Shackleton J.B. Herrmann R.G. Klösgen R.B. EMBO J. 1994; 13: 1317-1370Crossref Scopus (101) Google Scholar, 27Lorkovic Z.J. Schröder W.P. Pakrasi H.B. Irrgang K.-D. Herrmann R.G. Oelmüller R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8930-8934Crossref PubMed Scopus (83) Google Scholar, 28Shi L.-X. Schröder W.P. Photosynth. Res. 1997; 53: 45-53Crossref Scopus (13) Google Scholar). In this study we have developed a procedure by which a lumenal fraction can be isolated in a highly pure form from spinach thylakoids. We have carried out the first systematic characterization of this compartment, and we show that the thylakoid lumen contains a high concentration of proteins, among which at least 25 distinct polypeptides can be identified. Several have been characterized in terms of enzymatic activity or amino-terminal sequence. Spinach (Spinacia oleracea) was grown hydroponically for 6 weeks with alternating periods of 10 h light and 14 h darkness. The general approach applied consists of three principal steps as follows: (i), preparation of chloroplasts, (ii) purification of carefully washed thylakoids, and (iii) rupture of thylakoids by a Yeda press and isolation of the lumenal content (Fig. 1). Spinach leaves (200 g) were blended twice for 5–10 s in 330 mm sorbitol, 50 mm Hepes-KOH (pH 7.8), 10 mm KCl, 1 mm EDTA, 0.15% (w/v) bovine serum albumin, 4 mm sodium ascorbate, and 7 mmcysteine. The resulting slurry was filtered through four layers of nylon mesh (20 μm), and the filtrate was centrifuged 1 min at 1000 × g. The pellets were resuspended in 330 mm sorbitol, 50 mm Hepes-KOH (pH 7.8), 10 mm KCl, centrifuged for 1 min at 1000 × g, and resuspended in 12–18 ml of the same buffer. The yield was 60–70% intact chloroplasts containing 50–60 mg of chlorophyll. The chloroplasts were diluted with 10 mm sodium pyrophosphate buffer (pH 7.8) to a final chlorophyll concentration of 0.2 mg ml−1 and resuspended in a glass homogenizer. The thylakoids were collected by centrifugation for 5 min at 7500 ×g and sequentially washed in the same way twice with each of the following buffers: I, 10 mm sodium pyrophosphate (pH 7.8) to remove the soluble stromal proteins; II, 2 mmTricine (pH 7.8), 300 mm sucrose to partially remove ATP synthase, the membrane-attached fraction of Rubisco, 1The abbreviations used are: Rubisco, ribulose-bisphosphate carboxylase; AC, accession number; Chl, chlorophyll; FNR, ferredoxin-NADP+ reductase; LHCII, light harvesting complex II; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine. and unidentified extrinsic thylakoid membrane proteins; III, 30 mm sodium phosphate (pH 7.8), 50 mm NaCl, 5 mmMgCl2, 100 mm sucrose (fragmentation buffer) to equilibrate the thylakoids for Yeda press fragmentation. The thylakoid pellets were suspended in a small volume of fragmentation buffer to a concentration of 3.5–4.5 mg of chlorophyll ml−1 (total yield: 25–30 mg of chlorophyll). The washed thylakoids were then passed once through a Yeda press at a nitrogen pressure of 10 megapascals and centrifuged for 1 h at 200,000 ×g and 2 °C. The supernatant was separated from the pellet and centrifuged a second time under the same conditions to spin down residual membrane particles. The entire isolation procedure was performed on ice, and the chloroplasts and thylakoid membranes were purified under green light. The lumenal fraction was either used directly or stored in liquid nitrogen. The washed thylakoids (2 mg of chlorophyll ml−1) were incubated in the presence of 10 μm thermolysin (0.4 mg ml−1) for 2 min on ice in 100 mm sucrose, 30 mm Hepes (pH 7.8), 50 mm NaCl, 5 mm MgCl2, and 2 mm CaCl2. The digestion was stopped by adding EDTA to final concentration of 50 mm, and the thylakoids were washed twice with 100 mm sucrose, 30 mmHepes (pH 7.8), 50 mm NaCl, and 50 mm EDTA. These conditions were balanced to degrade the major part of peripheral proteins on the stromal side of the thylakoid membrane, without degrading too much of the integral membrane proteins. Harsher treatments were found to lead to leaky membranes followed by a loss of especially the lumenal