- Email: [email protected]
[30] Photosystem II-phycobilisome complex preparations
286
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N2 FIXATION
[30]
[30] P h o t o s y s t e m I I - P h y c o b i l i s o m e C o m p l e x P r e p a r a t i o n s By E. GANTT,J. D. CLEMENT-METRAL,and B. M. CHERESKIN Introduction Energy transfer measurements have suggested a direct structural attachment of phycobilisomes and photosystem II. The hydrophilic nature of the phycobiliproteins, and ready dissociation of phycobilisomes under thylakoid membrane isolation conditions, required development of a special isolation medium. A medium consisting of sucrose-phosphate-citrate developed for Anabaena variabilisI preserved intact the phycobilisomes, energy transfer to phqtosystem II, and light-driven 02 evolution. Adaptation of the medium to endocyanelles2 and red algae 3: proved useful for cell-free photosynthetic activity measurements in phycobilisomecontaining algae. The main objective was to solubilize the thylakoid membrane but leave the phycobilisomes together with photosystem II and the watersplitting enzyme complex, removing photosystem I. The initial procedure developed for the unicellular red alga Porphyridium cruentum,5 and since successfully modified for several cyanobacteria, 6,7 is included here. Methods for Preparation of Photosystem II-Phycobilisome Complexes
Preparation from Porphyridium Reagents SPCM: 0.5 M sucrose, 0.5 M potassium phosphate, 0.3 M potassium citrate, 15 m M MgCI2, pH 7.0 LDAO (lauryldimethylamine oxide) preparation: LDAO (30%), 10 ml; catalase, 10/.~g/ml (peroxide scavanger) Sucrose gradients: Prepared in 0.3 M potassium citrate, 0.5 M potassium phosphate, 15 mM MgCI2, pH 7.0, with final sucrose in the T. Katoh and E. Gantt, Biochim. Biophys. Acta 546, 383 (1979). 2 L. P. Vernon and S. Cardon, Plant Physiol. 70, 442 (1982). 3 M. F. Dilworth and E. Gantt, Plant Physiol. 67, 608 (1981). 4 A. C. Stewart and A. W. D. Larkum, Biochem. J. 210, 583 (1983). 5 j. D. Clement-Metrai and E. Gantt, FEBS Lett. 156, 185 (1983). 6 H. B. Pakrasi and L. A. Sherman, Plant Physiol. 74, 742 (1984). 7 M. Kura-Hotta, K. Satoh, and S. Katoh, Arch. Biochem. Biophys. 249, 1 (1986).
METHODS IN ENZYMOLOGY, VOL. 167
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[30]
PSII--PHYCOBILISOME COMPLEX PREPARATIONS
287
following three steps: 2.0 M sucrose, 6 ml; 1.0 M sucrose, 8 ml; 0.5 M sucrose, 8 ml
Procedure Step I. Cells grown at approximately 40 txE/mZ/sec in a defined medium, 3 at 18°, with continuous agitation and aeration (5% CO2, 95% air) are harvested in the late exponential phase of growth ( - 7 days) by centrifugation. Step 2. The pelleted cells are rinsed rapidly by being suspended in distilled water ( - 1 g wet weight per 10 ml). The pellet of rinsed cells is then dissolved in SPCM (4 ml/g wet weight). A glass homogenizer is used to facilitate suspension of the cells in SPCM. The following steps are carried out at 4 ° in subdued room light or in darkness when possible. Step 3. The cell suspension is passed through an Aminco French pressure cell at 18,000 psi (128 MPa). To the membrane fragments (-0.30 mg Chl/ml) 0.12% (v/v) lauryldimethylamine oxide (LDAO) is added to give a detergent-to-chlorophyll ratio of 3.5-4 : 1 (w/w). The detergent-to-chlorophyll