228
Biochimica et Biophysica Acta, 581 (1979) 228--236 © Elsevier/North-Holland Biomedical Press
BBA 38319
PURIFICATION AND CHARACTERIZATION OF 33 KILODALTON PROTEIN OF SPINACH CHLOROPLASTS
TOMOHIKO KUWABARA and NORIO MURATA *
Department of Biology, College of General Education, University of Tokyo, Komaba, Meguro-ku, Tokyo 153 (Japan) (Received April 9th, 1979)
Key words: 33 000 dalton protein; Membrane protein; (Chloroplast membrane)
Summary A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.
Introduction Recent studies [1--7] using polyacrylamide gel electrophoresis in the presence of SDS have shown a number of polypeptides present in spinach chloroplasts. Only a part of the polypeptides have been identified as subunits of chlorophyll-protein complexes I and II [1,2], CF1 [2--6], and ribulose-l,5bisphosphate carboxylase [5--7], while the other polypeptides are left unidentified. In order to understand the entire functioning of chloroplasts, it is indispensable to characterize the proteins that are constituted by the unidentified polypeptides. * T o w h o m correspondence should be addressed. Abbreviations; SDS, sodium dodecyl sulfate, Tricine, N-tris(hydroxymethyl)methyl glycine; Tris, tris(hydroxymethyl)aminomethane ~ C F 1, chloroplast coupling factor.
229 In the present paper, we report purification and a partial characterization of one of such proteins. The protein was provisionally named '33 kilodalton protein (33 kD protein)' according to its molecular weight. It was found only in System II particles, and thus considered to play a role in the functioning of System II. Materials and Methods Preparation procedures for the broken chloroplasts and the subchloroplast particles were all done at 4°C. Spinach was purchased from local market. Deveined spinach leaves were homogenized by a mixer in a preparation medium containing 0.2 M NaC1 and 0.05 M phosphate buffer, pH 7.8. After filtration through 16 layers of gauze, the green juice was centrifuged at 3000 X g for 5 min. The precipitate was resuspended in the preparation medium and centrifuged at 500 × g for 30 s. The supernatant was recentrifuged at 3000 X g for 5 min. The resulting precipitate was the 'broken chloroplast preparation'. Chlorophyll a to b ratio in this preparation was about 3.0. In order to remove ribulose-l,5-bisphosphate carboxylase, the broken chloroplasts were suspended in 0.01 M NaC1 [8]. After standing for 10 min, the suspension was centrifuged at 3000 X g for 5 min, and the precipitate was discarded. The chloroplasts were collected by centrifuging the supernatant at 12 000 × g for 10 min. The resulting precipitate was the 'hypotonically washed chloroplast preparation'. In order to prepare the System II particles, the broken chloroplasts were suspended in a medium containing 0.05 M NaC1, 0.3 M sucrose and 0.05 M phosphate buffer, pH 6.8. Chlorophyll concentration was adjusted to 3--4 mg/ ml. To the chloroplast suspension 20% Triton X-100 solution (w/v) was added in such a manner that the ratio of Triton X-100 to chlorophyll was 25. After a gentle stir for 30 min, the suspension was centrifuged at 5000 X g for 10 min. The supernatant was recentrifuged at 26 000 X g for 30 min. The obtained precipitate was suspended in 0.01 M Tricine/NaOH buffer, pH 7.6, and centrifuged at 26 000 X g for 30 min. The precipitate was washed with the same buffer by resuspension and recentrifugation. The precipitate thus obtained was the 'System II particle preparation'. Chlorophyll a to b ratio of this preparation was 2.0. System I particles and System II particles were also prepared according to Anderson and Boardman [9] using digitonin and differential centrifugation. Chlorophyll a to b ratio was 6.4 in System I particles, and 2.6 in System II particles. Isoelectric focusing was performed according to Vesterberg and Svensson [10] in 1% (w/v) Ampholine of pH range between 4.0 and 6.0. Electrophoresis was performed at a constant current of 4 mA for 48 h in the cold. Polyacrylamide gel electrophoresis in the presence of SDS and urea (SDS gel electrophoresis) was performed according to the method of H o o b e r [11] with minor modifications. A thin plate (0.1 X 14.8 X 15.7 cm) of polyacrylamide gel was made up in a m e t h o d similar to Chua and Bennoun [12] using an apparatus of slab gel electrophoresis (Toyo). The stacking gel, 1.2 cm in length, contained 5% polyacrylamide in pH 6.8, and the separation gel, 14.5 cm in
