Isolation and characterization of chloroplast Photosystem II antenna of ...

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Lello Zolla; Anna Maria Timperio; Maria Grazia Testi; Maria Bianchetti; Roberto Bassi; Francesco Manera; Danilo Corradini. Lello Zolla; Anna Maria Timperio ...
Photosynthesis Research 61: 281–290, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

281

Regular paper

Isolation and characterization of chloroplast Photosystem II antenna of spinach by reversed-phase liquid chromatography Lello Zolla1,∗ , Anna Maria Timperio1, Maria Grazia Testi3 , Maria Bianchetti1 , Roberto Bassi3 , Francesco Manera4 & Danilo Corradini2 1 Dipartimento

di Scienze Ambientali, Universit`a della Tuscia, via S. Camillo de Lellis, Blocco D, I-01100 Viterbo, Italy; 2 Istituto di Cromatografia del CNR, P.O. Box 10, I-00016 Monterotondo Stazione, (Rome), Italy; 3 Dipartimento Scientifico-tecnologico, Facolt` a di Scienze, Universit`a di Verona, Strada Le Grazie, 37134 Verona, Italy; 4 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Universit`a di Roma,La Sapienza 00185 Roma, Italy; ∗ Author for correspondence; (e-mail: [email protected]; fax: +39-761-357179) Received 18 February 1999; accepted in revised form 2 August 1999

Key words: antenna system, chlorophyll–proteins, HPLC, LHC II, Photosystem II, spinach

Abstract The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance liquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification has been accomplished by a combination of various SDS–PAGE systems employing either Comassie (or silver) staining or immunological detection using polyclonal antibodies raised against LHC II and against CP29, CP26 and CP24 proteins and by aminoacid microsequence. Moreover, peak identification is consistent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolution of all the protein components of the Photosystem II major Light Harvesting Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry in natural and stressed conditions, avoiding the expensive and time consuming separation procedure by sucrose-gradient ultracentrifugation and isoelectrofocusing. Abbreviations: chl – Chlorophyll; LHC II – Light-harvesting complex of PS II; PS – Photosystem,; TFA – Trifluoroacetic acid Introduction The Photosystem II (PS II) of green plants is a supramolecular complex intrinsic of the thylakoid membrane which is responsible for splitting water to form molecular oxygen, electrons and protons. PS II is composed of a core complex, containing chlorophyll a/β-carotene binding proteins which are chloroplast encoded, and of an antenna system composed by nuclear encoded polypeptides belonging to the Lhc protein family. Lhc proteins bind chl a, chl b and five different xantophylls. Among PS II proteins components, LHC IIb1,2 and 3 are the major antenna

and are encoded by the Lhcb1,2 and 3 genes (Jansson 1994). Up to 65% of the PS II chlorophyll is bound to this complex (Dainese and Bassi 1991). Minor components, binding together about 10% of PS II pigments, are the proteins CP29, CP26 and CP24 respectively encoded by the Lhcb 4, 5 and 6 genes. Besides the obvious role of harvesting light, antenna proteins are involved in several regulative mechanisms aimed at the distribution of excitation energy between PS I and PS II (Allen 1992), and at the dissipation of energy exceeding the capacity of the electron transport chain and thus it is potentially harmful to the PS II reaction centre. These photoprotecting