ratio is more critical than the percentage of LDAO. The mixture is incubated for 30 min in the dark with very gentle stirring. The incubation mixture is cleared of cell debris and starch by centrifugation at 27,000 gav for 30 min. Step 4. Two to three milliliters of supernatant is layered per tube on discontinuous sucrose step gradients, previously prepared, and centrifuged in an angle-head rotor for 11 hr at 130,000 gav. Step 5. The photosystem II-phycobilisome (PIIP) material is collected from the 1-2 M sucrose interface by a syringe with a flat-tipped canula (# 19), and diluted with 2 volumes of SPCM. The diluted material is centrifuged at 27,000 gav for 30 min to remove any contaminating membrane fragments. The supernatant is carefully removed with a syringe, taking care not to disturb the pellet. The PIIP in the supernatant can be assayed directly, or it can be concentrated further by centrifugation for 3 hr at 270,000 gav. Assays. The density of PIIP particles is only slightly lower than that of isolated phycobilisomes. It is important not to exceed the LDAO treatment time or concentration. The PIIP preparation retains a small amount of chlorophyll (Fig. 1A), but no detectable photosystem I components (P700). 5,8 A rapid determination of the fluorescence emission (-196°), with excitation through phycoerythrin (545 nm), reveals a maximum of 688 nm (Fig. 1B). On removal of the phycobilisome components the maxi8 j. D. Clement-Metral, E. Gantt, and T. Redlinger, Arch. Biochem. Biophys. 238, 10 (1985).
288
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND l
I
I
A
I
--
PS Tr- phycobilisome particles
///'~l I
---
Thylokoid-phycobllieome froctlon
[30]
I
688-
440
0.8
N2 FIXATION
>I---
550r563 I/~
I/
Z W I---
W CD
0.6
Z hl ~_) CO W nO
0.4
.J LL W
I 625
0.2
F< _J W ne
II Exc 4 4 0 nm
k.ll~\J
E x c ~ 3.3x
0
i
400
i
500
i
600
~\
I
I
700
6OO
7OO
WAVELENGTH ( n m )
FIG. 1. (A) Porphyridium cruentum absorption spectra of PIIP particles and unfractionated thylakoids in SPCM medium at 20 ° normalized at 550 nm. (B) Fluorescence emission of PIIP particles at - 1 9 6 ° with excitation of phycoerythrin (545 nm) and chlorophyll (440 nm).
mum becomes 698 nm, 9 a value typical of photosystem II reaction center cores. When chlorophyll is excited directly (440 nm) the PIIP preparations lack the photosystem I emission band at 715-720 nm. Examination by electron microscopy is recommended since it is an excellent indicator of contamination especially of thylakoid membranes lacking phycobilisomes. The sample in SPCM (A545nm, 1.5) is applied to carbon film-coated grids which have been freshly "ionized" by glow discharge treatment. Fixation in 0.25% glutaraldehyde for 2 min is followed by 5 rinses with distilled water and staining in 1% uranyl sulfate for 30 sec. The majority of phycobilisomes retain small putative thylakoid fragments.8 Photosystem II assays can be made by measuring 02 evolution or by the reduction of 2,6-dichlorophenolindophenol (DCPIP). ]°,11 Good PIIP preparations have typical 02 evolution rates of 900-1500/zM O2/mg Chl/ 9 B. M. Chereskin and E. Gantt, Arch. Biochem. Biophys. 250, 286 (1986). 10 B. M. Chereskin and E. Gantt, Plant Cell Physiol. 27, 751 (1986). i1 B. M. Chereskin, J. D. Clement-Metral, and E. Gantt, Plant Physiol. 77, 626 (1985).
