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Physicochemical studies of bean and wheat chloroplast structural protein
132
BIOCI-IIMICAET BIOPHYSICAACTA
BBA 355O 3 PHYSICOCHEMICAL S T U D I E S OF BEAN AND W H E A T C H L O R O P L A S T STRUCTURAL PROTEIN
R. S. M A N I AND S A U L Z A L I K
Department of Plant Science, University of Alberta, Edmonton, Alberta (Canada) (Received A u g u s t i 4 t h , 1969)
SUMMARY
Chloroplasts from wheat and bean seedlings were isolated, and after removing lipid and pigments the protein was solubilized. (NH,)2SO 4 was used to precipitate the structural protein fraction. Sedimentation velocity studies showed homogeneous profiles in both species with s values lower than reported for spinach. The amino acid composition of this protein fraction from bean and wheat was similar. Optical rotatory dispersion measurements indicated the presence of a-helix in both. Gel electrophoresis showed that with respect to charge the structural protein fraction of wheat and bean chloroplasts was different and not homogeneous.
INTRODUCTION
The term "structural protein" was initially given by CRIDDLE et al. 1 to a protein fraction they isolated from beef heart mitochondria. It had no detectable enzyme activity and represented about 6O~o of the total mitochondrial protein. In addition it formed complexes with mitochondrial cytochromes and was viewed as a possible site for enzyme alignment. Later CRIDDLE AND PARK2 isolated a protein component from spinach chloroplasts which they designated as structural protein. It constituted about 40% of the total chloroplast protein and was homogeneous with a molecular weight of 23 ooo and a sedimentation coefficient of 2.2 S. MENKE AND JORDAN 3 who isolated proteins associated with chloroplast lamellae designated these as lamellar proteins. BIGGINS AND PARK4 and JI et al. 5 obtained an s value of 2.2 for the lamellar protein from spinach chloroplasts. For the lamellar lipoprotein of bean, MOLCHANOV AND BEZINGER 6 reported an s value of 2. 9. The chloroplast structural protein studied by CRIDDLE7 had a relatively high content of glutamate, aspartate and arginine, and a low value for phenylalanine compared to chloroplast lamellar protein. Recently GOFFEAUs reported the amino acid composition of Acetabularia chloroplast structural protein. It differed from that of spinach in the levels of aspartic and glutamic acids. As chloroplast structural protein is a maj or component of lamellar protein ~, the differences m a y have been a reflection of species. HALDAR et al. 9, TuPPY gt al. 1° and SCHATZ Biochim. Biophys. Aeta, 2oo (I97 o) I32 137
CHLOROPLAST
STRUCTURAL
133
PROTEIN
AND SALTZGABER11 found that on the basis of gel electrophoresis mitochondrial structural protein was heterogeneous. Also WARD AND POLLAKTMnoted that microsomal structural protein was heterogeneous. In view of our findings for chloroplast structural protein and in light of the cited T M results the term structural protein fraction is being used in this paper instead of structural protein. The study being reported was undertaken to characterize and compare chloroplast structural protein fractions from bean and wheat by sedimentation velocity studies, optical rotatory dispersion measurements, amino acid analysis and gel electrophoresis. MATERIALS AND METHODS
Seedlings of Stewart 63 wheat (Triticum durum Desf.) and Kinghorn wax bean (Phaseolus vulgaris L.) were grown in soil in the glasshouse under natural illumination. Wheat leaves were harvested on the 5th day after planting whereas bean leaves were harvested on the 7th day, following a I2-h dark period. Chilled, fresh wheat leaves were ground for 2 rain in a mortar in a medium consisting of 0.44 M sucrose; o.I M Tris (pH 7.8); 50 mM KC1; IO mM MgC12 and 4 mM mercaptoethanol. The ratio of flesh leaves to buffer was 1:2 (w/v). The grinding medium for bean leaves was 0.33 M sucrose in o.I M phosphate buffer (pH 6.8) ; o.oi M KC1 and o.oi M MgC12 (I :1.5, w/v). After filtering through cheesecloth, chloroplasts were isolated from the filtrates as diagramed. All operations were at 4 °, and are shown in Scheme I. Wheat ieaf filtrate