282 mechanisms are elicited by low lumenal pH and they closely correlate with the process of violaxanthin to zeaxanthin de-epoxidation. This suggests they are associated to minor chlorophyll–proteins since these bind dicychlohexylcarbodiimide (DCCD) (Walters et al. 1994, 1997; Pesaresi et al. 1997) and contain 80% of PS II violaxanthin (Bassi et al. 1993). Antenna proteins undergo long term adaptation in their relative abundance (Brugnoli et al. 1994) when exposed to different environmental conditions including low and high light intensity (Anderson and Anderson 1988; Flachmann 1997), heat stress (Larsson et al. 1987) water stress (Hao et al. 1996) and cold stress (Hayden et al. 1986); this is consistent with their different roles in PS II function.These adaptation processes are of interest in both basic and applied research. However, the available methods for separation and identification of antenna proteins are both expensive and technically demanding (Dainese et al. 1990; Dainese and Bassi 1991), while traditional approaches by SDS–PAGE are not only cumbersome but also rather ineffective for evaluating differences in the relative quantity of each component unless time consuming antibody titration is used (Bassi et al. 1995). Thus, it seems necessary to develop a rapid and sensitive method to identify and determine quantitatively the various components of the two photosystems. With this aim in mind, the electrophoretic migration behaviour of three closely related hydrophobic intrinsic membrane proteins of the Photosystem II light-harvesting complex (LHC II) was investigated in capillary zone electrophoresis (CZE) at pH 8.0–10 with running electrolyte solutions containing either anionic, zwitterionic or non-ionic detergents (Zolla et al. 1996). Moreover, a study was performed to obtain the rapid resolution of the protein components of LHC II by reversed phase HPLC, which is more suitable than CZE for the micropreparative scale separation, in view of protein sequencing or peptide mapping of the isolated LHC II proteins (Zolla et al. 1997). To our knowledge, very few other studies have reported the application of HPLC to isolate the protein components of either photosystem I or Photosystem II. These include the RP-HPLC partial resolution of LHC II proteins and the separation of the PS II reaction centre proteins reported by Damm and Green (1994) and by Sharma et al (1997), respectively. The isolation of both oxygen-evolving PS II complexes and PS II reaction centres by anion exchange perfusion chromatography has been reported (Roobol-Boza and Anderson 1996). Perfusion chromatography has also been applied to isolate the PS

I core complexes in spinach leaves (Roobol-Boza et al. 1995) and from various deletion mutants of the mesophylic cyanobacterium Synechocystis PCC 6803 (Kruip et al. 1997). In this study, we have extended the previously reported HPLC method (Zolla et al. 1997) to the resolution of both major and minor antenna of the PS II system protein components, showing that the method is suitable for their identification even upon direct injection of the grana membrane preparation (BBY particles). In addition, the resolution of these proteins from solubilized thylakoid membranes allows accurate estimation of the subunit stoichiometry of Photosystem II.

Materials and methods Instrument and column The experiments were carried out on two different liquid chromatograph units: a Beckman (Fullerton, CA, USA) System Gold system, consisting of two Model 126 solvent delivery pumps and a Model 166 UV detector, interfaced with a Kbyte Informatica (Viterbo, Italy) computer utilizing Version 6.0 System Gold software for instrument control, data acquisition and analysis. Samples were introduced onto the column by a Model 210A sample injection valve with either a 250-µl or a 50-µl sample loop. Fractions eluted were collected by a Gilson (Villiers le Bel, France) Model 201 sample collector. A second HPLC unit consisted of a Perkin Elmer (Norwalk, CT, USA) Model 200 C system equipped with a Mode l785 A UV detector, and a Model LC 240 Florescence detectors connected in series. Samples were loaded onto the column by a Model 7125NS-005 Rheodyne (Cotati, CA, USA) sample injection valve with either a 250-µl or 50 µl sample loop. The experiments were performed using two Vydac (The Separation Group, Hesperia, CA, USA) Protein C-4 columns of either 250 × 4.6 mm I.D. or 250 × 10 mm I.D., both containing 5-µm porous butyl silica. All solutions were filtered through a Millipore (Milan, Italy) type FH 0.22-µm membrane filter and degassed by bubbling with helium before use. Chemicals Reagent-grade phosphoric acid, magnesium chloride, sodium chloride, silver nitrate, sodium carbonate, trifluoroacetic acid, methanol, ethanol, form-