230 length, contained a linear gradient of polyacrylamide concentration from 7.5 to 20% in pH 8.4. Unless otherwise indicated, the electrode buffer did not contain thioglycolic acid. Before the electrophoresis, the membrane preparations were solubilized at room temperature in a medium containing 0.05 M Na2CO3, 2% SDS, 0.01% EDTA, 0.5 M urea and 10% sucrose. The protein preparations were also treated in the same medium. After standing for 30 min, they were loaded on the polyacrylamide gel. Electrophoresis was performed at a constant voltage of 200 V for about 12 h in a cold room. Discontinuous polyacrylamide gel electrophoresis was performed according to Davis [13] using the slab gel apparatus. The concentration of polyacrylamide for the separation gel was 8.5%. Electrophoresis was performed at a constant current of 15 mA for about 5 h in a cold room. After the electrophoresis, gel plates were stained for 2 h in 50% methanol and 10% acetic acid containing 0.04% Coomassie brilliant blue R-250, and destained in 35% methanol and 10% acetic acid. Densitogram of the electrophoretic pattern was measured using a dual-wavelength TLC scanner CS-900 (Shimadzu) equipped with a 25 pm slit. Absorbance at 560 nm was traced with 700 nm as a reference beam. The molecular weight of protein was estimated by SDS gel electrophoresis with a uniform concentration of polyacrylamide at 10%. Conditions for the pretreatment and the electrophoresis were the same as described above. The molecular weight of protein was estimated also by gel filtration chromatography with Sephadex G-100 with a column size of 2.5 cm in diameter and 60 cm in length. Eluent contained 1.0 M NaC1 and 0.1 M Tris-HC1 buffer, pH 8.0. Molecular weight standards in the SDS gel electrophoresis and the gel filtration chromatography were bovine serum albumin, ovalbumin, chymotrypsinogen A and cytochrome c. Chlorophyll concentration was determined according to Arnon [14 ]. Protein concentration was determined according to Lowry e t a l . [15]. Absorption spectrum was measured with a double beam spectrophotometer UV-200 (Shimadzu). Tricine buffer was purchased from Sigma. Ampholine, pH 4.0--6.0 was from LKB. Sephadex G-50 and G-100 were from Pharmacia. Proteins of molecular weight standard were from Boehringer Mannheim. Other chemicals were from Wako Pure Chemical Industries, Ltd. and of guaranteed grade. Results
Purification of 33 kD protein SDS gel electrophoresis of the hypotonically washed chloroplasts (Fig. la) indicated that there were more than 25 polypeptides in this preparation. Two green-colored polypeptide bands appeared, and were identified as the P-700 chlorophyll a-protein complex (or CP I) and the light-harvesting chlorophyll a/b-protein complex (or CP II) [1,2,16,17]. The a - a n d /3-subunits of CF1 [ 2 - 6 ] were seen in the densitogram, while the large and small subunits of ribulose-l,5-bisphosphate carboxylase [5--7] were not. The 33 kD protein was found as one of the major polypeptide bands in the densitogram. System II particles prepared with Triton X-100 contained a much less number of
231 U
(a)
(b) g
g
gli • I
Z: (c)
8
(d)
m
~
~
~
15
6
Migration distance (cm)
mm
~
~
i
9
i
12
F i g . 1. D e n s i t o g J r a m s o f S D S gel electrophoresis of spinach c h l o r o p l a s t s and the 33 k D protein. ( a ) H y p o t o n i c a l l y w a s h e d c h l o r o p l a s t s . ( b ) S y s t e m I I particles prepared w i t h T r i t o n X - 1 0 0 . ( c ) C r u d e 3 3 k D p r o t e i n preparation. (d) 3 3 k D p r o t e i n preparation after isoelectric focusing. Bands m a r k e d a s C P I and C P I I w e r e green c o l o r e d . N u m b e r 33 indicates 33 k D protein. ~ and fl stand for ~- and fl-subunits of C F 1 , respectively.