part of the exposed stromal lamellae. Chlorophyll concentrations were measured as described (29Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4697) Google Scholar). Determination of soluble proteins was carried out according to Ref. 30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar and that of membrane proteins was performed essentially as described (31Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5333) Google Scholar). The standard used was bovine serum albumin. Oxygen evolution activities and intactness of the chloroplasts were measured with a Clarke-type electrode at 20 °C using potassium hexacyanoferrate(III) as the electron acceptor (32Leegood R.C. Malkin R. Hipkins M.F. Baker N.R. Photosynthesis Energy Transduction: A Practical Approach. IRL Press at Oxford University Press, Oxford1986: 9-26Google Scholar). SDS-PAGE was performed by the method described in Ref. 33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar in slab gels containing 18% (w/v) polyacrylamide and 2 m urea. Determination of molecular masses were performed by using the low molecular weight electrophoresis calibration kit from Pharmacia Biotech Inc. For immunoblotting analyses proteins were transferred onto a polyvinylidene difluoride membrane in a semidry electroblotter system (Millipore). The antisera used were raised in rabbits against the following spinach proteins: phosphoribulokinase (K.-H. Süss, Institute for Plant Genetics and Crop Plant Research, Gatersleben); violaxanthin de-epoxidase (H.-E. Åkerlund, University of Lund); plastocyanin (P.-Å. Albertsson, University of Lund); PsbO, PsbP, PsbQ, the lumen fraction, and Rubisco (our own production). The immunoreactivity was detected using goat anti-rabbit IgG-conjugated horseradish peroxidase in combination with an enhanced chemiluminescence detection. Quantification was performed using a Fast Scan Personal Densitometer and the ImageQuant software from Molecular Dynamics. To be able to compare directly the results for the chlorophyll-free lumenal fractions with those of the chloroplast and thylakoids, a chlorophyll equivalent was used for the lumenal fraction. It was calculated as follows: [Lumeneq] = [Chl]thyl ×Vthyl/Vlumen. However, the amount of plastocyanin and ferredoxin-NADP reductase (FNR) in the lumenal fraction was determined from the difference between the content in the washed thylakoids and their residual membrane fragments. Mitochondrial cytochrome coxidase was assayed according to Ref. 34Errede E. Kamen M.D. Hatefi Y. Methods Enzymol. 1978; 53: 45-46Google Scholar and malate dehydrogenase according to the Worthington manual. 2Available on-line at the following address: http://www.worthington-biochem.com/manual/manIndex.html As controls mitochondria from potato and spinach were used (kindly provided by P. Pavlov and X. Zhang, Stockholm University). Catalase activity was determined by oxygen evolution in the presence of 60 mmhydrogen peroxide (32Leegood R.C. Malkin R. Hipkins M.F. Baker N.R. Photosynthesis Energy Transduction: A Practical Approach. IRL Press at Oxford University Press, Oxford1986: 9-26Google Scholar). Phosphoribulokinase was assayed according to Ref. 35Porter M.A. Milanez S. Stringer C.D. Hartman F.C. Arch. Biochem. Biophys. 1986; 245: 14-23Crossref PubMed Scopus (65) Google Scholar. To prevent oxidative inactivation of this enzyme during the lumenal isolation, all buffers contained 5 mmdithiothreitol. Diphenolase activity of polyphenol oxidase was determined at pH 4.6 using the substrate 3,4-dihydroxyphenylpropionic acid (36Espı́n J.C. Morales M. Varón R. Tudela J. Garcı́a-Cánovas F. Anal. Biochem. 1995; 231: 237-246Crossref PubMed Scopus (113) Google Scholar). Peroxidase activity was measured using the substrate 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as described by Sigma. 3Available on-line at the following address:http://www.sigma.sial.com. Protease activity was tested using a dye-linked peptide (PepTag, Promega) and the in vitro translated, [35S]methionine-labeled β-subunit of mitochondrial ATP synthase of Nicotiana plumbaginifolia (37Boutry M. Chua N.