Physicochemical study of structural proteins of chloroplast from Zea mays L.

232 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

46184

PHYSICOCHEMICAL STUDY OF STRUCTURAL PROTEINS OF CHLOROPLAST FROM Z E A M A YS L. B. L A G O U T T E AND

J. D U R A N T O N

Service de Biochimie, Ddpartement de Biologie, Centre d'Etudes Nuclgaires de Saclay, B P No. 2, 9 ± Gif-sur- Yvette (France) (Received May 4th, 1971)

SUMMARY

I. Structural proteins from maize chloroplasts were obtained in aqueous solution by a simple method using the ionic detergent sodium dodeeyl sulfate. They were shown to be completely free from lipid and pigments by gas chromatography and spectrophotometry. 2. Gel electrophoresis, in the presence of the detergent, showed about ten bands, one being the most important with respect both to absorbance and nitrogen content (about 40 % of the total protein)' its molecular weight was 25 ooo. Sephadex filtration allowed a partial separation, thus providing a good purification of the main component. 3- Both the crude solution and the purified fraction seemed to be homogeneous by ultracentrifugation analysis, with sedimentation coefficients of 2.8 and 2.3 S, respectively. The crude solution exhibited a high diffusion coefficient, probably related to a polydisperse associated system.

INTRODUCTION

It is now well established that the photochemical reactions of photosynthesis occur in the lamellae and grana structures of the chloroplasts. The biological significance of these structures has given rise to much research in order to find out their biochemical composition1, 2. Although the various pigments and lipids of these structures are already well known, there is little information on their constituent proteins. However, the protein fraction plays an essential part, as judged by its quantitative importance (5o % of the mass of lamellae) and by the fundamental role allotted to it. This lack of information is, on tile whole, due to the insolubility of these proteins in an aqueous medium when they are entirely freed of their associated lipids and pigments. Although it was first considered that only one constituent protein existed in lamellae structures, it soon became evident that several proteins were present, at least ten according to the results of MENKE AND JORDANa and BOQUETet al. 4. Several methods have been described which enable a fraction freed of its lipids and pigments to be solubilized, but not all the lamellar proteins are obtained 5-7. The purpose of this work was to obtain the complete solubilisation of such proteins, and to establish some Biochim. Biophys. Acta, 253 (1971) 232-239

CHLOROPLAST STRUCTURAL PROTEINS

233

of their physicochemical characteristics in order to follow methodically the separation of the various components. MATERIALS AND METHODS

Zea mays L grains ( I N R A 260 variety) were made to g e r m i n a t e in m o u l d in a glass house at a t e m p e r a t u r e of 25 ° in d a y light. The y o u n g p l a n t l e t s were lifted 12 d a y s l a t e r at the three-leaf stage. The p l a s t i d structures were s e p a r a t e d at 4 ° b y grinding the limbs in a m o r t a r with s a n d in a solution of 0.35 M NaCI buffered with o.oi M p h o s p h a t e at p H 7.8. The g r o u n d m a t e r i a l was then filtered t h r o u g h cloth a n d centrifuged for 20 min at 3000 × g in order to o b t a i n a deposit containing m a i n l y p l a s t i d structures. The deposit was washed 3 times with the original buffer solution to eliminate the soluble substances still present s . The protein c o m p o n e n t s were then e x t r a c t e d from these structures b y the m e t h o d shown in Scheme I.

Pellet obtained after centrifugation at 3 0 0 0 x g , 2 O m i n Extraction

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20000xg,2min DI Remainder of lipids and p i g m e n t s

P r o t e i n e l l e t washed in a c e t o n e to r e m o v e t r a c e s of c h l o r o f o r m and m e t h a n o l 2 0 0 0 0 x g.2min Protein pellet Ambient temperoture--~-

S o l u b i l i s a t i o n in a s o l u t i o n of 1% s o d i u m dodecyl sulfate buffered with 001 M phosphate (pH7)

