Protein components of bacterial photosynthetic membranes

J. Mol. Bid.

(1972) 68, 97-105

Protein Components

of Bacterial Photosynthetic RODERICK

K.

Membranes

CLAYTON

Division of Biological Sciencesand Department of Applied Physics Cornell University, Ithaca, N. Y. ROBERT University

HASELKORN

Department of Biophysics of Chicago, Chicago, Ill., U.S.A.

(Received18 October1971, and in

revised

form 2 February 1972)

The pigmented membranes of photosynthetic bacteria, and fractions derived from them, have been analyzed by electrophoresis in polyacrylamide gels, following their dissolution by exposure to sodium dodecyl sulfate and mercaptoethanol. Purified photosynthetic reaction centers from Rhodqtwudomonas sphe~oidea yielded three components of apparent molecular weights 27, 22, and 19 kilodaltons, confbming the findings of Feher and collaborators. The density of staining suggested that RC’st contain these components in a molar ratio of 1: 1: 1. The RC protein comprised about 25% of the total chromatophore protein. Two major chromatophore proteins, distinct from RC protein, had apparent molecular weights of 46 and 11 kdaltons. The RC proteins could not be found in any preparation made from the pigmented but non-photosynthetic mutant strain PM8 of Rhodopseudomonus q&r&es. Triads of bands suggesting the three RC proteins could be discerned in preparations from Rhodospirillum TUbTUm, Rps. cqwsuiata, and Rps. pa&u&is, but not Rps. gelatinosa or Rps. wiridk. Antibody specific toward RC’s from Rps. qnheroidee did not react with any component from non-photosynthetic mutant Rps. q~heroide~, nor from any of the other species of photosynthetic bacteria mentioned here. The Coomassie Brilliant Blue stain was observed to be fluorescent on some bands in the gels and not on others. With the RC triad the band of highest molecular weight was fluorescent and the others were not; this set of bands was accordingly very easy to recognize in crude preparations.

1. Introduction The photochemical activities of photosynthetic bacteria reside in a pigmented membrane, the chromatophore membrane, seemingly derived from the cytoplasmic membrane by invagination (seeClayton, 1971a). Fragments of the pigmented membrane, isolated from broken cells by differential centrifugation, comprise a chromatophore preparation. Such preparations from certain photosynthetic bacteria, notably a oarotenoidless mutant strain of Rhodopseudomo~ spheroides, can be separated further into components that serve the functions of light harvesting and photochemistry respectively. In particular, one can isolate a “reaction center” particle that performs the primary photochemistry. Reaction center particles from Rps. spheroide.9(Clayton & Wang, 1971; Feher, 1971) 7 Abbreviations sodium dodecyl 7

used : RC, photosynthetic reaction sulfate; LDAO, lanryl dimethyl amine 97

center oxide.

; BChl,

bacterioohlorophyll

; SDS,

98

R. K. CLAYTOX

AND

R. HASELKORN

consist mainly (at least 90%) of protein bearing two kinds of chromophore, bacteriochlorophyll and bacteriopheophytin. During photochemistry electrons are displaced from the BChlt to someother part of the system. This oxidation of the BChl is manifested (Clayton, 19716) by bleaching of an absorption band at 865 nm (P870) and blue-shift of a band at 800 nm (P800). The light-harvesting function in chromatophores of photosynthetic bacteria is served by BChl and carotenoid pigments. Chromatophores of carotenoidless mutant Rps. spheroides contain only light-harvesting BChl (maximum absorption 860 to 870 nm) and the characteristic chromophores of the RC. The absorption at 800 nm and the reversible bleaching at 865 nm serve to distinguish the RC BChl from a much larger amount of light-harvesting BChl. The isolation of RC’s from carotenoidless Rps. spheroides is effected by treating the chromatophore fraction with a detergent such as lauryl dimethyl amine oxide, centrifuging, and then fractionating the supernatant phase with ammonium sulfate. This results in a complete separation of RC’s from the light harvesting BChl, much of the latter remaining bound to other components of the chromatophore membrane. When this procedure is applied to chromatophores from wild-type Rps. spheroides, which contain colored carotenoids, the attempted separation of RC’s from the lightharvesting pigments fails. To gain further insight into the architecture of the chromatophore membrane, and especially the role of the characteristic RC protein in this architecture, we have analyzed the proteins of photosynthetic bacteria by electrophoresisin polyacrylamide gels. The samples, obtained by fractionations applied to a variety of speciesand mutant strains of photosynthetic bacteria (especially Rps. spheroides), were dissociated by exposure to sodium dodecyl sulfate and mercaptoethanol before electrophoresis. This technique has already been applied to purified RC’s from Rps. spheroides by Feher, Okamura, Raymond & Steiner (1971). They found that under strong denaturation (1% SDS) RC’s yielded three components of apparent molecular weights 28, 23 and 21 kdaltons. Milder denaturation (O*1o/oSDS) yielded only two components, nominally 28 and 37 kdaltons, the latter apparently a conjunction of the 23- and 21-kdalton components. The 37-kdalton component held all the BChl of the RC’s and was photochemically active. We have confirmed that RC’s yield three components, as described above. These RC proteins are prominent constituents of the chromatophore membrane, detected easily in various fractions that may or may not contain light-harvesting pigments. The RC proteins appear to be missing in the non-photosynthetic strain PM8 of Rps. spheroides; in this strain the light-harvesting pigments are bound exclusively to other membrane components. This supplements earlier information (Sistrom & Clayton, 1964) that P800 and P870 are missing in PM8. Proteins similar in molecular weight to those of the RC proteins from Rps. spheroides were found in chromatophores of Rps. palustris, Rps. u~psulatu, and R~ospirillum r&urn, but not in chromatophores of Rps. viridis and Rps. gelatinma.