[30]
PSII-PHYCOBILISOME COMPLEX PREPARATIONS
289
hr. Oxygen evolution is measured at 25° with a Clark-type electrode, with light from a slide projector and heat filters consisting of a 10-cm cooled water column and a Schott KG filter. The assay volume of 3.0 ml typically contains 2 m M FeCN, 1 mM dimethylbenzoquinone (DMBQ), and 0.671% ethanol. The electrode is calibrated by the method of Robinson and Cooper. lz It is important to note that the solubility of 02 in air-saturated SPCM is lower (55%) than in water. 1~ When activity is measured by dye reduction, characteristic values range from 250-450 /zM DCPIP/mg Chl/hr. DCPIP measurements (40 /xM DCPIP in a 3-ml sample in SPCM) are made in an Aminco DW2 spectrophotometer with side illumination from a Dolan Jenner Model 170-D illuminator. The absorbance change of 560 minus 520 nm is measured with a 3-nm slit width and at an extinction coefficient of 5.75 mM/ cm. Complete inhibition of activity occurs with 3,4-dichlorophenyl-l,1dimethylurea (DCMU) on the acceptor side and with NH2OH on the donor side of photosystem II. TM Oxygen-evolving activity is often inhibited 50-80% by aging, dilution, low pH, and salt washing. Bovine serum albumin (0.05%) and dithiothreitol (0.5 mM) can stimulate activity in all but salt-washed preparations.I°
Preparations from Synechococcus Several detergents have been successfully applied for PSII-enriched preparations from two cyanobacteria. Triton X-100 was used with Anacystis nidulans R2. 6 For Synechococcus sp. digitonin was found to produce PIIP preparations of greater purity than/3-octylglucoside. The protocol for PIIP preparation from Synechococcus with digitonin, provided below, is according to Kura-Hotta et al. 7
Reagents SPC incubation medium for PIIP: 0.5 M sucrose, 0.5 M phosphate, 0.3 M citrate, 5 m M MgClz, 20 m M CaCI2, 1 m M phenylmethylsulfonyl fluoride, 0.10% DNase (w/v), 1.5% bovine serum albumin, pH 6.9
Procedure Step 1. Cells grown for 2 days at 55° are harvested. Since these cells are difficult to break, spheroplasts are produced to enhance the yield. The incubation medium consists of 0.5 M sucrose, 0.1 M phosphate (pH 7.2), and 0.15% (w/v) lysozyme. Incubation for 2 hr at 40 ° with gentle stirring is followed by centrifugation at 10,000 gay for 10 min. 12 j. Robinson and J. M. Cooper, Anal. Biochem. 33, 390 (1970).
290
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N 2 FIXATION
[30]
A V)
~r
T
4
0
a W n,1.1.1 IJ_ if) Z ne
Z 0 nl---w _J W
I
0
I I
0 LIGHT
I 2 QUANTA
I 3 ABSORBED
I 4
5
(lO-"Einstein/s)
FIG. 2. Synechococcus sp. PIIP-digitonin preparations: ferricyanide photoreduction as a function of light quanta absorbed by phycobiliproteins.
Step 2. Spheroplasts (0.4 mg Chl/ml) are suspended in SPC incubation medium, broken in a French pressure cell at approximately 980 psi (7 MPa), and incubated for 30-60 min at 25 ° following addition of 0.5% digitonin with a final detergent-to-chlorophyll ratio (w/w) of 12.5. Step 3. On removal of large membrane fragments, by centrifugation at 25,000 gay for 10 min, the supernatant is layered on discontinuous sucrose step gradients in SPC medium. The sucrose gradient steps range from 0.5, 0.75, 1.0, to 1.5 M sucrose. Step 4. The dark-green PIIP material is collected from the 1.0-1.5 M sucrose interface after centrifugation for 70 min at 300,000 gav. Assay. By their absorption spectra the phycobilisome composition characteristics of the PIIP complexes are identical to those in unfractionated thylakoids and those in isolated phycobilisomes. The energetic coupling between PSII and phycobilisomes, according to their emission at -196 °, is comparable to whole cells. 7 A high quantum yield (0.85), according to measurements of ferricyanide photoreduction and quanta absorbed by phycobilisomes (Fig. 2), shows a complete coupling to PSII reaction centers. Note. Sulfobetain 12 (0.35%) has been successfully used in isolation of PSII particles 13 and should be useful for preparing PIIP. t3 E. M6rschel and G. H. Schatz, Planta 172, 145 (1987).