Protein solution d i a l y s e d a g a i n s t 0.1°/o s o d i u m d o d e c y l s u l f a t e b u f f e r e d w i t h 0.01 M p h o s p h a t e ( p H 7 )

t~iochim. Biophys..4cta, 253 (i97i) 232-239

234

B . L . \ G ( ) I TTI~;, J. I)UR.kNT()N

Electrophoreses were carried out in p o l y a c r y l a m i d e gel b y the m e t h o d described by SHAPIRO el al. 9 and s y s t e m a t i c a l l y repeated by \VEBER AND ()SBORY,"1° for determining molecular weight.,< The gels which gave the hest separation contained io % acrylamide, o.25 % bisacrylamide and o.i % sodium dodecvl sulfate (the sodium d o d e c \ l sulfate was recrystallised in absolute ethanol, thus giving b e t t e r results). The gels were 5o m m long and 5 m m in diameter. Migration occured under a current , f 4 mA per tube for 4 11. The etectrophoresis buffer was a o.I M p h o s p h a t e hui'fer with o . 1 % sodium dodecyl sulfate at p H 7.2. The gels were stained with ('~,:mmssie blue, which was the most efficient stain, and destained with a m i x t u r e of 3o % methanol, 7 % acetic acid and w a t e r in the presence of a D o w e x - t y p e ion exchange resin. The same gels were used for a n a l y t i c a l purposes as well as for the molecular weight determinations. In the l a t t e r case, the m o b i l i t y of a component was calculated in relation to a migration indicator which in this case was bromophenol blue ~°. Mobility

distance of protein migration lenght after destaining

length before staining distance of dye migration

The s e d i m e n t a t i o n analyses were perforined in a Spinco E a n a l y t i c a l ultracentrifuge, fitted with an A N - D rotor. The deternfinations were made in a s y n t h e t i c b o u n d a r y cell at 59 78o rev./min and Io ~. Two t y p e s of fihn were taken sinmltaneouslv with a Schlieren optical system and by ultraviolet absorption. Diffusion coefficients were calculated according to the m e t h o d of EHRENBI~.RGII. Specific volumes were d e t e r m i n e d with an Antnn P A A R microdensitometer. Gas c h r o m a t o g r a p h y of the lipids was performed on a ~52o B aerograph. The protein solution was refluxed for 2.4 in c h l o r o f o r m - m e t h a n o l (2: I, v/v). After e v a p o r a t i o n of the solvent, the e x t r a c t thus o b t a i n e d was taken up in hexane, saponified a n d the f a t t y acids collected were then analysed. Detection of the reducing sugars was performed according to the m e t h o d of NEI.sox p', after solutions had been h y d r o l y z e d with I M HCI for periods of I - i 8 h at i o o . Protein nitrogen was d e t e r m i n e d after wet ashing hv the Kjeldhal inethod. RESULTS

The first objective was to solubilise in an aqueous m e d i u m all the proteins of tile plastid structures. Tile nitrogen d e t e r m i n a t i o n s of the protein solution o b t a i n e d bv the m e t h o d previously described showed t h a t almost all the proteins were thus solubilised, since over 95 % of the initial protein nitrogen was found in tile final solution. The absorption s p e c t r u m of the protein solution showed a complete absence of pigments. Fig. I shows the presence at p H 7 of an absorption m a x i m u m at 278 nm, followed b y a m a r k e d shoulder at 29I nm. A t p H I2, two m a x i m a a p p e a r at 275 a n d 283 nm and the shoulder at e 9 i n m is raised (tyrosine ionisation) ; a slight absorption is observed at 34 ° nm, a n d a h u m p at 4IO nm. These l a t t e r results are in agreement with the observations of BIGGINS AND PARKla on protein e x t r a c t s from spinach chloroplasts. Gas c h r o m a t o g r a p h i c analysis confirmed the almost complete absence of lipids in the p r e p a r a t i o n s . I n a solution containing 70 mg of proteins, a m a x i m u m 30o/~g Biochim. Biophys. Aota, 253 (I97I) 232-239

235

CHLOROPLAST STRUCTURAL P R O T E I N S

of f a t t y acids was detected, thus representing less than o.5 °o of the initial lipid content (based on LICHTENTHALER AND PARK'S~,2 calculations). A m a x i m u m content of 3 °6 of reducing sugars was found with regard to a glucose scale. The protein solution, brought to 2.5 mg of proteins per ml, was analysed directly bv electrophoresis on a polyacrylamide gel. The pattern obtained is shown in Fig. 2. It is important to stress that no stain remains at the start of the gel column. 10

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Biochim. Biophys. dcta, 253 (1971 ) 232 239