283 amide, as well as HPLC-grade water and acetonitrile were obtained from Carlo Erba (Milan, Italy). Acrylamide, N,N0 methylen-bis-acrylamide, and all other reagents for SDS–PAGE were purchased from Bio-Rad (Segrate, Italy). Sucrose, tricine, trishydroxymethylaminomethane (TRIS), n-octyl β-D glucopyranoside, n-dodecyl β-D maltoside, chlorophyll a and chlorophyll b, as well as 2[N-morpholino]ethanesulfonic acid (MES) were obtained from Sigma (Milan, Italy). Triton X-100, and n-octylsucrose were purchased from Calbiochem (San Diego, USA). Sequence-grade chemicals used for amino acid sequence were purchased from Perkin-Elmer Isolation of chloroplast thylakoid and PS II membranes Chloroplast thylakoid membranes (PS II membranes) were isolated from spinach leaves according to the Berthold method (Berthold et al. 1981) with the following modifications. Leaves were powdered in liquid nitrogen and subsequently homogenized in an ice-cold 20 mM tricine pH 7.8 buffer containing 0.3 M sucrose and 5.0 mM magnesium chloride (B1 buffer). The homogenization was followed by filtration through one layer of Miracloth (Calbiochem, San Diego, USA) and centrifuged at 4000 × g for 10 min at 4 ◦ C. Pellets were suspended in B1 buffer and centrifuged as above. This second pellet was resuspended in 20 mM tricine pH 7.8 buffer containing 70 mM sucrose and 5.0 mM magnesium chloride (B2 buffer) and centrifuged at 4500 × g for 10 min. The pellets containing the thylakoid membranes were then resuspended in 50 mM MES pH 6.3 buffer containing 15 mM sodium chloride and 5 mM magnesium chloride (B3 buffer) at 2.0 mg chlorophyll/ml for 15 min after adding Triton X-100 at a final ratio of 20 mg/mg chlorophyll. The concentration of chlorophyll was determined using the Porra method (Porra et al. 1989). The incubation was terminated by centrifugation at 40 000 × g for 30 min at 4 ◦ C. This pellet containing the PS II complex and corresponding to the BBY preparation described by Berthold (Berthold et al. 1981), was resuspended in B3 buffer containing 20% (v/v) glycerol and stored at –80 ◦ C. Isolation of the PS II major and minor antenna system by sucrose-gradient ultracentrifugation The light-harvesting complex was isolated from the PS II membranes as previously described (Bassi and Dainese 1992) with the following modifications: PS II

membranes were pelleted by centrifugation at 10 000 × g for 5.0 min at 4 ◦ C, suspended in B3 buffer at 1.0 mg/mg chlorophyll and then solubilized by adding 1% (w/v) n-dodecyl β-D maltoside. Unsolubilized material was removed by centrifugation at 10 000 × g for 10 min. The supernatant was rapidly loaded onto a 0.1–1.0 M sucrose gradient containing B3 buffer and 5.0 mM n-dodecyl β-D maltoside. The gradient was then spun on a Kontron Model Centricon T-1080 ultracentrifuge equipped with a Model TST 41.14 rotor at 39 000 rpm for 18 h at 4 ◦ C. Green bands were harvested with a syringe. The SDS–PAGE analysis of these green bands revealed that band 2 contained a mixture of the protein components of the major and minor PS II antenna system, whereas band 3 contained essentially the protein components of the major PS II antenna system, as previously reported (Bassi and Dainese 1992). These bands were used for HPLC analysis without any further treatment. HPLC The Vydac C-4 columns were pre-equilibrated with 38% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) TFA and samples were eluted by either gradient I or II with 0.1% (v/v) TFA, depending on the HPLC unit employed for the separation. Gradient I consisted of a first linear gradient from 38 to 55.4% (v/v) acetonitrile in 22 min, followed by 3 min isocratic elution with the eluent containing 55.4% acetonitrile, followed by a second gradient segment from 55.4 to 61.8% (v/v) acetonitrile in 8 min and by a third gradient segment from 61.8 to 95% acetonitrile in 1 min. Gradient II consisted of a first linear gradient from 38.0 to 61.8% (v/v) acetonitrile in 40 min, followed by a second gradient segment from 61.8 to 95% (v/v) acetonitrile in 1 min. For both gradients, the final part of the curve up to 95% acetonitrile was used for washing out hydrophobic contaminants of the PS II antenna system from the column. Gradient I was used to elute either the analytical or the semipreparative size column. The flow rate was 1.0 ml/min with the analytical column and 4.7 ml/min with the semi-preparative column. These conditions were selected in order to maintain the same gradient shape with both columns by keeping the ratio of the gradient volume to the column volume constant. Polyacrylamide gel electrophoresis In order to determinate the protein composition, the samples separated by HPLC were dried and solubil-