Bean leaf filtrate
500 x g, 1 0 m i n Supernatant
100 x g, 2 min
Nuclei, cell debris (discard)
Supernatant ]
2300 × g, 10 min
1000 × g, 15 min
l[ Crude
chloroplast pellet
Crude chloroplast pellet
Supernatant (discard)
Resuspended in sucrose phosphate buffer
Resuspended and washed 4 times in sucrose T r i s buffer
3000 x g, 20 min
2300 × g, 15 min
I Supernatant (discard) Purified chloroplasts (wheat)
h
Washed chloroplast pellet Layered on discontinuous sucrose density gradient (1.0 M, 1.5 M, 2.0 M sucrose in 0.1 M phosphate buffer) 25000 × g, 20 min Chloroplast band in 1.5 M layer collected Resuspended in sucrose free 0.1 M phosphate buffer
Scheme
I 3000 x g, 20 rain Purified chloroplasts (bean)
The isolated chloroplasts were freed from lipid and pigments by suspending in chloroform and acetone ( I : I , v/v) and subsequent washing with acetone. Finally, Biochim. Biophys. Acta,
2 0 0 ( 1 9 7 o) 1 3 2 - 1 3 7
134
R . S . MANI, S. ZALIK
they were washed with ether and dried under vacuum. The lipid-free protein was suspended in o.oo2 M Tris buffer (pH 8.5) containing o.o35 M NaC1, o.ooi M sodium ascorbate and a trace of Na~S204. Solubilization was effected by cholate (2 mg per mg protein) and deoxycholate (i mg per mg protein) and was followed by precipitation with I 2 - I 6 % (NH4)2SO 4 according to CRIDDLEAND PARK2. The precipitated protein was then extracted with cold acetone to remove the detergents. Sedimentation analyses were carried out on a Spinco Model E analytical ultracentrifuge at 20 °. The medium used for sedimentation consisted of 1% sodium dodecylsulphate, o.I M NaC1 and 5 mM 2-mercaptoethanol at pH II.O. Sodium dodecyl sulphate was required to solubilize the protein. NaC1 was employed to reduce the primary salt effect, whereas mercaptoethanol should have minimized any subunit association. Amino acid analysis of the structural protein was carried out on an amino acid analyzer according to SPACKMANet al. 13. The protein was hydrolyzed in oxygen-free sealed ampules with constant boiling HCI at IiO ° for 20 h. Optical rotatory dispersion measurements of bean and wheat chloroplast structural protein fractions in 1% sodium dodecyl sulphate, o.I M NaC1, 5 mM dithiothreitol at pH II.O were obtained using a Cary Model 60 recording spectropolarimeter. The solvent was filtered through a 0.45 /, Millipore filter and the protein solution through a 5.0 # filter. Readings were taken in the visible region with a I-cm path length cell and in the ultraviolet region a o. I-cm cell was used. Gel electrophoresis in acetic acid was carried out according to TAKAYAMAet al. 14 but the medium contained phenol-acetic acid-water ( i : i : i , w/v/v) to a final protein concentration of 7 mg/ml. Polymerization of the gel was carried out at 5°° for 15 rain. RESULTS AND DISCUSSION
The chloroplast structural protein fraction on the basis of isolation technique presumably was free of soluble and non-structural lamellar protein. The structural protein fractions from bean and wheat chloroplasts were homogeneous by analytical
1.6.
1.4-
!.2S
1.0-
0*8-
CONC. (mg/mJl
Fig. i. S e d i m e n t a t i o n v e l o c i t y p a t t e r n of w h e a t c h l o r o p l a s t s t r u c t u r a l p r o t e i n f r a c t i o n in I °. 0 s o d i u m d o d e c y l s u l p h a t e , o.i M NaC1, 5 mM m e r c a p t o e t h a n o t , p H II.O a t 2o °. P i c t u r e t a k e n a f t e r 48 m i n a t 59 780 r e v . / m i n w i t h a b a r a n g l e of 5 o°. Fig. 2. S e d i m e n t a t i o n coefficient of b e a n f r a c t i o n as a f u n c t i o n of c o n c e n t r a t i o n .
