... WILHELM MENKE, HANS CRAUBNER, GEORG H. SCHMID, and ALFONS RADUNZ. Max-Planck-Institut für Züchtungsforschung (Erwin-Baur-Institut), Köln- ...
CHLOROPLAST PROTEINS
1225
Photochemically Active Chlorophyll-Containing Proteins from Chloroplasts and their Localization in the Thylakoid Membrane FRIEDERIKE KOENIG, WILHELM MENKE, HANS CRAUBNER, GEORG H . SCHMID, a n d ALFONS R A D U N Z Max-Planck-Institut für Züchtungsforschung (Erwin-Baur-Institut), Köln-Vogelsang (Z. Naturforsch. 27 b . 1225—1238 11972] ; received July 5, 1972)
Chloroplasts, membranes, proteins, photosynthesis, antibodies After solubilization of stroma-freed chloroplasts with deoxycholate, the lipids and the detergent used are separated from the proteins by gel filtration. In this way not denatured pigment-containing protein preparations were obtained. The particles in fraction 1 exhibited a molecular weight of 600 000 and contained an average of 25 chlorophyll molecules. The circular dichroism spectrum showed exciton splitting of the red band. The particles in fraction 2 contained 1 chlorophyll molecule and exhibited a molecular weight of 110 000. The particles in fraction 3 also contained only 1 chlorophyll molecule and had a molecular weight of between 80 000 and 100 000. Pure preparations of fraction 1 only carried out the methylviologen M e h 1 e r reaction with the dichlorophenol indophenol/ascorbate couple as electron donor. Fraction 3 only reduced ferricyanide with diphenylcarbazide as an electron donor in the light. Fraction 2 exhibited both the photosystem I reaction and the photosystem II reaction. An antiserum to extracted fraction 1 does not inhibit electron transport in the intact lamellar system. The photoreduction of methylviologen is only inhibited after disruption of the thylakoids. The antiserum to fraction 2 inhibits the photoreduction of methylviologen in the intact lamellar system. Consequently, one inhibition site for this photosystem I reaction must be located on the inner and another on the outer surface of the thylakoid membrane. In addition, antibodies to fraction 1 are specifically adsorbed onto the lamellar system without any effect on electron transport and without a concomitant agglutination. Antibodies to fraction 3 partially inhibit the photoreduction of ferricyanide with diphenylcarbazide as an electron donor in the intact lamellar system. Hence, the inhibition site of this system II reaction is located on the outer surface of the thylakoids. We have reason to believe that the inhibition sites not reacting are located in the partitions, which are not accessible to antibodies.
Abbreviations: EDTA, ethylenediamine tetraacetate; FMN, flavin mononucleotide; PMS, phenazine methosulphate; CMU, N- (p-chlorophenyl) -A, A'-dimethylurea; DCMU, 3- (3,4-dichlorophenyl) -1,1-dimethylurea; DPIP, 2,6-dichlorophenol indophenol. If the lipids are extracted f r o m thylakoids organic
solvents,
a
residue
is
obtained
with
which
m o s t l y i n s o l u b l e in w a t e r . T h i s r e s i d u e is o n l y completely
solubilized
even
in
the
presence
is
f o r m o n l y w h e n l i p i d s h a v e b e e n e x t r a c t e d . T h e isolations acetic
the m e m b r a n e
alterations
It h a s
been
separation presents
the r e s i d u e is n o t well
suited
for
cannot
be
proteins
with
disadvantage avoided.
formic
that
These
or
chemical
preparations
known
for
some
time
that
chloro-
plast material can b e d i s s o l v e d b y detergents 4. T h e
6 — 8 M u r e a o r 6 M g u a n i d i n e - h y d r o c h l o r i d e . It c o n its i n s o l u b i l i t y ,
the
tial r e a c t i o n s o f p h o t o s y n t h e s i s .