284 ized in 4% LDS, 120 mM DTT and 120 mM Tris/HCl pH 8.45, 5 M urea and were run in SDS–PAGE of uniform polyacrilamide concentration (13%) with the Tris/tricine system (Shagger and von Jagow 1987), using a BRL (Bethesda Research Laboratories, Gaithersburg, USA) Model V16 vertical gel electrophoresis system. The gels were fixed and stained for 2 h in a 5:1:4 (v/v) methanol-glacial acetic acid-water mixture, containing 0.1% (w/v) Comassie brilliant blue. For silver-staining , the gels were fixed in 50% (v/v) methanol-water and 10% (v/v) ethanol-water solutions, stained with 0.1% (w/v) silver nitrate-water solution and developed in 3.5% (w/v) aqueous sodium carbonate containing 0.05% (v/v) formamide. Immunoblotting Following electrophoresis, gels were blotted to nitrocellulose filters (Towbin et al. 1979; Burnette et al. 1981) and the antenna proteins were detected by using antisera which was directed to individual PS II antenna proteins. Antibodies were raised in rabbits using native proteins isolated from Zea mays (Di Paolo et al. 1990). Antibody binding was detected with Alkaline phosphatase coupled anti-immunoglobulin antibodies. Amino acid analysis The amino acid sequence was determined by automated Edman degradation using a Perkin-Elmer model AB476A sequencer. The samples separated on a Tris-tricine SDS–PAGE were electroblotted onto a polyvinylidene difluoride membrane (Problott, Perkin-Elmer) and stained with Comassie Brillant Blue. The bands of interest were excised and directly analyzed. The proteins found to be N-terminally blocked were treated with trypsin at room temperature with a chlorophyll concentration of 400 µg/ml, in 25 mM n-octyl b-D-glucopyranoside, 10 mM NaCl and 10 mM Tricine (pH 7.5). A trypsin (Sigma, type III) to chlorophyll ratio of 0.05 (w/w) was used. The reaction was terminated after 10 min by heating (70 ◦ C) and peptide mixture was processed as reported above.

Results and discussion Photosystem II membranes were isolated from thylakoid membranes of spinach leaves by Triton X100 extraction as reported in the experimental section.

This material, corresponding to the BBY preparation (Berthold et al. 1981), was then subjected to sucrose-gradient ultracentrifugation in order to isolate the protein components of the minor antenna system (band 2) from the major (band 3), as well as from the reaction-center complexes (band 3–4). In a previous paper (Zolla et al. 1997), the PS II major antenna system was resolved in the three main protein components Lhcb1, Lhcb2 and Lhcb3 by reversed-phase HPLC using a silica-based butyl stationary phase (Vydac C-4) packed into a 150 mm long column. In this study, we searched for the conditions to resolve the protein components of the major and the minor antenna system of PS II either as an isolated complex by sucrose gradient or as assembled complex in the grana membrane (BBY particle), using either an analytical (250 × 4.6 mm I.D.) or a semi-preparative (250 × 10 mm I.D.) size column, both packed with the same 5 µm spherical Vydac C-4 stationary phase. The use of a semi-preparative size column was needed in order to obtain an amount of purified polypeptide sufficient for peak identification by SDS–PAGE, immunoblotting and amino acid microsequence. Both analytical and semi-preparative scale separations were carried out using the same volatile mobile phase system consisting of trifluoroacetic acid in a water-acetonitrile mixture with increasing acetonitrile content during gradient elution. The optimal separation of the protein components of the PS II antenna system was obtained by a multisegments gradient which was slightly different in shape on the two HPLC units employed to perform this study, as reported in the ‘Material and methods’ section. The resolution obtained on the analytical column was retained while increasing the column diameter, as a consequence of having used gradients of the same shape, which was maintained by keeping the ratio of the gradient volume to the column volume constant. The chromatograms displayed in Figure 1 show that the material harvested from the second band of the sucrose-gradient ultracentrifugation, containing a mixture of the protein components of both the major and the minor PS II antenna system, was resolved in eight main peaks (panel A). Five of these peaks, with corresponding retention times, were also obtained separating, under the same conditions, the material harvested from the third band of the sucrose-gradient ultracentrifugation (Figure 1, panel B), containing essentially the protein components of the major PS II antenna system (Bassi and Dainese 1992). From these data, it can be inferred that the peaks labeled with the