236

i~. LAGOUTTE, J. DURANTON

The analysis of ~4C labeled proteins also showed that 9 ° % of tile radioactivity of the sample was found in the gel, confirming that the proteins had effectively entered. Of the ten bands observed, two, the a and b bands, were more densely stained. According to the absorbance readings, they represented 17 and 4 ° ?o, respectively, of the total proteins (staining with Coomassie blue being proportional to the amount of proteins). Band a is narrow, very clear and highly coloured; Band b the most important one, is large, its limits less clear and is equally intense. Appropriate tracers were used to deternfine the molecular weights of these various bands (Fig. 3). The main band, b, had a molecular weight in the order of 25 ooo, whereas that of Band a was estimated to be about 5o.ooo. The very fast front band, which migrates more rapidly than cytochrome c, has a molecular weight of about Io ooo. The protein solution was studied by analytical ultracentrifugation in order to confirm its heterogeneity, and the molecular weight of the various components. As will be seen from Fig. 4, the proteins migrated as a single peak. Its sedimentation coefficient varied with the protein concentration, and was estimated to be 2.8 S by extrapolation to zero concentration (Fig. 5). This sedimentation peak exhibits a high rate of diffusion. The diffusion coefficient, calculated from the Schlieren peaks by extrapolation at zero concentration was 18. IO-7 cmZ/sec. It was noted that, during the experiment, the area under the Schlieren peaks remained constant, indicating that no large aggregates sedimented from the boundary. By filtering through Sephadex G-2oo, the protein solution was separated into I

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Fig. 3 Curve of electrophoretic mobility as a function of molecular weight. The controls used at a concentration of 2 m g / m l are, i, bovine s e r u m albumin, 2, p y r u v a t e kinase, 3, ?,east alcohol dehydrogenase, 4, trypsin, 5, lactoglobulin. a and b represent the main bands of the plastid s t r u c t u r e s (Fig. 2). Fig. 4. Sedimentation velocity p a t t e r n of maize chloroplast structural proteins in o.i % sodium dodecyl sulfate, o.t % NaCI, p H 7 at 2o °. Picture taken in a synthetic b o u n d a r y cell after 24 rain at 59 78o rev./min.

Biochim. Biophys, ~qcla, 253 (I97 I) 232-239

237

CHLOROPLAST STRUCTURAL PROTEINS

two peaks, I and I I (Fig. 6). An examination of the elution profile at 28o nm showed these two peaks to be asymmetrical, which probably reflects their heterogeneity. Electrophoresis on polyacrylamide gel confirmed the presence of the heaviest molecules in the first elution peak, whereas the lighter molecules, Band b and following, were in the second peak (Fig. 2B) By taking the top part only of the second peak, it was possible to obtain a very slightly contaminated band b. iI

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Fig. 5. Sedimentation coefficient curves as a function of the protein content, for the crude solution (O) and for Fraction If obtained after filtering with Sephadex G-2oo (A). Fig. 6. Elution profile obtained by filtering the crude solution of the s t r u c t u r a l proteins with Sephadex G-2oo. The elution buffer was o.I °..o s o d i u m dodecyl sulfate, o.i % NaC1 and o.oi M p h o s p h a t e buffer at p H 7.

Ultracentrifugation analyses were made on Fraction I I of the crude solution (second peak obtained with G-2oo). They gave an s°2o,w = 2.3, less therefore than that of the crude solution. The diffusion coefficient, which was also less, was IO. Io -7 cm2/sec (Fig. 5). DISCUSSION

Since the work of SMITH 14, the use of detergents for studying structural proteins, either free or associated with lipids and pigments, has grown considerably. The method developed here makes use of sodium dodecyl sulfate, and is akin to that described by BIGGINS AND PARK 13, however, we obtained complete solubilisation of the precipitated proteins, whereas BIGGINS AND PARK only attained 75 %. Our result m a y be explained by the very good lipid extraction by the chloroform-methanol step, since, if this stage is omitted, solubilisation drops to around 80 %. With all the plastid structural proteins in aqueous solution at our disposal, we were able to make a valid analytical study of all of them. On the assumption that Biochim. Biophys. Acta, 253 (197 I) 232-239