polypeptides in SDS gel electrophoresis (Fig. l b ) . The 33 kD protein was concentrated in System II particles which were, therefore, employed as a starting material for purification of the 33 kD protein. System II particles were suspended in 0.01 M Tricine/NaOH buffer, pH 7.6, and were treated with sonic oscillation (Toyoriko, 2N-100, at full power) for 10 min at 0°C. The suspension was centrifuged at 220 000 × g for 10 h. The green precipitate was discarded and the colorless supernatant was collected as crude 33 kD protein preparation. In SDS gel electrophoresis of the crude 33 kD protein preparation, there were a few minor bands in addition to the 33 kD protein band (Fig. lc). The crude 33 kD protein preparation underwent isoelectric focusing. 33 kD protein appeared at pH 5.2 (Fig. 2). SDS and disc gel electrophoresis (Fig. l d and Fig. 4a) indicated that there was no detectable impurity in the 33 kD protein preparation. In this m e t h o d a b o u t 2.5 mg of 33 kD protein was prepared from the broken chloroplasts that contained 1.5 g chlorophyll.
Characterization of 33 kD protein The molecular weight of the protein estimated by SDS gel electrophoresis was 33 000 (average of 5 experiments). According to this value of molecular
232
O'4[~ 6
8.0
/
ZO
6.0
~
pN 5.0
1.2
300 1.0
E
2'0
Jo
40
~o
Fraction numbeP
60
~5o
650
:oo
e ~
9.6 ~
o,2 lb
~oo
Wavelength (nm)
9.8
~° pH
4,0
3.0
0
70
0.4
~ ~1
I ~ I
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Biochimica et Biophysica Acta, 581 (1979) 228--236 © Elsevier/North-Holland Biomedical Press
BBA 38319
PURIFICATION AND CHARACTERIZATION OF 33 KILODALTON PROTEIN OF SPINACH CHLOROPLASTS
TOMOHIKO KUWABARA and NORIO MURATA *
Department of Biology, College of General Education, University of Tokyo, Komaba, Meguro-ku, Tokyo 153 (Japan) (Received April 9th, 1979)
Key words: 33 000 dalton protein; Membrane protein; (Chloroplast membrane)
Summary A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.
Introduction Recent studies [1--7] using polyacrylamide gel electrophoresis in the presence of SDS have shown a number of polypeptides present in spinach chloroplasts. Only a part of the polypeptides have been identified as subunits of chlorophyll-protein complexes I and II [1,2], CF1 [2--6], and ribulose-l,5bisphosphate carboxylase [5--7], while the other polypeptides are left unidentified. In order to understand the entire functioning of chloroplasts, it is indispensable to characterize the proteins that are constituted by the unidentified polypeptides. * T o w h o m correspondence should be addressed. Abbreviations; SDS, sodium dodecyl sulfate, Tricine, N-tris(hydroxymethyl)methyl glycine; Tris, tris(hydroxymethyl)aminomethane ~ C F 1, chloroplast coupling factor.