285

Figure 1. Reversed phase HPLC separation of spinach chlorophyll a/b binding proteins contained in band 2 (A) and in band 3 (B) of BBY proteins of spinach fractionated by sucrose-gradient ultracentrifugation. Column, Vydac Protein C-4 (250 × 10 mm I.D.) eluted by the increasing acetonitrile concentration gradient described as gradient I in the M. and M. section; flow rate, 4.7 mL/min; detection, 214 nm; size sample loop, 250 µl. Chlorophyll concentration of band 2 and band 3 was respectively 0.07 mg/ml , 0.12 mg/ml.

numbers 1, 2, 5, 6 and 7 correspond to components present in both the sucrose-band 2 and 3, whereas peaks 3, 4 and 8 are essentially due to the component only present into the sucrose band2: this can be tentatively identified with the minor antenna complexes CP29, CP26 and CP24 (Bassi and Dainese 1992). Contents of the band 3 from sucrose-gradient ultracentrifugation were treated with 80% (v/v) aqueous acetone at room temperature in order to extract the pigment and lipid components, and then chromatographed by a second HPLC unit, having UV and fluorescence detectors connected in series. The chromatogram is reported in Figure 2 and is comparable to that of Figure 1, but the peak #1 is missing, whereas the peaks labeled with the numbers 2, 5, 6 and 7 were detected both by UV adsorption at 214 nm (panel A) and by fluorescence emission at 330 nm upon excitation at 280 nm (panels B). This indicates that peak 1 is due to pigments that are removed by the treat-

Figure 2. Comparison of the reversed phase HPLC separation of spinach LHC II proteins contained in band 3 of BBY detected by absorbance at 214 nm (panel A) and by fluorescence at 330 nm upon excitation at 280 nm. In both cases, the detection signal is displayed in mVolts. Column, Vydac Protein C-4 (250 × 4.6 mm I.D.) eluted by the increasing acetonitrile concentration gradient described as gradient II in the ‘Materials and methods’ section; flow rate, 1.0 mL/min; size sample loop, 100 µl. Chlorophyll concentration of band 3 was 0.12 mg/ml.

ment of the sample with acetone. This interpretation was further confirmed by the SDS–PAGE and amino acid microsequence analysis of the material collected throughout the chromatogram that hightlighted the absence of proteic bands in the fraction corresponding to peak 1 (see the following discussion). Moreover, analysis by mass spectroscopy HPLC ESI MS of peak 1 reveals the presence of organic material with a molecular weight of 512 Da, probably detergent and phospholipids (data not shown). Finally, injection to the column, under the same experimental conditions of band 1 from sucrose gradient, which is known to contain free pigments, yielded only one peak with the elution time of peak 1.

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Figure 3. Gel electrophoresis and immunoblotting of each protein collected throughout the chromatographic run reported in Figure 1A. Lanes corresponds to the peak labels in Figure 1A. C represents the LHC II from Zea mais used as control. (A) SDS-PAGE of uniform polyacrilamide concentration (13%) with Tris/tricine system of each collected peak, which were dried and than solubilized in 4% LDS, 120 mM DTT and 120 mM Tris/HCl pH 8.45, containing 5 M urea. (B) Immunoblotting of the gel reported in (A) detected by using antisera directed to CP29. (C) Immunoblotting of the gel reported in (A) detected by using antisera directed to CP26. (D) Immunoblotting of the gel reported in (A) detected by using antisera directed to CP24. Experimental conditions are reported in the Material and methods section.