-H. EMBO J. 1985; 4: 2159-2165Crossref PubMed Scopus (215) Google Scholar) as artificial substrates. The activity of violaxanthin de-epoxidase was determined as in Ref. 38Arvidsson P.-O. Bratt C.E. Carlsson M. Åkerlund H.-E. Photosynth. Res. 1996; 46: 141-149Google Scholar. All enzymatic activities were measured on freshly prepared samples (except those for violaxanthin de-epoxidase) at 25 °C in the presence of saturating substrate concentrations. ATP was detected using the firefly luciferase system (39Lundin A. Rickardsson A. Thore A. Anal. Biochem. 1976; 75: 611-620Crossref PubMed Scopus (235) Google Scholar) and the ATP monitoring reagent from BioOrbit. ATPase activity was tested according to Ref. 40Fromme P. Gräber P. Biochim. Biophys. Acta. 1990; 1016: 29-42Crossref PubMed Scopus (41) Google Scholar. Proteins were sequenced from polyvinylidene difluoride membrane following resolution by SDS-PAGE essentially as described (41Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). The amino-terminal sequence analyses were performed by P. I. Ohlsson (University of Umeå) using an Applied Biosystems pulsed liquid phase sequenator (ABS 477A). Searches in the data bases of EMBL and SwissProt as well as sequence alignments were carried out using the Wisconsin GCG software (42Genetics Computer Group (1996) Program Manual for the Wisconsin Package, Version 9, Madison, WI.Google Scholar). The purpose of the present study was to obtain a preparation of thylakoid lumen in a yield and purity sufficiently high to make it generally useful for characterizing this chloroplast compartment. The developed method, as outlined in the scheme of Fig.1, starts with the isolation of intact spinach chloroplasts. The chloroplasts obtained were normally 60–70% intact and had an oxygen evolving activity of 140–185 μmol of O2 mg Chl−1 h−1. In the next step the chloroplasts were disrupted by osmotic shock, and the thylakoids were collected and purified by several washing steps. Soluble stromal proteins were effectively removed by 10 mm sodium pyrophosphate (pH 7.8), and 2 mm Tricine (pH 7.8) containing 300 mm sucrose was used to remove ATP synthase, membrane-attached Rubisco, and other unidentified peripheral thylakoid proteins. Finally, the thylakoids were equilibrated in fragmentation buffer; these thylakoids retained an oxygen evolution rate of 90–165 μmol of O2 mg Chl−1 h−1. The washed thylakoids were then fragmented by a Yeda press, and the released lumenal content was separated from the membrane fragments by two ultracentrifugation steps. The final fraction was free of chlorophyll and had a protein concentration of 0.3–0.5 mg ml−1, giving a total yield of 2–3 mg of protein from 200 g of spinach leaves. The separation during the course of the preparation was followed by electrophoretic analysis of the polypeptide pattern of the starting chloroplasts, the stromal fraction, the washed thylakoids, and the final lumenal preparation (Fig. 2). The dominant bands in the isolated chloroplasts (lane 1) were the large and small subunits of Rubisco and the LHCII. The two subunits of Rubisco and other soluble proteins were found in the stromal fraction (lane 2). The membrane-bound LHCII and other integral membrane proteins were recovered in the thylakoid fraction (lane 3). Finally, lane 4 shows the soluble lumenal proteins released after the disruption of the washed thylakoids and removal of the membrane fragments. As summarized in Table I, there were more than 25 polypeptides in the purified lumenal fraction. Furthermore, the polypeptide pattern (Fig. 2, lane 4) is clearly different from that of the other chloroplast subfractions. These polypeptides range in molecular mass from 14 to 100 kDa. There are 5 dominant, 10 to 12 medium abundance, and 8 to 10 low abundance polypeptides. Silver staining the polypeptides led to the detection of five additional bands in the molecular mass range of 20–45 kDa. Furthermore, an investigation of the low molecular mass polypeptides of the lumenal fraction by Tricine/SDS-PAGE (43Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10481) Google