sists o f u p to 8 0 p e r c e n t o f p o l y p e p t i d e s
D u e to
have
a r e t h e r e f o r e n o t s u i t e d f o r the i n v e s t i g a t i o n o f p a r -
inof
of
acid3
of
proteins
difficulties,
from
lipids
however,
and
in
this
detergents method
of
m e m b r a n e p r o t e i n i s o l a t i o n . K the s o l u b i l i z a t i o n
is
i n v e s t i g a t i o n s o f the m e m b r a n e p r o t e i n s . WEBER first
achieved
s u c c e e d e d i n b r i n g i n g the p r o t e i n s o f the t h y l a k o i d
m i c e l l s a n d l i p i d d e t e r g e n t m i c e l l s , it s h o u l d b e p o s -
m e m b r a n e into a water-soluble f o r m b y the use
sible
concentrated these
formic
experiments
acid2. that
it
It b e c a m e is
possible
clear to
of
from
of
obtain
so
to
by
obtain
membrane when
a q u e o u s s o l u t i o n s o f m e m b r a n e p r o t e i n s . It a p p e a r s
HELENIUS proteins
membranes
dium
into a
water-insoluble
lipids5. Requests for reprints should be sent to Prof. Dr. W . MENKE, Max-Planck-Institut für Züchtungsforschung (Erwin-Baur-Institut), D-5000 Köln 30.
formation
lipid-
and
break
SIMONS down
Lipids
and
f r o m proteins by two
of
protein
detergent
detergent-free
solutions
I n d e e d , this a p p e a r s t o
sodium
deoxycholate
columns. As
and
proteins.
using
that the p r o t e i n s f r o m the t h y l a k o i d s a n d f r o m o t h e r are t r a n s f o r m e d
the
deoxycholate. reported
in
that
concentrated
solutions
into
deoxycholate filtrations
be
Recently, serum-lipo-
alkaline proteins
soand
were
separated
through
Sephadex
e a r l y a s 1 9 4 1 , SMITH a n d PICKELS a n d
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F. KOENIG, W. MENKE, H. CRAUBNER, G. H. SCHMID, AND A. RADUNZ
1226
later BRIL et al. reported that chloroplasts are solubilized by deoxycholate 6 ' 7 . It therefore appeared reasonable to us to apply the method of HELENIUS a n d SIMONS to c h l o r o p l a s t s .
Materials and Methods Chloroplast preparations: Stroma-freed chloroplasts of Antirrhinum ma jus strain 50 and small fragments of the thylakoid membrane, termed ultrasonic supernatant, were prepared according to K A N N A N G A R A et al.8. Stroma-containing chloroplasts from Nicotiana tabacum var. John William's Broadleaf and from the tobacco aurea mutant Su/su2 were prepared as described earlier 9 . Preparation of the chloroplast fractions: Amongst several variations the following procedure yielded the best results: Immediately after isolation an amount of stroma-freed chloroplasts corresponding to 2.38 g dry weight (volume 27 ml) was admixed to 27 ml of a solution composed of 0.1 mole NaCl and 0.1 mole Na 2 HP0 4 per liter. The mixture was made up to 172 ml with a 0.05 M NaCl, 0.05 M Na 2 HP0 4 solution. 26.5 g sodium deoxycholate (Schwarz/Mann) were added under stirring. The final volume was 200 ml. The concentration of stroma-freed chloroplasts in the mixture was 11.9 mg/ml and the concentration of sodium deoxycholate 133 mg/ml. 11.2 mg sodium deoxycholate were used per mg of lamellar system. The mixture was stirred at room temperature in the dark for 15 hours and then centrifuged for 30 min at 31 000 g at 22 °C, which yielded a green sediment and a clear dark green supernatant. The sediment was lyophylized and was used later for nitrogen determina-
100
150
50
J00 Fraction
50
100
Number
Fig. 1. Isolation of the fractions 1, 2 and 3. Elution diagrams : a. Separation of the lipids (L) from the proteins (P) ; b. separation of the proteins into three fractions; c. separation of deoxycholate from the three fractions; D start of the detergent-containing fractions. Fraction volumes a and b : 17.0 m l ; c. fraction 1 : 18.6 ml, fraction 2 and 3 : 9.8 ml. The arrows indicate the cutting of the fractions.
