THB JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 13, Issue of July 10, PD. 4568-4573, 1973

Printed in U.S.A.

Chloroplast Membrane Proteins

II. SOLUBILIZATION OF THE LIPOPHILIC COMPONENTS*

(Received for publication, December 26, 1972)

FINBAR A. MCEVOYS AND WILLIAM S. LYNN

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 277iO

SUMMARY

The membrane of spinach chloroplast grana has been solubilized by a method which does not involve use of deter- gents. This method is based on the fact that most of the membrane components are soluble in acidic ethanol. The alcoholic extract is chromatographed on a hydroxylapatite column and the pigments are removed quantitatively by washing with alcohol. The membrane proteins can then be eluted by successive washing with phosphate buffer and dilute ammonium hydroxide. Some protein remains on the column and requires sodium dodecyl sulfate for elution. The major component of the membrane is eluted in the am- monia fraction, and is freely soluble in aqueous buffers at pH 7.5. This protein was shown to bind p-carotene. None of the major cbloroplast proteins are glycoproteins.

The fraction which was insoluble in acidic alcohol had a bright pink color due to the presence of p-carotene. This complex was highly insoluble, even in high sodium dodecyl sulfate concentrations unless the p-carotene was first re- moved by extraction with an organic solvent. This com- plex was shown to be a precipitate of p-carotene and several peptides.

Progress in understanding of the structure and mechanisms of action of membrane-bound constituents of cells has been slow due to the difficulties of isolating and characterizing these water- insoluble components. This has been particularly true in the case of the energy-conserving reactions carried out by the inner membrane of mitochondria and the thylakoid membrane of chloroplasts. It has long been known that detergents, such as sodium dodecyl sulfate and digitonin cause partial solubilization of the chloroplast membrane (1, 2) and more recently, progress has been made in isolating various components of the membrane by this method (3-6). Also, polyacrylamide gel electrophoresis in sodium dodecyl sulfate has been used to analyze the protein constituents of chloroplasts (3, 7), bacterial chromatophores (8), and of both the inner (9) and outer membranes of mitochondria

* This research was suppored by Grant GM1402203 from the National Institutes of Health and by Grant GB-17440 from the National Science Foundation.

$ Present address, Institute of Child Health, University of Bir- mingham, Birmingham B16 8ET, U. K.

(9,lO). The “structural” proteins of photosynthetic membranes have been isolated and characterized (11). These proteins are highIy insoluble and are thought to be part of the hydrophobic core of the membrane (12, 13). A “structural” protein of the mitochondrial inner membrane was isolated but this has been shown to consist of several denatured proteins, including the coupling factor Frl (14). Indeed, current theories of membrane structure do not include a protein with a function which is simply structural (15).

Our current knowledge of membrane protein structure has largely been obtained through solubilization of the protein com- ponents by replacement of the lipids with detergents. However, these methods are limited by the fact that the detergents which are most effective in solubilizing membrane proteins remain bound to protein. In this communication a method is described for obtaining 90% solubilization of membrane proteins of chloro- plast without the use of detergents. This method is based on the fact that the core proteins of the membrane will dissolve in acidified ethanol-acetic acid and can be quantitatively removed from pigment by chromatography on hydroxylapatite. The proteins can be eluted from the column in a form which is soluble in aqueous solvents.

METHODS

Albumin-washed chloroplasts were prepared as previously described (17). The particles were washed once with 0.4 M

sucrose to remove any albumin. The coupling factor enzyme (CFJ was removed by washing with dilute EDTA as previously described (18) except that the chloroplasts were incubated with EDTA for a longer time (30 min). Alcohol extraction of the EDTA chloroplasts and hydroxylapatite chromatography was carried out at room temperature in subdued light. Generally, hydroxylapatite chromatography was carried out immediately after preparation of the ethanol extract. However, it was found that the alcohol extract could be stored in the dark at -20” under nitrogen for several days without change in its properties or solubility. Thus, this preparation differs from the butanol- soluble preparation of isoprenoid phosphokinase which was re- ported to come out of solution at low temperatures (19).

Chromatography was carried out on columns, 1.5 x 10 cm, of hydroxylapatite (Hypatite C, Clarkson Chemical Co., Williams- port, Pa.). The gel was first washed sequentially with 0.4 M

1 The abbreviations used are: R, mitochondrial coupling factor 1; CFI, chloroplast coupling factor 1; PAS reagent, periodic acid- Schiff reagent.

