Free Flow Electrophoresis of Chloroplasts - NCBI

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illuminated forI or 2 hr in the cold chamber before use, with 300- w lamps and frequently wetted with cold distilled H20. These leaves (50-100 g, fresh wt) were ...
Plant Physiol. (1968) 61, 465-468

Free Flow Electrophoresis of Chloroplasts Received for publication August 2, 1977 and in revised form November 15, 1977

JEAN-PAUL DUBACQ AND JEAN-CLAUDE KADER Laboratoire de Physiologie Cellularie, 12 rue Cuvier 75005 Paris, France ABSTRACT

Highly purified intact chloroplasts were isolated from spinach (Spinacis oleracea L.) leaves by free flow electrophoresis. Morphological and biochemical studies showed that the fraction enriched in intact chloroplasts has a higher protein to chlorophyll ratio and a higher linolenic acid content than the broken organelles of the other fraction. The intact chloroplasts prepared by electrophoresis retained their capacity for CO2 fixation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that this fraction was rich in stroma and lameDlae proteins. Free flow electrophoresis, which separates organelles and molecules according to their surface charges, is a good technique for producing purified chloroplasts with complete physiological activities.

Free flow electrophoresis, a technique which separates biological structures according to their surface charges, has been used successfully to separate lysosomes and mitochondria (26), mitochondrial membranes (10), and other subcellular fractions (8, 9). It was tempting to apply this technique to chloroplasts for which preliminary assays had been done (14, 15). This paper reports the properties of two chloroplast classes (intact and broken) separated by free flow electrophoresis.

metric measurement (3). The values obtained were confirmed by spectrophotometric titration of pheophytins after HCI addition. Lipids were extracted according to Bligh and Dyer (4). Total fatty acids were analyzed by GLC after methylation by the methoxide ion according to the technique of Loury (19), adapted to the microscale by J. P. Carreau and J. P. Dubacq (unpublished). Lipids were separated by TLC (17) and quantitatively estimated by their fatty acid content. SDS Polyacrylamide Gel Electrophoresis. The plastid fractions were incubated for 3 min at 100 C with the following solubilizing solution (100 l d of solution/mg of protein): Tris (125 mm, pH 6.8); SDS (4%); fl-mercaptoethanol (10%o). The material solubilized (100-200 ,ug of protein) to which bromophenol blue was added as an electrophoretic marker, was then electrophoresed in polyacrylamide gels (7 mm diameter) where the respective concentrations of acrylamide and bis-acrylamide were 10 and 2.6% in the upper gel and 3 and 2.6% in the lower gel (16). The two gels, respectively 0.5 and 7 cm long, contained 0.1% SDS and were buffered with 300 mM Tris (pH 8.8). The electrophoresis was performed at 4 mamp/gel with Tris (125 mM)/glycine (190 mM) (pH 8.3) containing 1% SDS as electrode buffer. Staining was done with 0.04% Coomassie blue in methanol-acetic acid-water (2.5:1:6.5, v/v/v) and destaining in methanol-acetic acid-water (2:1:7, v/v/v). The gels were read at 592 nm with a Joyce-Loebl chromoscan densitometer. C02-dependent 02 Evolution. The crude plastid pellet was isolated under the same conditions as above except that the isolation medium was that recommended by Robinson and Wiskich (23): 50 mM MES; 400 mM sorbitol; 2 mm EDTA; I mM MgCl2; 2 mm NaNO3; 20 mM NaCl; 0.4% BSA; 0.5 mM Pi; and 2 mM isoascorbate (pH 6.1). The pellet was carefully suspended in the isolation medium and injected in the FF5 apparatus. The intact plastids, located by means of phase contrast microscopy, were centrifuged at 2,000g for 1 min and resuspended in 1 ml of the isolation medium, adjusted to pH 6.7, where MES was replaced by HEPES buffer and where isoascorbate was omitted. The pellets were assayed for 02 evolution in an oxygraph apparatus fitted with a Clark electrode, in the 3-ml chamber, calibrated with H202 and catalase. Temperature was maintained at 20 C by a very rapid circulation of water. Oxygen evolution was initiated by addition of sodium bicarbonate (final concentrations, 10 mM). The Chl concentration in the chamber was about 50 ,ug/ml. Electron Microscopy. Pellets obtained by centrifugation after electrophoresis were fixed in 1% glutaraldehyde, then postosmiated and embedded in "Spurr."