238

B. LAGOUTTE, J. I)URANT~)N

each b a n d was pure, since the entire sample enters the gel, there should be about ten different proteins. This n u m b e r is higher t h a n those found in the literature. The electrophoresis m a d e b y Ma.'q A~D ZALIK7 on w h e a t and bean chlor(@asts e x h i b i t e d two and five components, respectively. Many investigations have also been m a d e on lipid-containing protein structures; THORNBERG ¢'1 al. 1'5 ill p a r t i c u l a r o b t a i n e d a picture close to ours for spinach chloroplasts, with two p r e d o m i n a t i n g complexes. During a previous investigation, TnORNBER(; t't al. 1G found six bands after electrophoresis of a fraction of lamellae proteins of the same origin. A part of these proteins was ascribed to fraction I of the chloroplasts. BRAtXlrZI~;I¢ .\N1) ]{.xt i.:I~~: essentially' found three protein bands in spinach and observed a great similarity between bands from higher plants. \VEI~E~ 5, working on :l,¢lirrhilzum m a j u s , fimnd only one m i g r a t i o n band. I t should be emphasised that, in most of these ext)eriments, solubilisation is only p a r t i a l and p a r t of the sample is retained at the s t a r t of the gel, thus a complete picture of all the s t r u c t u r a l proteins is not given. In our case, two of the c o m p o n e n t s were more imt)ortant, one of which, clearly predominating, r e p r e s e n t e d 4o % of total proteins. TiLe studies previously u n d e r t a k e n on maize s t r u c t u r a l proteins b y BOQUET d al. 4 and COLLOT cl al. ~s showed t h a t there was a p a r t i c u l a r frequency in the d i s t r i b u t i o n of the N- and C-terminal amino acids, alanine and glycine, respectively, being the most a b u n d a n t with frequencies in tile order of 4o %. I t therefore seems likely t h a t these two amino acids in fact represent the ends of this p r e d o m i n a n t protein. TILe more or less purified " s t r u c t u r a l p r o t e i n " from both m i t o c h o n d r i a and chloroplasts, f r e q u e n t l y represent, q u a n t i t a t i v e l y , 5o % of the t o t a l s t r u c t u r a l proteins (CRIDDI.E~9). This result agrees well with our result. The d a t a o b t a i n e d with u l t r a c e n t r i f u g a t i o n showed a fall in the s e d i m e n t a t i o n coefficient from 2.8 to 2.3 S ,from tile crude solution to fraction II. This tallies with the electrophoretic data, since it is logical to assume t h a t mean molecular weight of F r a c t i o n I I , which contains the lightest components, is less t h a n t h a t of the total solution. The small discrepancy between the two s e d i m e n t a t i o n coefficients m a y be explained (a) b y the absence of high molecular weight substances and (b) by the pred o m i n a n t protein in b o t h solutions. A l t h o u g h the crude solution is heterogeneous u n d e r electrophoresis, it sedim e n t s as a single peak. This could be due to the strong association of the various con> ponents, as well as the presence of a d o m i n a n t protein which, because of its importance, m a s k s the weaker protein peaks. I t should be n o t e d t h a t tile very' high diffusion coefficient of this solution (I8. I o v cm2/sec) p r o b a b l y reflects this associated heterogeneity. The diffusion coefficient of F r a c t i o n I I is lower (zo- Io 7 cnF-/sec) showing t h a t a p u r e r p r o d u c t is present, which is confirmed b y electrophoresis. The specific w)lume is o.7ee cma/g, which gives a molecular weight of ee ooo ~ eooog/M b y S v e d b e r g ' s equation. This value agrees perfectly with the results of BIGGINS AND PARK 1:' who found a molecular weight of 22 ooo g/M for the proteins of spinach chloroplasts, and also with those of CRIDDL~EAND PARK6 who used a different m e t h o d of p r e p a r a t i o n of the same material, a n d o b t a i n e d a molecular weight of 23 ooo g/M. The s e d i m e n t a t i o n coet~Scients found b y MANI AND ZALIK7 for w h e a t and bean of I. 3 and 1.2 S, respectively, are v e r y different. These authors used a m e d i u m containing r % sodium dodecyl sulfate and the significant effect of the detergent concentration on sedil]iochim, tetiophys..4eta, 25~ (I97~) 232 239