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phosphate buffer, pH 7.0, and then with 2 M ammonium hy- droxide. Small particles were removed by decantation and the column was packed, and washed extensively with water before application of the sample. Five-milliliter fractions were col- lected. Protein-containing fractions were located by their 280- nm absorbance and were immediately brought to neutral pH and dialyzed against 5 rnM Tris-HCl, pH 7.5 at 4”. In all other cases, protein was determined by the method of Lowry et al. (20). Carbohydrate was measured by the phenolsulfuric acid method (21), with glucose as standard.

Subsequent manipulations of these proteins were carried out at 4’ and the proteins were stored at -20”. Spectra were obtained with a Cary 11 spectrophotometer. Amino acid analyses were carried out by Dr. H. Steinman (Duke University) on a Beckman analyzer model 120. Analyses given are based on 24-hour hy- drolyses in 6 M HCl, with corrections for destruction of serine and threonine. Coomassie blue-stained gels were scanned at 560 nm on a Gilford 2000 spectrophotometer equipped with a scanning attachment. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate was carried out by a standard method

(22). Reagents were the best grade available from Sigma (St.

Louis) or else were analytical grade.

FIG. 1. Sodium dodecyl sulfate gel electrophoresis patterns of CFI and of the proteins of whole chloroplasts stained with Coomas- sie blue. CF, from chloroplasts, prepared as previously described (23), and whole chloroplast proteins, prepared by extracting chloroplasts twice in 80% acetone, were dissolved in 1% sodium dodecyl sulfate containing 0.01 M phosphate and 1% 2-mercapto- ethanol, pH 7.0, for 1 hour at 40”. The bands described as Red Bands refer to those bands giving a bright red color when viewed in indirect light against a dark background (see text for details). PAS-positive bands refer to those stained by the periodic acid- Schiff reagent.

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RESULTS

Treatment of Chloroplasts with EDTA-The chloroplast particles used in this study were the most purified preparation available which retained energy-conserving activities, i.e., they were capable of carrying out high rates of phosphorylation and could transport electrons to both ferricyanide and NADP+ (17). The particles were thought to be substantially free of soluble proteins, since they had been washed several times with sucrose, and once with a hypotonic Tris-albumin-ascorbate buffer. As shown in Fig. 1 this membrane, when solubilized and submitted to sodium dodecyl sulfate gel electrophoresis (22) contained two peptides of molecular weight approximately 60,000, a minor component at 45,000, and about six bands ranging in size from 32,000 down to about 15,000. The principal component was a rather diffuse band of molecular weight 26,000. Occasionally Coomassie-positive material was observed at the origin of the gels (see Fig. 1). This material was inconstant in amounts and not present at all if reduction of the peptides was for 2 hours or longer and followed by alkylation with iodoacetamide. It was concluded that no peptides of molecular weight greater than 62,000 exist in these lamellar membranes. Treatment of the membrane with hypotonic EDTA removes most of the 60,000 peptides, which correspond to the coupling factor, CF1 (Fig. 2). The isolated CFI, when purified by ion exchange chromatog- raphy, contains three minor peptides in addition to the two major bands (see also Refs. 23 and 24). These minor bands do not correspond with any of the major components of the chloro- plast membrane (Fig. 1). The EDTA-washed chloroplast thus consists almost entirely of proteins with subunits varying in molecular weight between 32,000 and 15,000 (Fig. 2). These gels were stained with the Schiff stain to test for the presence of

WHOLE CHLOROPLASl PROTEIN

ll I” ETHANOL d\ pRET’ n 2%THANOL

PRECIPITATE

FIG. 2. Densitometer scans of sodium dodecyl sulfate gels of whole chloroplasts, EDTA-treated chloroplasts, and the protein precipitated by 70% ethanol and 90% ethanol. The distance to which the marker dye migrated in each gel is shown by the arrow.

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glycoprotein and none of the protein bands were found to give a positive stain with this reagent. However, two diffuse bands of Schiff-positive material were observed with mobilities of greater than 0.8 relative to the marker dye. Since these bands did not stain with either Amido black or Coomassie blue, they were presumed to be glycolipid. These data do not support the earlier proposal of Weber (25) that a major protein in chloro- plasts is a glycoprotein of subunit molecular weight 35,000.