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N2 FIXATION
[30]
[30] P h o t o s y s t e m I I - P h y c o b i l i s o m e C o m p l e x P r e p a r a t i o n s By E. GANTT,J. D. CLEMENT-METRAL,and B. M. CHERESKIN Introduction Energy transfer measurements have suggested a direct structural attachment of phycobilisomes and photosystem II. The hydrophilic nature of the phycobiliproteins, and ready dissociation of phycobilisomes under thylakoid membrane isolation conditions, required development of a special isolation medium. A medium consisting of sucrose-phosphate-citrate developed for Anabaena variabilisI preserved intact the phycobilisomes, energy transfer to phqtosystem II, and light-driven 02 evolution. Adaptation of the medium to endocyanelles2 and red algae 3: proved useful for cell-free photosynthetic activity measurements in phycobilisomecontaining algae. The main objective was to solubilize the thylakoid membrane but leave the phycobilisomes together with photosystem II and the watersplitting enzyme complex, removing photosystem I. The initial procedure developed for the unicellular red alga Porphyridium cruentum,5 and since successfully modified for several cyanobacteria, 6,7 is included here. Methods for Preparation of Photosystem II-Phycobilisome Complexes
Preparation from Porphyridium Reagents SPCM: 0.5 M sucrose, 0.5 M potassium phosphate, 0.3 M potassium citrate, 15 m M MgCI2, pH 7.0 LDAO (lauryldimethylamine oxide) preparation: LDAO (30%), 10 ml; catalase, 10/.~g/ml (peroxide scavanger) Sucrose gradients: Prepared in 0.3 M potassium citrate, 0.5 M potassium phosphate, 15 mM MgCI2, pH 7.0, with final sucrose in the T. Katoh and E. Gantt, Biochim. Biophys. Acta 546, 383 (1979). 2 L. P. Vernon and S. Cardon, Plant Physiol. 70, 442 (1982). 3 M. F. Dilworth and E. Gantt, Plant Physiol. 67, 608 (1981). 4 A. C. Stewart and A. W. D. Larkum, Biochem. J. 210, 583 (1983). 5 j. D. Clement-Metrai and E. Gantt, FEBS Lett. 156, 185 (1983). 6 H. B. Pakrasi and L. A. Sherman, Plant Physiol. 74, 742 (1984). 7 M. Kura-Hotta, K. Satoh, and S. Katoh, Arch. Biochem. Biophys. 249, 1 (1986).
METHODS IN ENZYMOLOGY, VOL. 167
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[30]
PSII--PHYCOBILISOME COMPLEX PREPARATIONS
287
following three steps: 2.0 M sucrose, 6 ml; 1.0 M sucrose, 8 ml; 0.5 M sucrose, 8 ml
Procedure Step I. Cells grown at approximately 40 txE/mZ/sec in a defined medium, 3 at 18°, with continuous agitation and aeration (5% CO2, 95% air) are harvested in the late exponential phase of growth ( - 7 days) by centrifugation. Step 2. The pelleted cells are rinsed rapidly by being suspended in distilled water ( - 1 g wet weight per 10 ml). The pellet of rinsed cells is then dissolved in SPCM (4 ml/g wet weight). A glass homogenizer is used to facilitate suspension of the cells in SPCM. The following steps are carried out at 4 ° in subdued room light or in darkness when possible. Step 3. The cell suspension is passed through an Aminco French pressure cell at 18,000 psi (128 MPa). To the membrane fragments (-0.30 mg Chl/ml) 0.12% (v/v) lauryldimethylamine oxide (LDAO) is added to give a detergent-to-chlorophyll ratio of 3.5-4 : 1 (w/w). The detergent-to-chlorophyll ratio is more critical than the percentage