Biochim. Biophys. Acta, 200 (197 o) 132-137
(©)
and wheat
(O) c h l o r o p l a s t s t r u c t u r a l p r o t e i n
CHLOROPLAST STRUCTURAL PROTEIN
135
ultracentrifuge analysis having s°20,w values of 1.2 and 1.3, respectively (Fig. i). There was no indication of any slower moving component after 4 h at 59 780 rev./min. The sedimentation coefficients decreased with increasing concentration (Fig. 2) indicating no association or dissociation of the protein in the concentration range studied. As seen, the s values obtained by us differ considerably from those reported by CRIDDLE AND PARK2 for spinach. This m a y be attributed to differences in species, isolation techniques and solvent systems used. Wheat chloroplasts were subjected to four washings during isolation and bean chloroplasts were purified on a discontinuous sucrose density gradient whereas the solvent system used for sedimentation studies included mercaptoethanol, unlike the preparations of CRIDDLE AND PARK2. Based upon the relatively low s values obtained for the structural protein fraction it is unlikely that it contained significant amounts of non-structural lamellar protein.
TABLE
[
AMINO ACID COMPOSITION
A m i n o acid
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Cysteic acid
OF CHLOROPLAST
STRUCTURAL
PROTEINS
Mole % Wheat
Bean
Acetabularia s
Spi nac h ~
Spi nac h lamellar protein ~
8.6 5.5 7.7 5.9 lO. 4 lO.9 lO.4 6. 7 1.4 4.5 9.4 1.8 4.2 6.5 1.7 4.4 --
8.2 5-8 7. i 5.4 lO.2 I i .7 9.9 6.2 i. i 4 .8 lO.2 2.4 5.o 6.3 1.7 4 .0
9.3 5-6 6.o 5.3 i i .o I I .o I i .o 7-4 Trace 5 .8 io. i 1.6 5.8 4.9 1.5 3-9 --
14-o 3.7 5.5 5. i 13- t 9.2 lO. 4 7.4 o.z 6.6 i 1.4 o.7 o.2 8.2 2.I i.o 1.3
8.8 4.7 5.7 5.9 9.2 io. 5 9.6 6.5 1.7 5-3 I i.o 3.8 6.5 5.5 1.4 4.2 --
Table I compares amino acid composition of the structural protein fractions from bean and wheat chloroplasts with that of other species. Essentially these fractions from wheat and bean had like composition. The major differences from structural protein of spinach were in the levels of aspartate, glutamate, phenylalanine and arginine. However, the values for bean and wheat were similar to chloroplast structural protein from Acetabularia. The amino acid composition was similar to spinach lamellar protein as well as lamellar protein fractions from other species 15,16. The relatively high levels of non-polar amino acids in the chloroplast structural protein fraction could give rise to extensive hydrophobic regions which might account for protein stabilitylL Biochim. Biophys. Acta, 200 (197 o) 1 3 2 - 1 3 7
136
R.S. MANI, S. ZALIK
Conformation of the structural protein fraction was studied by optical r o t a t o r y dispersion using a solvent system similar to t h a t employed for sedimentation analysis. The one-term Drude equation was used to calculate the dispersion constants. In the visible region the constant was 245 m/~ for wheat and 223 m/, for bean, indicating a-helix content of 23 and 8 % , respectively. In the ultraviolet region a Cotton trough at 233 m # was obtained for both species, additional evidence for the presence of a-helix in these proteins. A
B m
m
I
m
m m