The protein pattern obtained by this method is very similar to that shown by a preparation of Vicia fuba lamellae (7), except that the latter preparation had a pigment-binding band with a mobility corresponding to a molecular weight of 100,000. This band, which has been reported to be the Photosystem I reaction center (26) had been dissociated to smaller components in our preparation, due to the fact that our procedure, which involves more rigorous detergent action and a delipidation step, breaks the components down to their monomeric form.

Solubilization of EDTA-treated Chloroplasts in Acidic Alcohol- Chloroplast membranes have frequently been treated with or- ganic solvents to disrupt their structure and solubilize their lipids (16, 27). Although Zill and Harmon (28) found that 4% of the chloroplast protein could be solubilized in chloroform- methanol as a proteolipid, generally it has been reported that the effect of organic solvents is to extract the lipids and leave the lamellar protein as a precipitate. However, when the mem- brane was extracted with ethanol containing 1% acetic acid, most of the protein went into solution (Table I). The protein in this clear green viscous solution could not be sedimented by centrifugation at 35,000 x g for 30 min. The protein could readily be precipitated by adjusting the pH to 7.0, or by adding trichloroacetic acid to a final concentration of 10%. When EDTA-chloroplasts were treated with 70% ethanol containing

TATJLE I

Praclionation of chloroplast membrane proteins

Whole chloroplast membranes (100 mg)

EDTA supernatant

--j

Hypotonic EDTA (CFd (15 me)

4 EDTA-treated membranes

Precipitate (10 mg) -I

70% Acid-ethanol

4 Supernatant (75 mg)

I Precipitate (2 mg) ‘-1 85% Acid-ethanol

SupernataL (70 mg)

I

0.4 M phosphate eluate (2 me)

2 M NHbOH eluate

(45 md

0.2% sodium dodecyl sulfate eluate

(17 ms)

1% acetic acid, the small amount of insoluble material was bright pink. A further small pink pellet was obtained when the ethanol concentration in the supernatant was increased to 8501,. These pink pellets corresponded to the carotene-protein com- plexes originally described by Menke (27,29).

Isolation of Proteins from Alcohol Solution-The proteins could be isolated almost free from the lipid in a water-soluble state by column chromatography on hydroxylapatite. The green alco- holic solution was applied to the column, and the column washed with ethanol until all of the pigment was removed. The column was then washed sequentially with water, 0.4 M phosphate buffer, 2 M ammonium hydroxide, and 0.2% sodium dodecyl sulfate. Water eluted a small amount of material which contained some sugar but did not contain any detectable protein (Fig. 3). Most of the carbohydrate and all the pigments were eluted with the ethanol, Fig. 3. The phosphate buffer (31) eluted only a small amount of protein and by sodium dodecyl sulfate gel electrophore- sis, only one peptide of molecular weight of approximately 18,000 was observed (Fig. 4).

The major peptide in the chloroplast, which on sodium dodecyl sulfate gels had molecular weight of 26,000, was eluted from the hydroxylapatite with 2 M NH*OH at pH 10.3. It was found that this peptide could not be eluted from the column in any buffer at a lower pH. After elution the protein-containing frac- tions were immediately pooled, brought to pH 8.0 with HCI, and dialyzed against 0.005 M Tris-HCl buffer, pH 7.5 at 4”, for 24 hours. A small amount of protein (about ,30/, of the sample) precipitated during dialysis and this was removed by centrifuga- tion at 10,000 X g for 10 min. The remaining protein was water-soluble at neutral pH.

Elution of the column with 0.2% sodium dodecyl sulfate re- moved all the remaining protein from the column, which con- sisted of a mixture of bands, including some of the residual coupling factor peptides (not shown).