of LDAO. The mixture is incubated for 30 min in the dark with very gentle stirring. The incubation mixture is cleared of cell debris and starch by centrifugation at 27,000 gav for 30 min. Step 4. Two to three milliliters of supernatant is layered per tube on discontinuous sucrose step gradients, previously prepared, and centrifuged in an angle-head rotor for 11 hr at 130,000 gav. Step 5. The photosystem II-phycobilisome (PIIP) material is collected from the 1-2 M sucrose interface by a syringe with a flat-tipped canula (# 19), and diluted with 2 volumes of SPCM. The diluted material is centrifuged at 27,000 gav for 30 min to remove any contaminating membrane fragments. The supernatant is carefully removed with a syringe, taking care not to disturb the pellet. The PIIP in the supernatant can be assayed directly, or it can be concentrated further by centrifugation for 3 hr at 270,000 gav. Assays. The density of PIIP particles is only slightly lower than that of isolated phycobilisomes. It is important not to exceed the LDAO treatment time or concentration. The PIIP preparation retains a small amount of chlorophyll (Fig. 1A), but no detectable photosystem I components (P700). 5,8 A rapid determination of the fluorescence emission (-196°), with excitation through phycoerythrin (545 nm), reveals a maximum of 688 nm (Fig. 1B). On removal of the phycobilisome components the maxi8 j. D. Clement-Metral, E. Gantt, and T. Redlinger, Arch. Biochem. Biophys. 238, 10 (1985).
288
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND l
I
I
A
I
--
PS Tr- phycobilisome particles
///'~l I
---
Thylokoid-phycobllieome froctlon
[30]
I
688-
440
0.8
N2 FIXATION
>I---
550r563 I/~
I/
Z W I---
W CD
0.6
Z hl ~_) CO W nO
0.4
.J LL W
I 625
0.2
F< _J W ne
II Exc 4 4 0 nm
k.ll~\J
E x c ~ 3.3x
0
i
400
i
500
i
600
~\
I
I
700
6OO
7OO
WAVELENGTH ( n m )
FIG. 1. (A) Porphyridium cruentum absorption spectra of PIIP particles and unfractionated thylakoids in SPCM medium at 20 ° normalized at 550 nm. (B) Fluorescence emission of PIIP particles at - 1 9 6 ° with excitation of phycoerythrin (545 nm) and chlorophyll (440 nm).
mum becomes 698 nm, 9 a value typical of photosystem II reaction center cores. When chlorophyll is excited directly (440 nm) the PIIP preparations lack the photosystem I emission band at 715-720 nm. Examination by electron microscopy is recommended since it is an excellent indicator of contamination especially of thylakoid membranes lacking phycobilisomes. The sample in SPCM (A545nm, 1.5) is applied to carbon film-coated grids which have been freshly "ionized" by glow discharge treatment. Fixation in 0.25% glutaraldehyde for 2 min is followed by 5 rinses with distilled water and staining in 1% uranyl sulfate for 30 sec. The majority of phycobilisomes retain small putative thylakoid fragments.8 Photosystem II assays can be made by measuring 02 evolution or by the reduction of 2,6-dichlorophenolindophenol (DCPIP). ]°,11 Good PIIP preparations have typical 02 evolution rates of 900-1500/zM O2/mg Chl/ 9 B. M. Chereskin and E. Gantt, Arch. Biochem. Biophys. 250, 286 (1986). 10 B. M. Chereskin and E. Gantt, Plant Cell Physiol. 27, 751 (1986). i1 B. M. Chereskin, J. D. Clement-Metral, and E. Gantt, Plant Physiol. 77, 626 (1985).