Fig. 3- Diagrams of polyacrylamide gel electrophoretic profiles of chloroplast structural protein fractions. Electrophoresis carried out in lO% acetic acid with a constant current of 5 mA per gel at 21° for i h. Cathode at bottom; application point at top. A. Wheat. B. Bean. Whereas the structural protein fraction of these species was homogeneous b y analytical ultracentrifuge analysis, 2 bands were obtained b y electrophoresis of wheat and 5 bands for bean (Fig. 3). Therefore, t h e y were heterogeneous at least with respect to charge. This finding differs from t h a t reported b y CRIDDLE ~ who got a single boundary for spinach chloroplast structural protein using b o u n d a r y electrophoresis. However, like mitochondrial and microsomal structural proteins 9-1~ the chloroplast structural protein fractions investigated b y us were heterogeneous in nature. In conclusion, it is seen t h a t the chloroplast structural protein fractions from bean and wheat were similar in their sedimentation coefficients and amino acid composition. On both these bases the findings were different t h a n those reported for spinach chloroplast structulal protein. However, it cannot be concluded t h a t the structural protein fractions of chloroplasts used in these studies were homogeneous since on gel electrophoresis more than one band was obtained. ACKNOWLEDGEMENTS It is a pleasure to acknowledge Dr. Cyril M. Kay, D e p a r t m e n t of Biochemistry for optical r o t a t o r y dispersion analyses. This work was supported by a grant to S. Z. from the National Research Council of Canada. One of the authors (R.S.M.) is a Recipient of Province of Alberta Graduate Fellowship. REFERENCES I R. S. CRIDDLE, R. M. BOCK, E. D. GREEN AND H. TISDALE, Biochemistry, I (1962) 827. 2 R. S. CRIDDLE AND L. PARK, Biochem. Biophys. Res. Commun., 17 (1964) 74-
Biochim. Biophys. Acta, 200 (197 o) 1 3 2 - 1 3 7
CHLOROPLAST STRUCTURAL PROTEIN 3 4 5 6 7 8 9 io 11 12 13 14 15 16 17
137
W. MENKE AND E. JORDAN, Z. Naturforsch., I4b (1959) 393J. BIGGINS AND ]~_. B. PARK, Plant Physiol., 4 ° (1965) 11o9. T. H. JI, J. L. HESS AND A. A. BENSON, Biochim. Biophys..4cta, 15o (1968) 676. M. I. MOLCHANOV AND E. N. BEZlNGER, Dokl. Akad. Nauh S S S R , 178 (1968) 475. R. S. CRIDDLE, in T. W. GOODWlN, Biochemistry of Chloroplasts, Academic Press, New York, 1966, p. 203. A. GOFFEAU, Biochim. Biophys. Acta, 174 (1969) 34 o. D. I-IALDAR, K. FREEMAN AND T. S. WORK, Nature, 211 (1966) 9. H. TuPPY, P. SWETLY AND I. WOLFF, European J. Biochem., 5 (1968) 339. G. SCHATZ AND J. SALTZGABER, Biochim. Biophys. Acta, 18o (1969) 186. K. A. WARD AND J. M. POLLAK, Bioch. J., 114 (1969) 41. D. H. SPACKMAN, W . I-~. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. K. TAKAYAMA, D. H. MACLENNAN, A. TZAGOLOFFAND C. D. STONER, Arch. Biochem. Biophys., 114 (1966) 223. P. WEBER, Z. Naturforsch., i 8 b (1963) 11o5. A. LOCKSHIN AND I~.. H. BURRIS, Proc. Natl. Acad. Sci. U.S., 56 (I966) 1564. D. E. GREEN, H. D. TISDALE, R. S. CRIDDLE AND R. M. BOCK, Biochem. Biophys. Res. Commun., 5 (1961) 81.
Biochim. Biophys. Acta, 200 (197 o) 132-137
BIOCI-IIMICAET BIOPHYSICAACTA
BBA 355O 3 PHYSICOCHEMICAL S T U D I E S OF BEAN AND W H E A T C H L O R O P L A S T STRUCTURAL PROTEIN
R. S. M A N I AND S A U L Z A L I K
Department of Plant Science, University of Alberta, Edmonton, Alberta (Canada) (Received A u g u s t i 4 t h , 1969)
SUMMARY
Chloroplasts from wheat and bean seedlings were isolated, and after removing lipid and pigments the protein was solubilized. (NH,)2SO 4 was used to precipitate the structural protein fraction. Sedimentation velocity studies showed homogeneous profiles in both species with s values lower than reported for spinach. The amino acid composition of this protein fraction from bean and wheat was similar. Optical rotatory dispersion measurements indicated the presence of a-helix in both. Gel electrophoresis showed that with respect to charge the structural protein fraction of wheat and bean chloroplasts was different and not homogeneous.