Properties of Isolated Membrane Peptides-The above frac- tionation procedure gives a purification in water-soluble form of t.wo of the chloroplast peptides. One of these (a minor compo- nent) is eluted with the phosphate buffer and is of unknown function. The other protein, which is the major peptide of the chloroplast lamella, appears to be the same as the Photosystem II reaction center protein previously described (3, 7, 26, 32)

-Pigment M Protein ----- sugar

? 0.6 2

E

100 200 300 400 Eluote Volume (ml)

FIG. 3. Hydroxylapatite chromatography of the acid alcohol- soluble components of the chloroplast membrane. Fifty mil- liliters of the alcohol solution were applied to the column, and the column was washed with ethanol until all the pigments were re- moved. The column was then washed sequentially with 50-ml aliquots of water, 0.4 M sodium phosphate, pH 7.0, 2 M NH,OH, and 0.2% sodium dodecyl sulfate (SDS) in 0.01 M sodium phos- phate, pH 7.0.

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FIG. 4. Densitometer scans at 560 nm of sodium dodecyl sulfate gels of the proteins of the ethanol-soluble fraction, and of the frac- tions eluted from the hydroxylapatite column.

This appeared likely from the high yield of the peptide, but also there was evidence that this peptide binds chlorophyll, since when chloroplast membranes were dissolved in sodium dodecyl sulfate buffer and subjected to electrophoresis without delipida- tion with organic solvents, some chlorophyll could be seen to migrate with this band although this tended to dissociate with longer running times. The amino acid analysis of this prepara- tion (Table III) is very similar to that described by Thorber et al. for their preparation of the Photosystem II reaction center (32).

As shown in Table II, this peptide is completely free of glyco- lipid and pigments. It contains a trace amount of organically bound phosphorus which may be phospholipid. In aqueous solution this peptide exists as a large oxidized polymer, since it is completely excluded by G-100 Sephadex columns and it does not migrate beyond 0.5 cm into acrylamide gels with electrophoresis for 8 hours if it is not first reduced with 2-mercaptoethanol. Addition of alcohol to the purified peptide causes immediate precipitation showing that the earlier solubility in organic sol- vents is a function of its lipoprotein nature. Efforts to frac- tionate these 26,000 molecular weight peptides using gels con- taining 15% acrylamide and 1% sodium dodecyl sulfate were unsuccessful. It is possible that each of the peptide bands ob- served in Fig. 1 may represent more than one peptide of the same molecular weights. NHZ termini have not been successfully determined.

The small amount of protein eluted from the hydroxylapat,ite column with the phosphate buffer is not completely pure on sodium dodecyl sulfate gel electrophoresis. It contains small amounts of the 32,000 molecular weight peptide, the 26,000 molecular weight peptide, and a Coomassie blue-staining band corresponding to 10,000 or less (Fig. 4). One interesting prop- erty of this protein on sodium dodecyl sulfate gels is that when

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TABLE II Properties of proteins isolated from chloroplast membranes by ethanol

fractionation and hydroxylapatite chromatography

I- mg/mg prolt?in~

jO.003 N.D.b 0 Phosphate. 0 1.33 0

Ammonia. 4 1.42 10.003 / 0.001 / 0 0

a Estimated by the phenolsulfuric acid method (21), with glu- cose as standard.

* Not determined.

Sugar Organic Chloro- CW3- phosphorus phyll tenoid

stained with Coomassie blue, it gives a bright pink glow when viewed in indirect white light against a black background. In direct light, it is blue. A similar observation has been made with bacterial chromatophore membrane proteins (8), and in this case the authors have explained this phenomenon as fluores- cence of the Coomassie blue. This is not the case, in this in- stance however, since on illumination of the gel with monochro- matic indirect light of 600 nm or lower, the red sunset glow is lost, but the blue band remains. Attempts to observe fluores- cence of Coomassie dye and the proteins in solution were all negative. Elution of the material from the electrophoretically treated gels yielded material which was also nonfluorescent. Also, as indicated, no fluorescence is visible with direct light. The glow is not due to lipids, since no extractable lipids are pres- ent. Therefore, the source of the red glow results from light scattering rather than from fluorescence. Of a large number of standard proteins tested for this phenomenon, only one, mam- malian cytochrome c, was found to give this red color in indirect light. This was not due to absorbance by the heme group since small amounts of the cytochrome (less than 2 PC(g) which were not detectable on gels before staining, were still bright pink in in- direct light on Coomassie blue-stained gels. Demetallized cytochrome c (33) also gave the same pink color.