tions. The supernatant (197 ml) was divided into two equal parts which were separated into two fractions each on two columns of Sephadex G 50 Fine (Pharmacia) . The first fraction was eluted with the void volume (Fig. 1 a). For equilibration of the columns (length 90 cm, diameter 10 cm) and for the elution of the fractions, a solution was used which contained 0.05 mole NaCl, 0.05 mole Na 2 HP0 4 and lOmmoles sodium deoxycholate per liter. The first fraction was concentrated from 2030 ml to 148 ml by ultrafiltration using a Sartorius apparatus SM 16525 and filter SM 12136. Gel filtration of this fraction through Sephadex G 100 yielded a separation into the three fractions 1, 2 and 3, from which the first dark green fraction was eluted with the void volume (Fig. 1 b). Fractions 2 and 3 were yellowish green in colour. The size of the column and the composition of the solutions used for equilibration and elution were the same as described above. Fraction 1 was concentrated by ultrafiltration from 740 ml to 142 ml. In 108 ml of this concentrated solution the phosphate was substituted by carbonate, by dialysis for 21 hours. Dialysis was carried out against a solution consisting of 0.05 mole NaCl, 0.05 mole Na 2 C0 3 and 10 mmoles sodium deoxycholate per liter. The detergent was removed from fraction 1 in carbonate solution by gel filtration through Sephadex G 75 (Fig. 1 c). Elution was carried out with a solution containing 0.05 mole NaCl and 0.05 mole Na2CO:) per liter, but containing no detergent. The dimensions of the column corresponded to that used above. Fraction 2 was concentrated by ultrafiltration from 1445 to 65 ml, and fraction 3 from 1920 ml to 61 ml. The concentration procedures led to large losses in fractions 2 and 3. Both fractions were separated from detergent by gel filtration through Sephadex G 75 columns (length 90 cm, diameter 5 cm) (Fig. 1 c). Elution was performed with a solution containing 0.05 mole NaCl and 0.05 mole Na 2 HP0 4 per liter. All fractions were cut as shown in the diagrams (Fig. 1 a, b, c). The fractions described in the following were all eluted with the void volume. The remainder (34 ml) of the above mentioned concentrated solution of fraction 1 in phosphate was treated, without dialysis against carbonate solution, as for fractions 2 and 3. When removing the detergent, fraction 1 aggregated on the column and could only be partially eluted. In all cases, gel filtration was carried out at 20 °C. The fractions obtained were dialysed against running deionized water. Part of the preparations was lyophylized and used for chemical analyses. The results from 5 preparations are listed in the tables, where possible. Values are given together with their mean errors of the average value. The large variations are partially due to the fact that slightly different concentrations of sodium deoxycholate were used in the individual preparations for solubilization of the stroma-freed chloroplasts. In addition, different amounts of stroma-freed chloroplasts were used. The determination of chlorophyll and carotenoids and the analysis of colourless lipids were performed as described earlier10. The test for sodium deoxycholate was carried out by means of thinlayer chro-
CHLOROPLAST
matography by comparing Rf-\alues, using precoated plastic sheets (Macherey, Nagel & Co., Polygram SIL N-HR). The solvent in all cases was a mixture of chloroform-methanol-ethylacetate 2 per cent ammonium hydroxide 50 : 25 : 25 : 1 —1.5 (v/v). In some cases chloroform-methanol-acetic acid-water 85 : 15 : 10 : 3.5 (v/v) was also used. A 5 per cent solution of phosphomolybdic acid in ethanol or conc. sulfuric acid were used for spraying. Nitrogen determinations were carried out according to K j e 1 d a hi. The conversion of nitrogen to protein was obtained using a factor of 6.0 For the determination of carbohydrates, 2 — 3 mg of material were suspended in 0.1 N NaOH and the sugars determined
according
to
the
LUSTIG
and
LANGER
procedure, modified according to WEIMER and MOSHIN 12. We heated the assay mixture in a boiling waterbath for 20 min, and used galactose as a standard. The molecular weight determinations were carried out at 20 °C in an analytical ultracentrifuge (Spinco Model E, Beckman), equipped with a photoelectric scanning system. Measurements of sedimentation speeds and the diffusion were carried out in double sector cells with the photoelectric scanning system at 280 and 670 nm. For determination of the apparent partial specific volumes a digital precision density measuring apparatus DMA 02 (Paar) according to STABINGER, LEOPOLD, and KRATKY was used. The solvents used were water, carbonate solution (0.05 M NaCl, 0.05 M Na 2 C0 3 ) or 0.05 M phosphate buffer pH 8.0 according to S e r e n s e n . The determination of particle size by electron microscopy was as described earlier 13. The techniques used for obtaining the absorption spectra and determination of the circular dichroism have also been described earlier 14> 8 . Preparation of the antisera: 1 mg antigen was suspended in 1 ml physiological sodium chloride solution (0.8 per cent) and emulsified with 1 ml of F r e u n d's adjuvant (complete, Difco). The emulsion was injected subcutaneously into the hindlegs of rabbits. 21 days later, 1 to 3 times 1 mg antigen was suspended in physiological sodium chloride and intravenously injected. To test for antibody formation, blood was withdrawn from the test animals before treatment and on the 7 th , 14th and 21 st days after the last injection. Precipitation tests with the antisera: The agglutination reactions were carried out as slide or tube tests. The agglutination titer was determined by the tube dilution method (dilution ratio 1/2) 15. The amount of antigen was fixed at 1.5 mg/ml. Precipitation reactions were carried out on slides, in microtubes and in the gel diffusion test according to OUCHTERLONY 16. The gel used consisted of 0.8 per cent agarose (L'lndust. Biologique Frangaise) in 0.06 M S e r e n s e n phosphate buffer pH 7.4. Partial protein decomposition with proteases and the treatment of chloroplasts with EDTA has been described in earlier papers I7, 8 .