CHLOROPLAST STRUCTURAL PROTEINS

239

m e n t a t i o n m u s t be s t r e s s e d h e r e . SMITH AND PICKELS 2°, u s i n g s o d i u m d o d e c y l s u l f a t e c o n t ' e n t r a t i o n s of 0.25 t o 2.5 %, f o u n d a v a r i a t i o n of 2.32 t o 1.69 S in t h e s e d i m e n t a ti{)n {'(}efficient. L i k e w i s e fTOH et al. 2~ n o t i c e d , in t h e case of a s p i n a c h p r o t e i n - p i g m e n t c )mt)lex, a c h a n g e of 3.5 to r. 9 S in t h e s e d i m e n t a t i o n coefficient w i t h d o d e c y l b e n z e n e stflf(}nate c o n c e n t r a t i o n s of 2. 9. I o -a M t o 2- IO-" M. F u r t h e r m o r e , a l i g h t e r c o m p o n e n t apl)~ared (1.2 S) a t c o n c e n t r a t i o n s b e l o w 2. 5. lO -2 M. I t is t h e r e f o r e n e c e s s a r y to i n t e r p r e t t h e s e c l i m e n t a t i o n coefficient v a l u e s o b t a i n e d in t h e p r e s e n c e of d e t e r g e n t s with extreme caution. T h e r e s u l t s f r o m t h i s s t u d y s h o w t h a t t h e p l a s t i d s t r u c t u r e s of m a i z e c o n t a i n a large n u m b e r of d i s t i n c t p r o t e i n s w h i c h h a v e a s t r o n g t e n d e n c y t o a s s o c i a t e , as s h o w n b y u l t r a c e n t r i f u g a t i o n . T h i s p r o p e r t y m u s t p l a y a p a r t in t h e b u i l d i n g of t h e pla~t s t r u c t u r e s . T h e r e is, h o w e v e r , a p r e d o m i n a n t a m o u n t of a p a r t i a l l y p u r i f i e d c o m p o n e n t w i t h a r e l a t i v e l y low w e i g h t in t h e r e g i o n of 25 ooo g/3I. ACKNOXVLEDGEMENTS W e are g r a t e f u l t o Drs. Y. A r n a u d a n d M. L. L e r o a x for p e r f o r m i n g t h e a n a l y tical u l t r a c e n t r i f u g a t i o n . W e also w i s h t o t h a n k Dr. P. F r o m a g e o t for his h e l p a n d e n c { } u r a g e m e n t d u r i n g t h e c o u r s e of t h i s w o r k .

R 1'2I: f'; RENCES I 2 3 4 5 6 7 S 9 1o II I2 13 14 15 16 t7 i8 t9 20 2I

I-3[.i(. LICHTENTHALER AND R. B. PARK, Nature, 198 (1963) to7o. R. B. PARK AND J. Bm~r.~s, Selectees, 144 (1964) lOO9. \V. M'ENKE AND E. JORDAN, Z. Naturforsch., I4b (1959) 234. M. Bo?u~r, O. OUIGNERY AND J . DURANTON, Bull. Soc. Chim. Biol., 28 (t968) 531P- \VEBER, Z. Naturforsch., 18b (t963) 11o 5. [{. S. CRIDDLE AND L. PARK, Biochem. Biophys. Res. Commun., 17 (1964) 74. }~..~. ~[ANI AND S. ZALIK, Biochim. Biophys. Acta, 200 (197o) 132. I?. I~. V~THATLEYAND D. [. ARNON, Methods Enzymol., 6 (1963) 308. -\. L. SHAPIRO, E. VrNUELA AND J. V. MAIZEL, Biochem. Biophys. Res. Comm,, 28, 5 (1967) 815. I'(. \¥EBER AND M. OSBORN, J. Biol. Chem., 244 (1969) 44{16. A. EHRENBERG, Acta Chem. Scan&, 11 (1957) t257. N. NELSON, J. Biol. Chem., I53 (1944) 375J. BIGGtNS AND R. B. PARK, Plant Physiol., 4o, 6 (1965) 111o9 . l'2. L. SMITH, .J. GeE. Physiol., 24 (1941) 565 . J. P. THORNBERG, R. P. F. GREGORY, C. A. SMITH AND J. L. BAILEY, Biochemistry, 6 (1967) 39 1. J. P. THORNBERG, J. L. BAILEY AND S. M. RIDLEY, Bioehem. J., iSP (1964) 92. ('. BRAUNITZER AND C. BAUER, Naturwissensehaflen, 54 (1967) 7TM D. COLLOT, G. GUIGNERY, J. DURANTON ANDT. TATSUNO, Bull. Soc. Chim. Biol., 52 (197 o) 241. l{. S. GRIDDLE, Ann. Rev. Plant Physiol., 20 (1969) 239. I:.. L. SMITH AND E. G. PICKLES, J. Gem Physiol., 24 (1941 ) 753. ~[. [TOH, .~. [ZAWA AND I~. SHIBATA, Biochim. Biophys. Acla, 69 (1963) 13o. Biochim. Biophys. Acta, 253 (1971) 232-239