Scholar) revealed five additional low abundance polypeptides in the range of 6–14 kDa (not shown).Table IPolypeptides of the isolated thylakoid lumenApparent molecular massIdentityMethod of identificationkDa100?64.5Polyphenol oxidaseProtein sequencing57.5?54.0Large subunit of Rubisco and ?Immunoblotting40.0New protein (see Table IV)Protein sequencing38.0 and 37.5Ferredoxin NADP+reductaseProtein sequencing32.5PsbO proteinImmunoblotting, protein sequencing29.0 and 28.5New protein (see Table IV)Protein sequencing28.0?27.5?25.0PsbP proteinImmunoblotting, protein sequencing24.0?23.0?21.0?20.0?18.5?18.0PsbQ proteinImmunoblotting, protein sequencing17.6?17.4New protein (see TableIV)Protein sequencing17.2?16.5New protein (see Table IV)Protein sequencing15.5?15.0PlastocyaninImmunoblotting, protein sequencing14.0?6–14Five protein bandsTricine/SDS-PAGE Open table in a new tab Following this general description of the protein content of the isolated lumenal fraction, several different approaches were used (summarized in Table I) to identify the individual polypeptides. Initially three of the four major polypeptides (Fig. 2, lane 4) at positions 32.5, 25, and 18 kDa were immunologically identified as the extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II. This is consistent with the observation that a soluble unassembled pool of these proteins occurs in the thylakoid lumen (9Ettinger W.F. Theg S.M. J. Cell Biol. 1991; 115: 321-328Crossref PubMed Scopus (54) Google Scholar). The fourth major protein of the lumenal fraction, with the apparent molecular mass of 15 kDa, was identified by immunoblotting as plastocyanin. The proteins of the lumenal fraction were systematically studied by amino-terminal protein sequencing. This confirmed the identification of plastocyanin, PsbO, PsbP, and PsbQ. Polyphenol oxidase was identified as a polypeptide with an apparent molecular mass of 64 kDa, and four new polypeptides were discovered at 40, 29, 17.4, and 16.5 kDa (see Tables I and IV). Finally, ferredoxin NADP+ reductase (FNR) was found to migrate at apparent molecular masses of 38 and 37.5 kDa.Table IVAmino-terminal sequences of four unknown proteins from the thylakoid lumenal fractionkDa1 3040.0VLISGPXIKD PEALLRYALP IDNKAIREVQ29.0ADLIQRRQRS EFQSDIKGIL YTVIKKNPDL17.4ANQRLPPLSN DPKRKE.... ..........16.5APLEDEDDLE LLEKVKRDRK KRLERQGAI. Open table in a new tab The detection of FNR, which is located on the stromal side of the thylakoid surface, prompted a more detailed analysis of contamination in the isolated lumenal fraction. Therefore, the washed thylakoids were treated by limited proteolysis with thermolysin prior to the Yeda press fragmentation. Lumenal proteins, unlike the contamination, should be protected against such proteolytic degradation. The conditions of proteolysis were designed to ensure degradation of most of the peripheral protein on the stromal side of the thylakoid membrane and to minimize membrane rupture. More extensive proteolysis could cause disruption of the thylakoid membrane, as was previously reported (44Carter D.P. Staehlin A. Arch. Biochem. Biophys. 1980; 2: 364-373Crossref Scopus (30) Google Scholar). The intensity of the polypeptide bands of the lumenal fraction following SDS-PAGE before (Fig. 2, lane 4) and after (Fig.2, lane 5) thermolysin treatment was compared by densitometric analysis of the Coomassie Blue-stained SDS gels using the PsbO protein as an internal reference protein. The data obtained from three different preparations demonstrated that most of the polypeptides of the lumenal fraction were not degraded, arguing that these proteins were located within the thylakoid lumen. Only in two cases were polypeptides significantly degraded (Fig. 2). A 40-kDa protein decreased in abundance by approximately 60%, whereas the 38-kDa FNR (Fig. 2) was degraded to 80–85%, which is consistent with its location at the stromal surface of the thylakoid membranes (1Andersson B. Barber J. Adv. Mol. Cell Biol. 1994; 10: 1-53Crossref Scopus (23) Google Scholar, 2Hall D.O. Rao K.K. Photosynthesis. 