229 In the present paper, we report purification and a partial characterization of one of such proteins. The protein was provisionally named '33 kilodalton protein (33 kD protein)' according to its molecular weight. It was found only in System II particles, and thus considered to play a role in the functioning of System II. Materials and Methods Preparation procedures for the broken chloroplasts and the subchloroplast particles were all done at 4°C. Spinach was purchased from local market. Deveined spinach leaves were homogenized by a mixer in a preparation medium containing 0.2 M NaC1 and 0.05 M phosphate buffer, pH 7.8. After filtration through 16 layers of gauze, the green juice was centrifuged at 3000 X g for 5 min. The precipitate was resuspended in the preparation medium and centrifuged at 500 × g for 30 s. The supernatant was recentrifuged at 3000 X g for 5 min. The resulting precipitate was the 'broken chloroplast preparation'. Chlorophyll a to b ratio in this preparation was about 3.0. In order to remove ribulose-l,5-bisphosphate carboxylase, the broken chloroplasts were suspended in 0.01 M NaC1 [8]. After standing for 10 min, the suspension was centrifuged at 3000 X g for 5 min, and the precipitate was discarded. The chloroplasts were collected by centrifuging the supernatant at 12 000 × g for 10 min. The resulting precipitate was the 'hypotonically washed chloroplast preparation'. In order to prepare the System II particles, the broken chloroplasts were suspended in a medium containing 0.05 M NaC1, 0.3 M sucrose and 0.05 M phosphate buffer, pH 6.8. Chlorophyll concentration was adjusted to 3--4 mg/ ml. To the chloroplast suspension 20% Triton X-100 solution (w/v) was added in such a manner that the ratio of Triton X-100 to chlorophyll was 25. After a gentle stir for 30 min, the suspension was centrifuged at 5000 X g for 10 min. The supernatant was recentrifuged at 26 000 X g for 30 min. The obtained precipitate was suspended in 0.01 M Tricine/NaOH buffer, pH 7.6, and centrifuged at 26 000 X g for 30 min. The precipitate was washed with the same buffer by resuspension and recentrifugation. The precipitate thus obtained was the 'System II particle preparation'. Chlorophyll a to b ratio of this preparation was 2.0. System I particles and System II particles were also prepared according to Anderson and Boardman [9] using digitonin and differential centrifugation. Chlorophyll a to b ratio was 6.4 in System I particles, and 2.6 in System II particles. Isoelectric focusing was performed according to Vesterberg and Svensson [10] in 1% (w/v) Ampholine of pH range between 4.0 and 6.0. Electrophoresis was performed at a constant current of 4 mA for 48 h in the cold. Polyacrylamide gel electrophoresis in the presence of SDS and urea (SDS gel electrophoresis) was performed according to the method of H o o b e r [11] with minor modifications. A thin plate (0.1 X 14.8 X 15.7 cm) of polyacrylamide gel was made up in a m e t h o d similar to Chua and Bennoun [12] using an apparatus of slab gel electrophoresis (Toyo). The stacking gel, 1.2 cm in length, contained 5% polyacrylamide in pH 6.8, and the separation gel, 14.5 cm in
230 length, contained a linear gradient of polyacrylamide concentration from 7.5 to 20% in pH 8.4. Unless otherwise indicated, the electrode buffer did not contain thioglycolic acid. Before the electrophoresis, the membrane preparations were solubilized at room temperature in a medium containing 0.05 M Na2CO3, 2% SDS, 0.01% EDTA, 0.5 M urea and 10% sucrose. The protein preparations were also treated in the same medium. After standing for 30 min, they were loaded on the polyacrylamide gel. Electrophoresis was performed at a constant voltage of 200 V for about 12 h in a cold room. Discontinuous polyacrylamide gel electrophoresis was performed according to Davis [13] using the slab gel apparatus. The concentration of polyacrylamide for the separation gel was 8.5%. Electrophoresis was performed at a constant current of 15 mA for about 5 h in a cold room. After the electrophoresis, gel plates were stained for 2 h in 50% methanol and 10% acetic acid containing 0.04% Coomassie brilliant blue