INTRODUCTION
The term "structural protein" was initially given by CRIDDLE et al. 1 to a protein fraction they isolated from beef heart mitochondria. It had no detectable enzyme activity and represented about 6O~o of the total mitochondrial protein. In addition it formed complexes with mitochondrial cytochromes and was viewed as a possible site for enzyme alignment. Later CRIDDLE AND PARK2 isolated a protein component from spinach chloroplasts which they designated as structural protein. It constituted about 40% of the total chloroplast protein and was homogeneous with a molecular weight of 23 ooo and a sedimentation coefficient of 2.2 S. MENKE AND JORDAN 3 who isolated proteins associated with chloroplast lamellae designated these as lamellar proteins. BIGGINS AND PARK4 and JI et al. 5 obtained an s value of 2.2 for the lamellar protein from spinach chloroplasts. For the lamellar lipoprotein of bean, MOLCHANOV AND BEZINGER 6 reported an s value of 2. 9. The chloroplast structural protein studied by CRIDDLE7 had a relatively high content of glutamate, aspartate and arginine, and a low value for phenylalanine compared to chloroplast lamellar protein. Recently GOFFEAUs reported the amino acid composition of Acetabularia chloroplast structural protein. It differed from that of spinach in the levels of aspartic and glutamic acids. As chloroplast structural protein is a maj or component of lamellar protein ~, the differences m a y have been a reflection of species. HALDAR et al. 9, TuPPY gt al. 1° and SCHATZ Biochim. Biophys. Aeta, 2oo (I97 o) I32 137
CHLOROPLAST
STRUCTURAL
133
PROTEIN
AND SALTZGABER11 found that on the basis of gel electrophoresis mitochondrial structural protein was heterogeneous. Also WARD AND POLLAKTMnoted that microsomal structural protein was heterogeneous. In view of our findings for chloroplast structural protein and in light of the cited T M results the term structural protein fraction is being used in this paper instead of structural protein. The study being reported was undertaken to characterize and compare chloroplast structural protein fractions from bean and wheat by sedimentation velocity studies, optical rotatory dispersion measurements, amino acid analysis and gel electrophoresis. MATERIALS AND METHODS
Seedlings of Stewart 63 wheat (Triticum durum Desf.) and Kinghorn wax bean (Phaseolus vulgaris L.) were grown in soil in the glasshouse under natural illumination. Wheat leaves were harvested on the 5th day after planting whereas bean leaves were harvested on the 7th day, following a I2-h dark period. Chilled, fresh wheat leaves were ground for 2 rain in a mortar in a medium consisting of 0.44 M sucrose; o.I M Tris (pH 7.8); 50 mM KC1; IO mM MgC12 and 4 mM mercaptoethanol. The ratio of flesh leaves to buffer was 1:2 (w/v). The grinding medium for bean leaves was 0.33 M sucrose in o.I M phosphate buffer (pH 6.8) ; o.oi M KC1 and o.oi M MgC12 (I :1.5, w/v). After filtering through cheesecloth, chloroplasts were isolated from the filtrates as diagramed. All operations were at 4 °, and are shown in Scheme I. Wheat ieaf filtrate
Bean leaf filtrate
500 x g, 1 0 m i n Supernatant
100 x g, 2 min
Nuclei, cell debris (discard)
Supernatant ]
2300 × g, 10 min
1000 × g, 15 min
l[ Crude
chloroplast pellet
Crude chloroplast pellet
Supernatant (discard)
Resuspended in sucrose phosphate buffer
Resuspended and washed 4 times in sucrose T r i s buffer
3000 x g, 20 min
2300 × g, 15 min
I Supernatant (discard) Purified chloroplasts (wheat)
h
Washed chloroplast pellet Layered on discontinuous sucrose density gradient (1.0 M, 1.5 M, 2.0 M sucrose in 0.1 M phosphate buffer) 25000 × g, 20 min Chloroplast band in 1.5 M layer collected Resuspended in sucrose free 0.1 M phosphate buffer
Scheme
I 3000 x g, 20 rain Purified chloroplasts (bean)
The isolated chloroplasts were freed from lipid and pigments by suspending in chloroform and acetone ( I : I , v/v) and subsequent washing with acetone. Finally, Biochim. Biophys. Acta,