PROTEINS
1227
Light sources and measurements were as described previously 18> 19. The measurement of photochemical reactions catalyzed by chloroplasts and the chloroplast fractions, namely the H i l l reaction, photophosphorylation and the photoreduction of methylviologen and anthraquinone-2-sulphonate, have been described in detail by RADUNZ et al.18. For the measurement of the photoreduction of methylviologen and anthraquinone-2-sulphonate, we used the manometric method described previously 18 and also a Pt-Ag oxygen electrode (Rank Bros. Bottisham, Cambridge, England) equipped with a Potentiometrie recorder (0 — 1 mV span, 1.3 sec response time) from Control Instruments Ltd., Birkenhead, Liverpool, England. The assay mixture contained: 2 ml buffer composed of 0.075 mole tricine and 0.2 mole KCl per liter, pH 7.8 — 8; 0.1ml 3.5-10~ 4 M DCMU; 0 . 5 - 1 ml chloroplast fraction; 0.1ml 0.6 M ascorbate, 0.1ml 2.58-10~ 2 M 2,6 dichlorophenol indophenol; 0.1 ml NaCN of a solution of 32.8 mg/20 ml H 2 0, and 0.1 ml 10~ 3 M methylviologen or anthraquinone-2-sulphonate. Preparation of the Chloroplast Fractions It proved principally possible to obtain soluble protein preparations from stroma-freed chloroplasts according to the procedure of H e 1 e n i u s and S i m o n s . A suspension of stroma-freed chloroplasts in an alkaline sodium chloride solution became clear upon the addition of a sufficient amount of deoxycholate. In order to minimize changes in the preparation, we substituted the sodium carbonate of H e 1 e n i u s by disodiumhydrogen phosphate. On an average, 87 per cent of the proteins were solubilized. The preparation was separated into two green fractions by filtration through Sephadex G 50 (Fig. I a). The second fraction contained the major part of the chlorophyll and lipids. This fraction was discarded. The first fraction running with the solvent front was separated into three fractions by filtration through Sephadex G 100 (Fig. l b ) . All three fractions were freed from detergent by means of Sephadex G 75 (Fig. l c ) . Fraction 1, which contained the bulk of the proteins, aggregated during this procedure and was largely retained on the column. In order to prevent this aggregation, sodium carbonate was substituted for the sodium phosphate by dialysis before the separation of fraction 1 from deoxycholate. The aggregation was avoided due to the higher pH and the detergent readily removed by gel filtration. Fractions 1, 2 and 3 were eluted from the Sephadex G 75 columns by the void volume. The elution patterns of fractions 2 and 3 showed a pronounced shoulder. The main fractions were separated
F. KOENIG, W. MENKE, H. CRAUBNER, G. H. SCHMID, AND A. RADUNZ
1228
according to the elution patterns (Fig. 1 ) . All fractions were dialysed against flowing deionized water. Part of the preparations were directly used, the remainder was lyophylized and used for analyses.
After dialysis fraction 1 consisted of a clear bluegreen solution, whereas fractions 2 and 3 were yellowish-green in colour. Comparison of the absorption spectrum of fraction 1 with that of stroma-freed chloroplasts showed that the pigment content of fraction 1 was essentially lower than that of the starting material. The maximum of the protein absorption between 270 and 295 nm, which is obscured by lipid absorption of the stroma-freed chloroplasts, was observed in spectra of fraction 1 (Fig. 2 ) .