MATERIALS AND METHODS Isolation of Plastidial Fractions. Several batches of spinach were purchased at a local market. Fresh dark green leaves were illuminated for I or 2 hr in the cold chamber before use, with 300w lamps and frequently wetted with cold distilled H20. These leaves (50- 100 g, fresh wt) were homogenized in 50 to 200 ml of a medium containing: 400 mm sucrose; 6 mm Tris-citrate (pH 7.3); with or without 4 mm cysteine chloride; 0.1% BSA for 5 sec in a Waring Blendor at maximum speed. After centrifugation at 600g for I min (without braking), the pellet was discarded and the supernatant was centrifuged at 2,000g for I min (with braking). The pellet obtained was carefully resuspended in 2 to 5 ml of medium and directly injected into the free flow electrophoresis apparatus. Free flow Electrophoresis. The electrophoresis was performed with a FF5 apparatus (Bender and Hobein, Munich, Germany) with 6 mm Tris-citrate and 400 mm sucrose (pH 7.3) as a chamber buffer and 25 mM Tris-citrate (pH 7.3) as an electrode buffer. During the electrophoretic run, the electric field was 100 v/cm and the temperature was fixed at 5 C. The chamber buffer (530 RESULTS ml/hr) was collected in 90 tubes. The flow rate of the injection of When a crude plastid fraction was separated by free flow plastid fractions was 4 ml/hr. Protein and Lipid Analysis. The protein and phospholipid con- electrophoresis, two main green fractions were seen in the sepatents of the fractions were, respectively, determined according to ration chamber. Reading the collected fractions at 280 nm located Lowry et al. (20) and Shibuya et al. (25). The Chl content was these two fractions precisely (Fig. 1). The most important peak (I) estimated according to Arnon (3) either by direct dilution of was collected in tubes 29 to 32 (near the anode), the second peak aliquots in acetone (final concentration of acetone = 80o) or by (II) had a maximum in tubes 45 to 46 (closer to the cathode). The extraction by light petroleum (45-60 C b.p.) and spectrophoto- relative positions were dependent on the electric field in the 465

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DUBACQ AND KADER

E

50

10

0.1

d

c

0

0 CL

c

Q065-

0.5

Q5.c 0

0

10

30

50

tube number

FIG. l. Separation of spinach chloroplasts by free flow electrophoresis. The experimental conditions are indicated under "Materials and Methods." The anode is at the left of the figure. The point of injection is indicated by an arrow. ( -): spectrophotometric reading at 280 nm; (E-U): protein concentration (mg/ml); (A---A): Chl concentration (mg/ml).

chamber, the flow rate of the buffer, and the surface charges of the chloroplasts. Nevertheless under the experimental conditions described, the two major peaks of plastids were separated at least by 10 tubes (this was observed in more than 20 chloroplast separations). If the fractions of the peak I or II were reelectrophoresed in the FF5 under the same experimental conditions, they were collected in the same tubes as in the first run. If the proportion of peak II increased, some aggregation occurred at the injection point after various times of injection. Neither EDTA nor EGTA1 (1-5 mM) introduced in the isolation medium or in the chamber buffer suppressed this phenomenon. Some assays designed to avoid this aggregation led us to conclude that the proportion of broken plastids reduced the separation and that too high a proportion of broken organelles at the injection may provoke an aggregation of the injected particles. The assays showed that a concentration of 10 mg of protein/ml was suitable to produce enough material without high aggregation. When the concentration of organelles was high, minor peaks were detected between the two major ones; they may reveal either particles not detected at low concentrations or more probably small stacks of organelles having different density to charge ratios. As examined by phase contrast microscopy, the two major peaks appeared to be very rich in chloroplasts, which were class 1 (7) in peak I and class II in peak 11 (7). The purity of peak II was close to 100%o, while only a few class II chloroplasts were found in peak I; some chloroplasts may have been broken during the microscopic examination. This observation suggested that peak I contained only intact chloroplasts with a well retained envelope; this property gave them a higher electronegative charge which allowed their separation from broken plastids. Electron microscopy confirmed this conclusion (Fig. 2). The chloroplasts of the peak I seemed intact, with a well preserved ultrastructure; neither mitochondrial nor other membrane contamination were observed. Biochemical Characteristics. The fractions of peak I (intact plastids) were characterized by their high protein to Chl ratio (Table I). Various ratios (from 1 I to 18.1 ,ug of protein/,ug of Chl) have been obtained on different batches of spinach. These values were in good agreement with the data from various authors (18) (Guillot-Salomon, personal communication) and the mean measure was close to those given by Joyard and Douce (11, 12) for intact plastids, isolated by sucrose gradients from spinach grown in the field and freshly harvested. The observed variations were related to the storage conditions of the leaves before their use in the laboratory. The protein to Chl ratio was lower (about half) in 1 Abbreviation: EGTA: ethylene ether)NNNN'N' tetraacetic acid.