[30]
PSII-PHYCOBILISOME COMPLEX PREPARATIONS
289
hr. Oxygen evolution is measured at 25° with a Clark-type electrode, with light from a slide projector and heat filters consisting of a 10-cm cooled water column and a Schott KG filter. The assay volume of 3.0 ml typically contains 2 m M FeCN, 1 mM dimethylbenzoquinone (DMBQ), and 0.671% ethanol. The electrode is calibrated by the method of Robinson and Cooper. lz It is important to note that the solubility of 02 in air-saturated SPCM is lower (55%) than in water. 1~ When activity is measured by dye reduction, characteristic values range from 250-450 /zM DCPIP/mg Chl/hr. DCPIP measurements (40 /xM DCPIP in a 3-ml sample in SPCM) are made in an Aminco DW2 spectrophotometer with side illumination from a Dolan Jenner Model 170-D illuminator. The absorbance change of 560 minus 520 nm is measured with a 3-nm slit width and at an extinction coefficient of 5.75 mM/ cm. Complete inhibition of activity occurs with 3,4-dichlorophenyl-l,1dimethylurea (DCMU) on the acceptor side and with NH2OH on the donor side of photosystem II. TM Oxygen-evolving activity is often inhibited 50-80% by aging, dilution, low pH, and salt washing. Bovine serum albumin (0.05%) and dithiothreitol (0.5 mM) can stimulate activity in all but salt-washed preparations.I°
Preparations from Synechococcus Several detergents have been successfully applied for PSII-enriched preparations from two cyanobacteria. Triton X-100 was used with Anacystis nidulans R2. 6 For Synechococcus sp. digitonin was found to produce PIIP preparations of greater purity than/3-octylglucoside. The protocol for PIIP preparation from Synechococcus with digitonin, provided below, is according to Kura-Hotta et al. 7
Reagents SPC incubation medium for PIIP: 0.5 M sucrose, 0.5 M phosphate, 0.3 M citrate, 5 m M MgClz, 20 m M CaCI2, 1 m M phenylmethylsulfonyl fluoride, 0.10% DNase (w/v), 1.5% bovine serum albumin, pH 6.9
Procedure Step 1. Cells grown for 2 days at 55° are harvested. Since these cells are difficult to break, spheroplasts are produced to enhance the yield. The incubation medium consists of 0.5 M sucrose, 0.1 M phosphate (pH 7.2), and 0.15% (w/v) lysozyme. Incubation for 2 hr at 40 ° with gentle stirring is followed by centrifugation at 10,000 gay for 10 min. 12 j. Robinson and J. M. Cooper, Anal. Biochem. 33, 390 (1970).
290
MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N 2 FIXATION
[30]
A V)
~r
T
4
0
a W n,1.1.1 IJ_ if) Z ne
Z 0 nl---w _J W
I
0
I I
0 LIGHT
I 2 QUANTA
I 3 ABSORBED
I 4
5
(lO-"Einstein/s)
FIG. 2. Synechococcus sp. PIIP-digitonin preparations: ferricyanide photoreduction as a function of light quanta absorbed by phycobiliproteins.
Step 2. Spheroplasts (0.4 mg Chl/ml) are suspended in SPC incubation medium, broken in a French pressure cell at approximately 980 psi (7 MPa), and incubated for 30-60 min at 25 ° following addition of 0.5% digitonin with a final detergent-to-chlorophyll ratio (w/w) of 12.5. Step 3. On removal of large membrane fragments, by centrifugation at 25,000 gay for 10 min, the supernatant is layered on discontinuous sucrose step gradients in SPC medium. The sucrose gradient steps range from 0.5, 0.75, 1.0, to 1.5 M sucrose. Step 4. The dark-green PIIP material is collected from the 1.0-1.5 M sucrose interface after centrifugation for 70 min at 300,000 gav. Assay. By their absorption spectra the phycobilisome composition characteristics of the PIIP complexes are identical to those in unfractionated thylakoids and those in isolated phycobilisomes. The energetic coupling between PSII and phycobilisomes, according to their emission at -196 °, is comparable to whole cells. 7 A high quantum yield (0.85), according to measurements of ferricyanide photoreduction and quanta absorbed by phycobilisomes (Fig. 2), shows a complete coupling to PSII reaction centers. Note. Sulfobetain 12 (0.35%) has been successfully used in isolation of PSII particles 13 and should be useful for preparing PIIP. t3 E. M6rschel and G. H. Schatz, Planta 172, 145 (1987).