5th Ed. Cambridge University Press, Cambridge, UK1994Google Scholar). The FNR may have been sheared off the outer thylakoid surface during the Yeda press treatment, thereby ending up with the lumenal proteins in the supernatant following centrifugation. In another test for purity an immunoblot analysis was performed to quantify marker proteins associated with specific chloroplast compartments: plastocyanin as a soluble lumenal protein; PsbO as a lumenal extrinsic thylakoid-bound protein; the D1 reaction center protein of photosystem II as an integral membrane protein; the FNR for the outer thylakoid surface; Rubisco as the major stromal protein, and phosphoribulokinase as typical soluble stromal enzyme. As presented in Table II, 60–65% of the total amount of plastocyanin and 10% that of the extrinsic PsbO protein were recovered in the lumenal fraction. Furthermore, the lumenal fraction was free of D1 protein demonstrating the absence of contaminating thylakoid membranes. In addition, less than 1% of the total amount of the large subunit of Rubisco was present in the lumenal fraction. The major contamination was FNR. Between 10 and 40% of this enzyme was found in the lumen; this represents approximately 10% of the total protein content of the lumenal fraction.Table IIImmunological analyses of chloroplast subcompartment marker enzymesChloroplastsWashed thylakoidsLumenal fractionResidual thylakoid fragments%Thylakoid lumen Plastocyanin10060–7560–652–10 PsbO protein10080–901070–80Thylakoid membrane D1 protein100100–1100100–105 Ferredoxin-NADR+ reductase10040–6010–405–30Chloroplast stroma Rubisco (large subunit)1008–20<1NDaPhosphoribulokinase100Under detection limitNDND, not determined. Open table in a new tab ND, not determined. As an additional indication of stromal contaminations as well as to find possible extra chloroplast contaminants, we assayed for a number of different marker enzymes. These marker activities included phosphoribulokinase from the chloroplast stroma, catalase from the cytoplasm, peroxisomes, and vacuole, and the mitochondrial enzymes cytochrome c oxidase (inner membrane) and malate dehydrogenase (matrix). As shown in TableIII, the contamination of the isolated lumenal fraction by phosphoribulokinase was only 0.3% of the total activity of the isolated chloroplasts and that of catalase was even lower. The activity of cytochrome c oxidase in the chloroplasts was 0.007 mmol of oxidized cytochrome cmin−1 mg protein−1 and that of spinach mitochondria was 8 mmol of oxidized cytochrome cmin−1 mg protein−1. Furthermore, the activity of malate dehydrogenase was 0.5 mmol of oxidized NADH min−1 mg protein−1 and that of the spinach mitochondria was 330 mmol of oxidized NADH min−1 mg protein−1. Based on these values the contamination of the lumenal fraction by soluble mitochondrial proteins was estimated to be lower than 0.2%. The activity of cytochrome c oxidase was not detectable in the lumenal fraction.Table IIIActivities of marker enzymesChloroplastsWashed thylakoidsLumenal fractionResidual thylakoid fragmentsThylakoid lumen Polyphenol oxidase3-aμmol oxidized 3,4-dihydroxyphenylpropionic acid min−1 mg Chl−1.0.01 ± 0.010.09 ± 0.050.25 ± 0.050.02 ± 0.005 ViolaxanthinNDND18 ± 1.8ND De-epoxidase3-bμmol of violaxanthin min−1 g protein−1.Chloroplast stroma Phosphoribulokinase3-cmmol oxidized NADPH min−1 mg Chl−1.7 ± 10.07 ± 0.0010.024 ± 0.002NDMitochondria and cytoplasm Cytochrome c oxidase3-dmmol oxidized cytochrome c min−1 mg Chl−1.0.06 ± 0.0050.02 ± 0.0070ND Cytochrome c oxidase3-emmol oxidized cytochrome c min−1 mg protein−1.0.007 ± 0.00060.003 ± 0.0010ND Malate dehydrogenase3-fmmol oxidized NADH min−1 mg Chl−1.0.18 ± 0.020.14 ± 0.0020.05 ± 0.04ND Malate dehydrogenase3-gmmol oxidized NADH min−1 mg protein−1.NDND0.5 ± 0.3ND Catalasei341 ± 24014 ± 70.7 ± 0.2ND3-h μmol O2 mg h−1 Chl−1.3-a μmol oxidized 3,4-dihydroxyphenylpropionic acid min−1 mg Chl−1.3-b μmol of violaxanthin min−1 g protein−1.3-c mmol oxidized NADPH min−1 mg Chl−1.3-d mmol oxidized cytochrome c min−1 mg Chl−1.3-e mmol oxidized cytochrome c min−1 mg protein−1.3-f mmol oxidized NADH min−1 mg Chl−1.3-g mmol oxidized NADH min−1 mg protein−1.