glycol

bis-(amino-2

ethyl

Plant Physiol. Vol. 61, 1978

the broken chloroplasts (peak II) than in the intact chloroplasts. This result was close to those obtained for thylakoids by several authors (11, 18). This may be correlated with the morphological observations: the high protein to Chl ratio of the intact chloroplasts was expected from the cytological studies which showed the presence in peak I of organelles with well preserved ultrastructure, facilitating the retention of stroma proteins. The maximum value obtained was a good confirmation of the curves published by Lilley et al. (18) who estimated 18.3 ,tg/,ug of Chl as the maximum protein content released from chloroplasts by osmotic shock. The Chl a/b ratios indicated an enrichment in PSI in peak I and a relative deficiency in Chl a in peak II. The SDS-polyacrylamide gel electrophoresis illustrated the richness in proteins of the peak I chloroplasts (Figs. 3 and 4). The proteins from the organelles of this peak show a pattern characterized by two intensively stained bands which may be attributed to the two subunits of ribulose-1,5-diP carboxylase (5, 6, 13). This stroma-protein was well retained in the chloroplasts of the peak I. In contrast, the organelles of peak II were poor in these two subunits and the electrophoretic pattem of this peak was similar to that of lamellar proteins (21, 22, 27, 28). The lipid content of peak I/mg of Chl was higher than that of peak II (Table I). The polar lipid level in these intact chloroplasts was in good agreement with the data given by Allen et al. (1), Sastry (24), and Joyard and Douce (11). No traces of phosphatidylethanolamine-a marker for contamination by other membranes-were detected. In a typical experiment presented in Table II, the percentages of each lipid class were identical to those found by Joyard and Douce (11). The polar lipid to protein ratio was close to that found by these authors. The fatty acid composition of the total lipids associated with peak I was typical of photosynthetic materials (Table III) (1, 2). The percentages of individual fatty acids in each lipid class were close to those found by Allen et al. (1, 2) except that phosphatidylinositol and sulfolipid were less unsaturated than those usually found in spinach plastids. In peak II (broken plastids), the total fatty acids were poorer in linolenic acid, as also indicated by others (1, 2). C02-dependent 02 Evolution. This reaction, which is a criterion of the intactness of chloroplasts of class A (7), has been checked on preparations obtained after free flow electrophoresis (peak I). An active fixation of CO2 was obtained: about 25 umol per mg of Chl/hr. No significant difference was noticed when the same crude pellet was washed by a centrifugation at 3,000g for I min and assayed without free flow electrophoresis for 02 evolution: 21 ,umol/mg Chl hr. Our values of CO2 fixation are lower than the data reported elsewhere (see ref. 18), but fixation depends essentially on the storage conditions.

CONCLUSIONS The cytological and biochemical studies on the two main plastid fractions separated by free-flow electrophoresis indicated that one of these fractions (peak I) was rich in intact chloroplasts, the other peak being rich in lamellae. All data are in agreement with other preparative methods. The separation may be explained by the negative charges which are more abundant at the surface of the intact plastids than in the broken ones. The same explanation has been proposed for the separation of other organelles (8). Except for earlier works performed on spinach leaves (14, 15) to separate subchloroplast vesicles with various Chl a/b ratios, this is the first time that different plastid populations have been isolated by free flow electrophoresis and their biochemical properties clearly established. This technique is rapid since in the experimental conditions used, a chloroplast is collected about 5 min after its injection. The yield in intact chloroplasts is maximum and the separation can be