Clayton and Haselkorn have suggested that this phenomenon may be the result of Coomassie binding to exceptionally hydro- phobic membrane proteins (8). The 18,000 peptide does not contain a large amount of hydrophobic residues (Table III). It is possible, however, that the peptide chain contains regions which are enriched in hydrophobic side chains (34).

The whole chloroplast has three “red” bands with apparent molecular weights of 23,000, 21,000, and 18,000 (Fig. 1). This latter peptide is the one which is isolated free of the other pep- tides by phosphate elution from hydroxylapatite. The function of this peptide is unclear. The protein appears to be intimately connected with the membrane, although its amino acid composi- tion shows that it is not exceptionally rich in hydrophobic groups (Table III).

Binding of B-Carotene to G’hloroplast Membrane Protein-The 26,000 molecular weight chloroplast protein was shown to have affinity for @-carotene by the following experiment. A sample of p-carotene containing 0.1 pmole in 0.1 ml of ether was added to 1 ml of the chloroplast protein solution, containing 0.5 mg of protein in 0.005 M Tris-HCl buffer, pH 7.5. The solution was mixed thoroughly by stirring on a Vortex mixer for 2 min and allowed to stand for 1 hour, after which the mixture was slightly turbid and had a reddish color. The mixture was centrifuged for

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Amino acid Phosphate &ate Ammonia &ate (18,000 mol wt) (26,000 mol wt)

Lysine ....................... 7.7 5.5 Histidine ..................... 0.95 1.6 Arginine ..................... 3.5 4.2 Aspartic acid. ................ 11.0 8.8 Threonine .................... 3.7 4.7 Serine ........................ 5.6 5.7 Glutamic acid. ............... 11.2 9.2 Proline ....................... 6.2 5.9 Glycine. ..................... 8.2 10.5 Alanine ...................... 7.4 9.6 Valine ........................ 7.7 6.5 Methionine ................... Trace 1.7 Isoleucine .................... 6.2 5.3 Leucine ...................... 9.4 11.0 Tyrosine ..................... 3.4 3.8 Phenylalanine ................ 4.0 6.5 7 o nonpolar residuesb. ........ 39.1 46.5

TABLE III Amino acid compositions of purified chloroplast membrane proteins”

u These analyses were made on 24.hour hydrolysates, with cor- rections for serine and threonine destruction.

b Calculated by the method of Guidotti (47).

10 min at 10,000 X g, which gave a clear yellow supernatant, and a brick-red precipitate. The supernatant solution, which con- tained all the protein, gave no further precipitate when centri- fuged at 25,000 X g for 30 min. The protein-carotenoid complex had the spectrum shown in Fig. 5, which is very similar to the spectrum of p-carotene in hexane, i.e. it is slightly shifted toward the blue compared to the spectrum of /?-carotene suspended in aqueous 0.2% sodium dodecyl sulfate. This suggests that the environment of the p-carotene is less polar on the protein complex than that in the sodium dodecyl sulfate micelles. The protein- carotenoid complex also has an extra absorbance peak at 518 nm. Intact chloroplast membranes have an absorption peak around 500 nM, also (16).

When p-carotene was added in a similar manner to Tris buffer in the absence of protein, the P-carotene was totally insoluble in the buffer and either adhered to the sides of the tube or else formed an oil globule at the bottom of the tube. When bovine serum albumin was substituted for the chloroplast protein, the p-carotene formed a reddish suspension. When centrifuged at 10,000 X g for 10 min, all of the protein, but no detectable pig- ment, remained in solution, thus showing the absence of binding of p-carotene to albumin under these conditions.

The pink material which was insoluble in the acidified ethanol had properties rather similar to those of the suspension of fl- carotene obtained with albumin. Both had poorly defined spectra which showed an extensive red shift (Fig. 6). The 70% ethanol-insoluble material also had an absorbance peak at about 420 nm which was not present in either the 90% ethanol precipi- tate or the albumin-carotene suspension. This peak may be due to cytochromes, as has been found with a similar prepara- tion (16). Red protein-carotene complexes have previously been isolated from chloroplasts by several different techniques (29) and these preparations have had absorbance maxima be- tween 537 and 560 nm. On the basis of binding studies with an insoluble chloroplast lamellar fraction, Ji et al. (16) proposed that this red-shifted form of p-carotene is indicative of a specific in- teraction between the pigment and lamellar protein. This con-

SOLUBLE CAROTENE-PROTEIN

COMPLEX

P-CAROTENE IN 0.2% SDS.