100
-
•
a
J
/ I
60
/
A
n
\
1 i 1
40
[-
b
20 A
300 Fig. 2.
1
A 400
/ 500
600
U
Xtnm]
Absorption spectra of a. stroma-freed chloroplasts after ultrasonic treatment, b. fraction 1.
As seen in Table I, the protein content of fraction 1 was the highest whereas that of fraction 3 was the lowest. On the other hand, fraction 1 contained the lowest amounts of ether soluble components, fraction 3 containing the highest. For comparison, we would like to note that stroma-freed
1
Fraction Ether soluble components Protein (N x 6.0) in the extraction residue
Chemical and Physical Properties of the Preparations
80
Table I. Average composition of the fractions in per cent dry weight. Deviations are given as mean error of the average value.
6.8i ± 0 . 9 87
±1
2
3
11 ± 2
25 ± 1
67 ± 4
45
chloroplasts consist of 46 per cent of proteins and 40 per cent of lipids 2 0 . Fraction 1 had a chlorophyll content of approximately 4 per cent (Table I I ) , whereas stroma-freed chloroplasts contained approximately 12 per cent chlorophyll 10 . Fractions 2 and 3 each contained only 1 per cent chlorophyll. The ratio of chlorophyll a to b varied between experiments. These variations are thought to be due to different loadings of the columns. In one preparation of fraction 1, chlorophyll b was barely detectable. By sharper fractionation it should be possible to obtain preparations which eventually only contain chlorophyll a. The ratio of chlorophyll a to b in fractions 2 and 3 was approximately the same as in the starting material. It may be mentioned that the chlorophyll extracted from fraction 1 was still positive in the phase test according to M o 1 i s c h 21 , indicating that the chlorophyll was not allomerized, despite the unusual conditions to which it had been exposed during the preparation of the fraction. The carotenoid content of fractions 1 and 2 was approximately the same, whereas fraction 3 contained roughly double the amount of carotenoids compared to the previous fractions. However, considerably less carotenoids were present in fraction 1 than in stroma-freed chloroplasts, which contain on average 2 per cent 10 . In addition, the weight ratio of chlorophyll to carotenoids was shifted in favour of the chlorophyll. In fractions 2 and 3, this ratio was approximately the same as in stroma-freed chloroplasts. The ratio of the different carotenoids amongst themselves, which varied considerably from preparation to preparation, was very different in the 3 fractions. In fraction 1 three times more lutein than /5-carotene was found, whereas fractions 2 and 3 contained approximately equal amounts of these pigments. The extraction residue of fraction 1 contained 2.5 per cent carbohydrates. Fractions 2 and 3 were not analysed for carbohydrates due to the low yields. Since these fractions contained less proteins than fraction 1, it may be assumed that higher amounts of carbohydrates
CHLOROPLAST PROTEINS
would be found in 2 and 3. No colourless chloroplast lipids were detectable by thin layer chromatography of ether extracts of fractions 1 and 2. In contrast, fraction 3 contained such lipids. Fraction 1 was always detergent-free and fractions 2 and 3 were detergent-free in most cases. The occasional occurrence of detergent may be due to the fact that when cutting the eluate into fractions, some samples containing detergent were erroneously combined with the fractions. We cannot exclude the possibility that our preparations contained small amounts of impurities which may originate from the deoxycholate used. Approximately 6 4 per cent of the deoxycholate solubilized proteins were recovered in fraction 1, 4 per cent were recovered in fraction 2 and about 0.4 per cent in fraction 3. About 30 to 4 0 per cent of the total soluble proteins were lost. Obviously, these were partially retained on the column. The possibility exists that single proteins have been selectively adsorbed. Molecular weights have been determined in the ultracentrifuge by measuring the sedimentation coefficients S (Fig. 3 ) , the diffusion coefficients D (Fig. 4) and the apparent partial specific volumes 20
a
1229
10
0
0.1
0
o
u
„
0
o
0.3
0.2
10
-
0 1
0.1
" '
1—
0.3
0.2
0.3
U
0.5 C-103[g/cm3]
Fig. 4. Concentration dependence of the diffusion coefficients of a. fraction 1 in carbonate solution, b. fraction 1 in water and c. fraction 2 in water. Measurements at X = 280 nm.
1.6
10 • 1
1
0.2
^i
20
\J
10
0 o>