Plant Physiol. Vol. 61, 1978

FREE FLOW ELECTROPHORESIS OF CHLOROPLASTS

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p-, FIG. 2. Electron microscopy of the peak I chloroplasts obtained after free flow electrophoresis of spinach plastid fractions. a: x 6,750; b: x 19,000. Table I Biochemical properties of the two chloroplast classes, separated by free flow

I.

electrophoresis

Protein/Chl, mg/al Fatty acid/Chl, mg/ml Phospholipid/protein, mg/ml Major fatty acids (in x of total

peak I intact plastids 13.4 ± 1.9 5.5 + 0.5 0.027 + 0.003

peak II broken plastids 7.5 + 2.4 5.4 ± 0.5 0.035 ± 0.004

15.1 1.5 8.0 5.3 6.5 7.6 55.4

21.0 1.3 7.2 12.4 9.8 9.9 36.4

fatty acids)

palaitic A3 trans hexadecenoic hexadecatrienoic stearic oleic linoleic linolenic

N 4

.. ..;... .;

4. .

0

1

3

5

7

electrophoretic mobility (cm) FIG.

3.

SDS-polyacrylamide gel electrophoresis

of the two main chlo-

roplast classes separated by free flow electrophoresis (peak I and peak II). arrows indicate the two subunits of ribulose- 1,5-diP carboxylase.

The

continuously performed starting from a crude fraction. The electrophoresis does not alter the physiological properties of chloroplasts since CO2 fixation is active in peak I. Thus the use of free flow electrophoresis for purifying chloroplasts, which are kept in

FIG. 4. Densitometer traces of SDS-polyacrylamide gels performed on the two chloroplast fractions (peak I and peak II) separated by free flow electrophoresis. The apparent mol wt (expressed in kilodaltons) is indicated at the top of each band. This mol wt was estimated from a calibration curve established with the following protein standards: RNA polymerase (160,000), BSA (68,000), ovalbumin (45,000), chymotrypsinogen A (25,000), trypsin inhibitor (21,500), myoglobin (17,800), Cyt c (12,500) (Boehringer Mannheim).

468

DUBACQ AND KADER Table I I Polar lipid content of the intact plastids from peak I The material was isolated by free flow electrophoresis. The total polar lipids were 353 og/mg protein, 8.077 Lomol/mg Chl, 6.4 mg/mg Chl. % weight of total polar lipids

Lipid class

49.6 25.8 4.8

Monogalactosyldiacylglycerol Digalactosyldiacvlglycerol Phosphatidylcholine Phosphatidylinositol Phosphatidylglycerol Sulfolipid

1.1 8.4 10.0

Table III Fatty acid composition of polar lipids of intact plastids (peak 1) Lipid species/ Fatty acids

Monogalactosyldiacylglvcerol Digalactosyldiacylglycerol Sulfolipid Phosphatidylglycerol Phosphatidylcholine Phosphatidylinositol

16

4.3 8.0 37.3 23.4 31.4 41.5

16:1

1.1 1.1 2.3 1.3 2.3 6.5

16:1,43

16:3

----

21.2 2.4 3.5

----

----

33.5

18

18:1

18:2

18:3

0.8 1.4 6.6 2.1 3.4 11.5

2.9 6.3 9.9 5.0 13.9 19.5

2.3 2.3

67.6 78.5 32.4 31.9 30.0 8.7

weight

----

----

----

----

----

7.5 2.9 19.0 12.4

the same isoosmotic medium from grinding to collecting in the analysis tube, may be developed in the future and may give new data on the properties of chloroplasts and subchloroplast particles. Acknowledgments The authors are indebted to T. Galliard (Food Research Institute. Norwich, U.K.), P. Mazliak and A. Tremolieres (Laboratoire de Physiologie cellulaire) for critical reading of the manuscript. The authors thank C. Lichtle for electron microscope examination.