The identification of the protein components of the other peaks (2–8) resolved by RP-HPLC was performed by: 1. SDS–PAGE systems employing different buffer systems and either Comassie or silver staining followed by immunoblotting detection with antisera directed to the individual antenna proteins; 2. amino acid microsequence. In the case of SDS–PAGE and immunoblotting, each fraction collected from the semi-preparative chromatographic separation was lyophilized, and then it was dissolved in 120 mM TRIS/HCl pH 8.45 buffer, containing 120 mM DTT, 5 M urea and 4% (w/v) SDS, and then analyzed by SDS–PAGE urea, according to the method reported by Shaegger (Schaegger and Von Jagow 1987) (Figure 3, panel A). It may be observed that the molecular weight range of 23–28 kDa allows one to exclude the presence of chlorophyll binding protein PS II S. Following electrophoresis, the gels were either silver stained or transferred to nitrocellulose. Replicates were assayed with antisera directed to LHC II, CP29, CP26 and CP24 (Di Paolo et al. 1990) (Figure 3 B–D). Because of the high degree of homology shared by all of the antenna polypeptides, it was essential for identification of the fractions to take in account both the immunoreactivity and the electrophoretic mobility of the SDS–PAGE bands. In Tris/Tricine electrophoresis, antenna proteins migrate in the following order of increasing mobility CP29>CP26>LHC II>CP24 and, within LHC II, Lhcb1>Lhcb2>Lhcb3 (Bassi et al. 1990). Accordingly, the slower migrating band was detected by the anti-CP29 in peak 4 (Figure 3B) while peak 8, containing a slightly more mobile band, was recognized by anti-CP26 (Figure 3C). The anti-CP24 detected several bands (Figure 3D), but the strongest signal, compared with to the intensity of the band in the silver stained gel, was obtained with the most mobile band in peak 3. Therefore, peaks 3, 4 and 8 seem to contain CP24, CP29 and CP26, respectively. The higher hydrophobicity of CP26 corresponds to the higher concentration of acetonitrile required to eluite this protein from RP-HPLC column. When the antiLHC II antibodies were used (data not shown), they strongly recognized bands in peaks 5 and 6 which showed the same mobility, slightly lower compared with the band detected in the slot from peak 2, while the band detected in peak 7 had even higher mobility. Moreover, Figure 4 shows that when the proteins collected in peaks 5 and 6 were subjected to Tris/Tricine electrophoresis on a 15 cm long gel, they migrate

287 Table 1. N-terminal amino acid sequences of native and trypsined fragments of the major and minor antenna proteins. Microsequence was performed directly on HPLC purified fraction, when possible, or after pretreatment of protein with trypsin Peak number

N-terminal blockage

Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8

yes no yes yes yes no yes

Microsequence

No protein XAPQSLXYGPD AAAPKKSWIPAVKG LGFSTDRPLWYPGA TVQSGSPXYGPDRVKYXL TVQSGSPXYGPDRVKY GNDLWYGPDRVKYL

as single resolved bands also in this gel, but show a significant difference in mobility. Thus, respecting the migration order of increasing mobility reported above, the fastest migrating band was identified as Lhcb3, the second fastest migrating band as Lhcb2, and the two newly resolved bands as Lhcb1 components of LHC II. This assignment is also consistent with the area of peaks 2, 5, 6 and 7 which reflects the abundance of the protein components of the PS II major in the order Lhcb1>Lhcb2> Lhcb3 (Jansson 1994). From the above data, we conclude that peak 1 does not contain protein components; peak 2, the Lhcb2 component of LHC II; peak 3, CP24 (Lhcb6); peak 4, CP29 (Lhcb4); peak 5 and 6, two Lhcb1 components of LHC II; peak 7, Lhcb3; peak 8, CP26 (Lhcb5). In order to support the identification previously assigned, the protein contained in each HPLC peak has undergone a amino acid microsequence. Table 1 reports the list of proteins found to be N-terminally blocked, the microsequence determined directly on HPLC purified fractions (without or after) pretreatment with trypsin and a comparison with the results reported in the literature for the spinach and ,when it is possible, with other species. It can be observed that the microsequence determined confirms the above assignment of each peak resolved by RP-HPLC, supporting the high resolution of the new separation method described. Moreover, the protein contained in peak 5 and 6 shows the same microsequence, in the fragment analyzed at least. Finally, the assignment of each peak resolved by RP-HPLC performed by electrophoresis, immunoblotting and amino acid sequence is corroborated from the

Similarity in literature

Reference

CP24 Spinach CP29 Arabidopsis Type I Spinach Type I Spinach Type III Barley

Jansson 1994 Green 1993 Mason 1989 Mason 1989 Jansson 1994

Figure 4. Denaturing SDS-PAGE of isolated LHC II proteins collected in correspondence of peaks 2, 4, 5 and 6 of the run reported in Figure 1B. Slab gel, 12–17% acrylamide gradient gel containing 7 M urea; electrolyte solution, 25 mM TRIS / 192 mM glycine (pH 8.8) containing 3.5 mM SDS; silver-staining, AgNO3 0.1% (w/v) in water.