2 0 0 ( 1 9 7 o) 1 3 2 - 1 3 7
134
R . S . MANI, S. ZALIK
they were washed with ether and dried under vacuum. The lipid-free protein was suspended in o.oo2 M Tris buffer (pH 8.5) containing o.o35 M NaC1, o.ooi M sodium ascorbate and a trace of Na~S204. Solubilization was effected by cholate (2 mg per mg protein) and deoxycholate (i mg per mg protein) and was followed by precipitation with I 2 - I 6 % (NH4)2SO 4 according to CRIDDLEAND PARK2. The precipitated protein was then extracted with cold acetone to remove the detergents. Sedimentation analyses were carried out on a Spinco Model E analytical ultracentrifuge at 20 °. The medium used for sedimentation consisted of 1% sodium dodecylsulphate, o.I M NaC1 and 5 mM 2-mercaptoethanol at pH II.O. Sodium dodecyl sulphate was required to solubilize the protein. NaC1 was employed to reduce the primary salt effect, whereas mercaptoethanol should have minimized any subunit association. Amino acid analysis of the structural protein was carried out on an amino acid analyzer according to SPACKMANet al. 13. The protein was hydrolyzed in oxygen-free sealed ampules with constant boiling HCI at IiO ° for 20 h. Optical rotatory dispersion measurements of bean and wheat chloroplast structural protein fractions in 1% sodium dodecyl sulphate, o.I M NaC1, 5 mM dithiothreitol at pH II.O were obtained using a Cary Model 60 recording spectropolarimeter. The solvent was filtered through a 0.45 /, Millipore filter and the protein solution through a 5.0 # filter. Readings were taken in the visible region with a I-cm path length cell and in the ultraviolet region a o. I-cm cell was used. Gel electrophoresis in acetic acid was carried out according to TAKAYAMAet al. 14 but the medium contained phenol-acetic acid-water ( i : i : i , w/v/v) to a final protein concentration of 7 mg/ml. Polymerization of the gel was carried out at 5°° for 15 rain. RESULTS AND DISCUSSION
The chloroplast structural protein fraction on the basis of isolation technique presumably was free of soluble and non-structural lamellar protein. The structural protein fractions from bean and wheat chloroplasts were homogeneous by analytical
1.6.
1.4-
!.2S
1.0-
0*8-
CONC. (mg/mJl
Fig. i. S e d i m e n t a t i o n v e l o c i t y p a t t e r n of w h e a t c h l o r o p l a s t s t r u c t u r a l p r o t e i n f r a c t i o n in I °. 0 s o d i u m d o d e c y l s u l p h a t e , o.i M NaC1, 5 mM m e r c a p t o e t h a n o t , p H II.O a t 2o °. P i c t u r e t a k e n a f t e r 48 m i n a t 59 780 r e v . / m i n w i t h a b a r a n g l e of 5 o°. Fig. 2. S e d i m e n t a t i o n coefficient of b e a n f r a c t i o n as a f u n c t i o n of c o n c e n t r a t i o n .
Biochim. Biophys. Acta, 200 (197 o) 132-137
(©)
and wheat
(O) c h l o r o p l a s t s t r u c t u r a l p r o t e i n
CHLOROPLAST STRUCTURAL PROTEIN
135
ultracentrifuge analysis having s°20,w values of 1.2 and 1.3, respectively (Fig. i). There was no indication of any slower moving component after 4 h at 59 780 rev./min. The sedimentation coefficients decreased with increasing concentration (Fig. 2) indicating no association or dissociation of the protein in the concentration range studied. As seen, the s values obtained by us differ considerably from those reported by CRIDDLE AND PARK2 for spinach. This m a y be attributed to differences in species, isolation techniques and solvent systems used. Wheat chloroplasts were subjected to four washings during isolation and bean chloroplasts were purified on a discontinuous sucrose density gradient whereas the solvent system used for sedimentation studies included mercaptoethanol, unlike the preparations of CRIDDLE AND PARK2. Based upon the relatively low s values obtained for the structural protein fraction it is unlikely that it contained significant amounts of non-structural lamellar protein.