R-250, and destained in 35% methanol and 10% acetic acid. Densitogram of the electrophoretic pattern was measured using a dual-wavelength TLC scanner CS-900 (Shimadzu) equipped with a 25 pm slit. Absorbance at 560 nm was traced with 700 nm as a reference beam. The molecular weight of protein was estimated by SDS gel electrophoresis with a uniform concentration of polyacrylamide at 10%. Conditions for the pretreatment and the electrophoresis were the same as described above. The molecular weight of protein was estimated also by gel filtration chromatography with Sephadex G-100 with a column size of 2.5 cm in diameter and 60 cm in length. Eluent contained 1.0 M NaC1 and 0.1 M Tris-HC1 buffer, pH 8.0. Molecular weight standards in the SDS gel electrophoresis and the gel filtration chromatography were bovine serum albumin, ovalbumin, chymotrypsinogen A and cytochrome c. Chlorophyll concentration was determined according to Arnon [14 ]. Protein concentration was determined according to Lowry e t a l . [15]. Absorption spectrum was measured with a double beam spectrophotometer UV-200 (Shimadzu). Tricine buffer was purchased from Sigma. Ampholine, pH 4.0--6.0 was from LKB. Sephadex G-50 and G-100 were from Pharmacia. Proteins of molecular weight standard were from Boehringer Mannheim. Other chemicals were from Wako Pure Chemical Industries, Ltd. and of guaranteed grade. Results
Purification of 33 kD protein SDS gel electrophoresis of the hypotonically washed chloroplasts (Fig. la) indicated that there were more than 25 polypeptides in this preparation. Two green-colored polypeptide bands appeared, and were identified as the P-700 chlorophyll a-protein complex (or CP I) and the light-harvesting chlorophyll a/b-protein complex (or CP II) [1,2,16,17]. The a - a n d /3-subunits of CF1 [ 2 - 6 ] were seen in the densitogram, while the large and small subunits of ribulose-l,5-bisphosphate carboxylase [5--7] were not. The 33 kD protein was found as one of the major polypeptide bands in the densitogram. System II particles prepared with Triton X-100 contained a much less number of
231 U
(a)
(b) g
g
gli • I
Z: (c)
8
(d)
m
~
~
~
15
6
Migration distance (cm)
mm
~
~
i
9
i
12
F i g . 1. D e n s i t o g J r a m s o f S D S gel electrophoresis of spinach c h l o r o p l a s t s and the 33 k D protein. ( a ) H y p o t o n i c a l l y w a s h e d c h l o r o p l a s t s . ( b ) S y s t e m I I particles prepared w i t h T r i t o n X - 1 0 0 . ( c ) C r u d e 3 3 k D p r o t e i n preparation. (d) 3 3 k D p r o t e i n preparation after isoelectric focusing. Bands m a r k e d a s C P I and C P I I w e r e green c o l o r e d . N u m b e r 33 indicates 33 k D protein. ~ and fl stand for ~- and fl-subunits of C F 1 , respectively.
polypeptides in SDS gel electrophoresis (Fig. l b ) . The 33 kD protein was concentrated in System II particles which were, therefore, employed as a starting material for purification of the 33 kD protein. System II particles were suspended in 0.01 M Tricine/NaOH buffer, pH 7.6, and were treated with sonic oscillation (Toyoriko, 2N-100, at full power) for 10 min at 0°C. The suspension was centrifuged at 220 000 × g for 10 h. The green precipitate was discarded and the colorless supernatant was collected as crude 33 kD protein preparation. In SDS gel electrophoresis of the crude 33 kD protein preparation, there were a few minor bands in addition to the 33 kD protein band (Fig. lc). The crude 33 kD protein preparation underwent isoelectric focusing. 33 kD protein appeared at pH 5.2 (Fig. 2). SDS and disc gel electrophoresis (Fig. l d and Fig. 4a) indicated that there was no detectable impurity in the 33 kD protein preparation. In this m e t h o d a b o u t 2.5 mg of 33 kD protein was prepared from the broken chloroplasts that contained 1.5 g chlorophyll.
Characterization of 33 kD protein The molecular weight of the protein estimated by SDS gel electrophoresis was 33 000 (average of 5 experiments). According to this value of molecular
232
O'4[~ 6
8.0
/
ZO
6.0
~
pN 5.0
1.2
300 1.0
E
2'0
Jo
40
~o
Fraction numbeP
60
~5o
650
:oo
e ~
9.6 ~
o,2 lb
~oo
Wavelength (nm)
9.8
~° pH
4,0
3.0
0
70
0.4
~ ~1
I ~ I
o
~D