LITERATURE CITED 1. ALLLN CF, P Goor), HF DAVIS, SD FOWLER 1964 Plant and chloroplast lipids. 1. Separation and composition of major spinach lipids. Biochem Biophys Res Commun 15: 424-430 2. ALLEN CF, 0 HIRAYAMA, P GOOD 1966 Lipid composition of photosynthetic systems. In TW Goodwin, ed, Biochemistry of Chloroplasts, Vol I Academic Press, New York, 195-200 3. ARNON DI 1949 Cooper enzymes in isolated chloroplasts. Polyphenoloxidase in Bela vulgaris. Plant Physiol 24: 1-14 4. BLItH EG, NJ DYER 1959 A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917 5. CAStIMORE AR 1976 Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins. J Biol Chem 251: 2848-2853 6. EA(GLESHAM ARJ, RJ ELLIS 1974 Protein synthesis in chloroplasts. 11. Light-driven synthesis

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of membrane proteins by isolated pea chloroplasts. Biochim Biophys Acta 335: 396 -407 7. HALL DO 1972 Nomenclature for isolated chloroplasts. Nature New Biol 235: 125-126 8. HANNi(; K, HG HEIDRICHi 1974 The use of continuous preparative free-flow electrophoresi.s for dissociating cell fractions and isolation of membranous components. Methods Enzymol 31: 746-761 9. HEIDRwtic HG. R KINNE. E KINNE-SAFFRAN, K HANNI(i 1972 The polarity of the proximal tubule cell in rat kidney different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings. J Cell Biol 54: 232-245 10. HEIDRicit HG. R STAHiN. K HANNIG 1970 The surface charge of rat liver mitochondria and their membranes. I Cell Biol 46: 137-150 11. JOYARD) J. R DOUCL 1976 Preparation et activites enzymatiques de 1'enveloppe des chloroplastes d'Epinard. Physiol Veg 14: 31-48 12. JOYARD J, R DOUCE 1977 Site of synthesis of phosphatidic acid and diacylglycerol in spinach chloroplasts. Biochim Biophys Acta 486: 273-285 13. KAWASHIMA N, SG WILDMAN 1970 Fraction I protein. Annu Rev Plant Physiol 21: 325-328 14. KLOFAT VW, K HANNIG; 1967 Elektrophoretische Isolierung von Chloroplasten. Z Phvsiol Chem 348, 739-741 15. KLOFAT VW, K HANNK; 1967 Elektrophoretische von chloroplasten Fragmenten mit unterschiedlichem Verhaltnis von Chlorophyll a = Chlorophyll b. Z Physiol Chem 348: 1332-1334 16. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227: 680-685 17. LEPAcGE M 1967 Identification and composition of turnip root lipids. Lipids 2: 244-250 18. LILLEY McCR, MP FITZERAL[). KG RIENITs. DA WALKER 1975 Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol 75: 1-10 19. LoURY M 1967 Une methode generale permettant la transformation rapide des corps gras en esters methyliques. Rev Fr Corps Gras 6: 383-389 20. LOWRY OH, NJ ROSEBRODJ(;H, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 21. REMY R 1971 Resolution of chloroplast lamellar proteins by electrophoresis in polyacrylamide gels. Different patterns obtained with fractions enriched in either chlorophyll a or chlorophyll b. FEBS Lett 13: 313-317 22. REMY R, G BEBEE 1974 Membrane proteins of higher plant chloroplasts related to photochemical systems and membrane stacking. In M Avron, ed, Proc 3rd Intern Congr Photosynthesis. Elsevier, Amsterdam, pp 1675-1684 23. ROBINSON SP, JT WISKICIl 1976 Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides. Plant Physiol 58: 156-162 24. SASTRY PS 1974 Glycosylglycerides. In R Paoletti, D Kritchevsky. eds. Advances in Lipid Research. Vol. 12. Academic Press, New York. pp 251-310 25. SHIBUYA 1, H HONDA, B MARUO 1967 A simplified colorimetry without incineration of phosphorus in phosphatides. Agric Biol. Chem 31: 111-114 26. STAHN R, KP MAIER, K HANNIG; 1970 A new method for the preparation of rat liver Iysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis. J Cell Biol 46: 576-591 27. THORNBER JP, RPF GREGORY, CA SMITH, JL BAILEY 1967 Studies on the nature of the chloroplast lamelIa. 1. Preparation and some properties of two chlorophyll-protein complexes. Biochemistry 6: 391-396 28. TIIORNBER JP, HR Hic;HKIN 1974 Composition of the photosynthetic apparatus of normal barley leaves and a mutant lacking chlorophyll b. Eur J Biochem 41: 109-- 116