values of molecular masses determined by the combined use of microbore HPLC coupled on-line with a mass spectrometer equipped with electrospray ion source (ESI-MS) (Corradini et al., submitted). The molecular masses, in fact, obtained by the deconvolution of the ESI-MS spectra collected during the chromatographic separation of the protein components

288 of the PS II give the following values: peak 2: 24761; peak 3: 22820; peak 4: 28076; peak 5: and peak 6 24936 and 25005, respectively; peak 7 24323 and peak 8: 27076. From the results presented, it can be conclude that the major antenna system of PS II isolated from spinach leaves contains two different Lhcb1 proteins which can be resolved by the RP-HPLC system employing a 250 mm long Vydac C-4 column eluted by a multisegments acetonitrile gradient, as well as by the high resolution Tris/Tricine SDS–PAGE system in gels of 15 cm of length. On the other hand, the small microsequence performed does not reveal any amino acid difference and the molecular weight measured by mass spectroscopy indicates that the difference is in the order of 60–80 Da. More experiments and a complete amino acid sequence are necessary to give an explanation to these different subpopulations of LHCb1, which show small difference in molecular weight and in electrophoretic mobility. Up to now, we are not able to asses that peak 6, containing the 25025 Da protein, may represent the phosphorylated form of LHCb1, since this does not agree with its higher hydrophobicity compared with peak 5, unless strong conformational changes of protein are induced by phosphorylation (Nilsson et al. 1997). However, the existence of more than one LHCb1 is in accordance with molecular genetic data reported in literature showing that higher plants have several Lhcb1 genes encoding different Lhcb1 proteins for each species (Allen et al. 1981; Dainese et al. 1990; Morishige and Tornber 1991; Sigrist and Staehelin 1992). However, it is important to remark that the resolution by SDS–PAGE of two Lhcb1 proteins usually requires special experimental conditions such as the use of polyclonal and monospecific antibodies (Sigrist and Staehelin 1992), dedicated electrolyte solutions and gel of extended length (Dainese et al. 1990 ), while the two different subpopulations may be easily separated using HPLC. Finally, once the peaks from partially purified preparations were identified, we investigated the possibility of resolving the protein components of the spinach PS II major and minor antenna system directly in the BBY preparation, avoiding the separation step by sucrose-gradient ultracentrifugation. The chromatogram reporting the separation by RP-HPLC of the BBY preparation from spinach leaves is displayed in Figure 5. It can be observed that the protein components of the PS II major and minor antenna system are well resolved without interference from the other

Figure 5. Reversed phase HPLC separation of the protein components present in the spinach BBY preparation directly injected onto the RP column without sucrose-gradient fractionation. Column and experimental conditions as in Figure 1. The amount of sample applied was 10 µg.

proteins components of PS II. Therefore, the use of the crude PS II membrane preparation does not affect resolution and retention times, while new and smaller peaks appear at longer elution time. Preliminary results suggest that these new peaks, recorded by fluorescence detector, represent the core proteins which are more hydrophobic and elute at higher concentration of acetonitrile. The injection of the BBY directly allows one to give an exact evaluation of the quantitative relations between chl a/b binding present in the Photosystem II, avoiding long separations, Comassie stain, quantification by densitometry and correction of the results according to the specific binding of Comassie to isolated proteins. Previous studies, attempting at developing of rapid and sensitive methods to identify and determine quantitatively the various components of PS II, have proven that capillary zone electrophoresis can be successfully applied for resolving the LHC II proteins (Zolla et al. 1996). Reversed phase HPLC (Zolla et al. 1997), besides being rapid, simple and precise, has proven to be effective in detecting differences in the protein components of LHC II isolated from different species that might be not evidenced by denaturing SDS–PAGE (data not shown). In addition, the possibility of separating all protein components of the PS II major and minor antenna system in samples not subjected to the sucrose-gradient ultracentrifugation, as in the case of injection of BBY, is expected to be advantageous for evaluating the relative content of the different protein components of PS II and their variation related to physiological adaptation to environmental conditions. The use of this method in screening photosynthetic mutants and plants adapted to different environmental

289 conditions will be useful in elucidating the composition and supramolecular organisation of LHC II and will possibly increase the understanding of the molecular mechanisms underlying the physiological adaptations.

Acknowledgement This work was supported in part by the CE INCOCOPERNICUS Project (no. IC15 CT98-0126).

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