TABLE
[
AMINO ACID COMPOSITION
A m i n o acid
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Cysteic acid
OF CHLOROPLAST
STRUCTURAL
PROTEINS
Mole % Wheat
Bean
Acetabularia s
Spi nac h ~
Spi nac h lamellar protein ~
8.6 5.5 7.7 5.9 lO. 4 lO.9 lO.4 6. 7 1.4 4.5 9.4 1.8 4.2 6.5 1.7 4.4 --
8.2 5-8 7. i 5.4 lO.2 I i .7 9.9 6.2 i. i 4 .8 lO.2 2.4 5.o 6.3 1.7 4 .0
9.3 5-6 6.o 5.3 i i .o I I .o I i .o 7-4 Trace 5 .8 io. i 1.6 5.8 4.9 1.5 3-9 --
14-o 3.7 5.5 5. i 13- t 9.2 lO. 4 7.4 o.z 6.6 i 1.4 o.7 o.2 8.2 2.I i.o 1.3
8.8 4.7 5.7 5.9 9.2 io. 5 9.6 6.5 1.7 5-3 I i.o 3.8 6.5 5.5 1.4 4.2 --
Table I compares amino acid composition of the structural protein fractions from bean and wheat chloroplasts with that of other species. Essentially these fractions from wheat and bean had like composition. The major differences from structural protein of spinach were in the levels of aspartate, glutamate, phenylalanine and arginine. However, the values for bean and wheat were similar to chloroplast structural protein from Acetabularia. The amino acid composition was similar to spinach lamellar protein as well as lamellar protein fractions from other species 15,16. The relatively high levels of non-polar amino acids in the chloroplast structural protein fraction could give rise to extensive hydrophobic regions which might account for protein stabilitylL Biochim. Biophys. Acta, 200 (197 o) 1 3 2 - 1 3 7
136
R.S. MANI, S. ZALIK
Conformation of the structural protein fraction was studied by optical r o t a t o r y dispersion using a solvent system similar to t h a t employed for sedimentation analysis. The one-term Drude equation was used to calculate the dispersion constants. In the visible region the constant was 245 m/~ for wheat and 223 m/, for bean, indicating a-helix content of 23 and 8 % , respectively. In the ultraviolet region a Cotton trough at 233 m # was obtained for both species, additional evidence for the presence of a-helix in these proteins. A
B m
m
I
m
m m
Fig. 3- Diagrams of polyacrylamide gel electrophoretic profiles of chloroplast structural protein fractions. Electrophoresis carried out in lO% acetic acid with a constant current of 5 mA per gel at 21° for i h. Cathode at bottom; application point at top. A. Wheat. B. Bean. Whereas the structural protein fraction of these species was homogeneous b y analytical ultracentrifuge analysis, 2 bands were obtained b y electrophoresis of wheat and 5 bands for bean (Fig. 3). Therefore, t h e y were heterogeneous at least with respect to charge. This finding differs from t h a t reported b y CRIDDLE ~ who got a single boundary for spinach chloroplast structural protein using b o u n d a r y electrophoresis. However, like mitochondrial and microsomal structural proteins 9-1~ the chloroplast structural protein fractions investigated b y us were heterogeneous in nature. In conclusion, it is seen t h a t the chloroplast structural protein fractions from bean and wheat were similar in their sedimentation coefficients and amino acid composition. On both these bases the findings were different t h a n those reported for spinach chloroplast structulal protein. However, it cannot be concluded t h a t the structural protein fractions of chloroplasts used in these studies were homogeneous since on gel electrophoresis more than one band was obtained. ACKNOWLEDGEMENTS It is a pleasure to acknowledge Dr. Cyril M. Kay, D e p a r t m e n t of Biochemistry for optical r o t a t o r y dispersion analyses. This work was supported by a grant to S. Z. from the National Research Council of Canada. One of the authors (R.S.M.) is a Recipient of Province of Alberta Graduate Fellowship. REFERENCES I R. S. CRIDDLE, R. M. BOCK, E. D. GREEN AND H. TISDALE, Biochemistry, I (1962) 827. 2 R. S. CRIDDLE AND L. PARK, Biochem. Biophys. Res. Commun., 17 (1964) 74-
Biochim. Biophys. Acta, 200 (197 o) 1 3 2 - 1 3 7
CHLOROPLAST STRUCTURAL PROTEIN 3 4 5 6 7 8 9 io 11 12 13 14 15 16 17
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