I

400 500 600 700

WAVELENGTH (nm)

FIG. 5. Absorption spectrum of the soluble complex of p-caro- tene 0.1 PM with the 26,000 molecular weight chloroplast protein compared with that of a suspension of pure p-carotene in 0.2% sodium dodecyl sulfate (SDS).

WAVELENGTH (nml

FIG. 6. Absorption spectra of the ethanol-insoluble fraction compared to that of the p-carotene-albumin suspension and the ethyl acetate extract of the ethanol-insoluble fraction. This latter spectrum is identical with that of pure p-carotene in the same solvent. The ethyl acetate extract was prepared by ex- tracting the 2% sodium dodecyl sulfate suspension of the 70% ethanol precipitate with an equal volume of ethyl acetate.

elusion is not supported by the data presented here, since p- carotene when suspended in aqueous buffer by bovine serum al- bumin is bright red, Fig. 6. The red ,&carotene is simply an aggregated form. The involvement of albumin in these aggre- gates is simply to stabilize the suspended carotenoid.

DISCUSSION

These data describe a fractionation technique in which the chloroplast membrane components are isolated in organic sol- vents and subsequently solublized and purified in aqueous sol- vents after delipidation. Recently there have been several re- ports of the solubility of membranes (3537) or membrane proteins (19, 38) in organic solvents. Gitler and Montal (38) have extensively studied the solubility of cytochrome c in organic solvents in the presence of phospholipids, and they have shown solubilization requires a low pH in order to ensure neutraliza- tion of the negative charges on the protein. These lipid-protein complexes are very stable in this state, since even boiling of the solution does not cause disruption of the protein conformation

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(19). We have also observed that boiling of the ethanolic chloro- plast solution for 10 min does not cause precipitation of the pro- tein. Solutions of chloroplast protein in acidified ethanol could be obtained in high yield only from chloroplasts which had been washed extensively, and then soaked for a long time in hypo- tonic EDTA to remove most of the soluble and peripherally bound hydrophilic proteins. Preparations of chloroplasts which still contained considerable amounts of CFI invariably gave low yields of ethanol-soluble protein.

The carotenoid and protein obtained as an insoluble precipi- tate clearly is not a specific lipoprotein complex and does not appear likely to be related to any physiological function. How- ever, the possibility of this type of aggregate being formed in viva cannot be ruled out. The known involvements of @-caro- tene in the maintenance of membrane structural integrity (34) and in the primary processes of Photosystem II (40) suggest that changes in the environment of the carotenoid on stimulation of electron transport might give rise to the observed changes in ab- sorbance attributed to the chromophore C-550 (41, 42). C-550 has not yet been chemically identified, but the observations that it is inhibited by lipases (42) and chaotropic and other agents which affect the polarity of the membranes (44) are consistent with this possibility, as is the observation that the effect of oxida- tion and reduction on the spectrum of C-550 at -196” appears to be a band-shift (43).

This fractionation procedure results in the preparation of the major membrane peptides in a purified but probably aggregated state which is water-soluble, free of pigments, and essentially free of lipid. Preliminary experiments showed that this peptide binds chlorophylls, although a shift in absorption spectrum was not observed in this case.

In agreement with the observation of Thornber et al. (3) we find that preparations enriched in Photosystem II activity (i.e. oxygen evolution) contain mainly the insoluble, intrinsic mem- brane components, while fractions enriched in Photosystem I activities are also enriched in the more easily solubilized mem- brane constituents, such as the coupling factor, CFl.a Thus, it is difficult to ascertain which polypeptides are uniquely associated with each photosystem (45, 46). However, it is clear that es- sentially the major proteins of the chloroplast are soluble in water, once lipids are properly removed.

Acknowledgments-We are grateful to Mary Lynn who pre- pared the chloroplasts, to Dr. S. Munoz who carried out the carotenoid chromatography, and to Dr. H. Steinman who carried out the amino acid analyses.

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Finbar A. McEvoy and William S. LynnCOMPONENTS

Chloroplast Membrane Proteins: II. SOLUBILIZATION OF THE LIPOPHILIC

1973, 248:4568-4573.J. Biol. Chem. 

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