2. Materials and Methods (a) Cell cultures and subcellula~ preparations Cultures of photosynthetic bacteria were grown in completely filled glass-stoppered bottles under about 5 mW/cma illumination from household tungsten lamps, at 27 to t See footnote on p. 97.

iiNALYSIS

OF

PHOTOSYNTHETIC

MEMBRANES

99

32°C. Some cultures were grown aerobically in darkness, in flasks on a shaker at 30°C. The culture medium was modified Hutner medium (Cohen-Bazire, Sistrom & Stanier, 1957) in some cases and YCCSu in others. YCCSu, devised by W. R. Sistrom (personal communication), contained 5% yeast extract, 3% casein hydrolysate, 0.2% potassium succinate, and 2% (v/v) of the concentrated base used in Hutner medium. The choice between these media did not seem to matter. The following organisms were grown: Rhodoapirillum 8 sl3heroides: wild-type strain ATH 2.4.1 rubrum, strain S- 1 (van Niel) ; Rhodop8eudomonu (van Niel), cerotenoidless mutant strain R26 (Clayton & Smith, 1960), and Sistrom’s nonphotosynthetic mutant strain PM8 (Sistrom & Clayton, 1964) ; the lineage of both mutant strains begins with ATH 2.4.1. Rpa. palustrie, strain 11168, donated by Dr A. Jane Gibson. Rps. Gridis, strain NTHC 133 (Eimhjellen, Aasmundrud & Jensen, 1963). Rpe. capsulatu and gelatinosa, strains TCl and TG5, respectively, isolated in 1970 by R. K. Clayton end B. J. Clayton from mud in Ithaca, N.Y. Cells were broken with a Fench pressure cell (R. rubrum and Rps. spheroides) or by sonic disruption (the other species). The resulting extracts were centrifuged at 10,000 g for 20 min, the pellets discarded, and the supernatant phases centrifuged at about 100,000 g for 2 hr to sediment the chromatophore fraction (and ribosomes, cell-wall fragments, etc.). The supernatant from this high-speed centrifugation, containing soluble proteins, was saved for analysis. The pellet was washed twice by repeated centrifugation and redispersed in 0.01 aa-Tris-Cl, pH 7.5, to yield a washed chrometophore preparation. Some of the chromatophores were placed over sucrose, 1.0 M and 1.2 M layers, and centrifuged to yield “light chromatophores” (Worden & Sistrom, 1964) at the 1.0/1.2 M-SUCPOSB interface. For the isolation of RC’s, chromatophore preparations were adjusted so that the maximum optical density due to BChl in the near infrared was 50. Aqueous lauryl dimethyl amine oxide (1/30th vol. of 30%) (donated by Onyx Chemical Co., Jersey City, N.J.) was then added, and the mixture centrifuged at about 170,000 g for 3 hr. The supernrttant phase contained the RC’s in cases where these could be recognized. Some of this “LDAOupper phase” (code letter U in the Figures) was set aside for analysis. The sedimenting material could be separated into two fractions by placing the LDAO-treated chromatophores over sucrose (0.5 M and 1.0 M layers) for the centrifugation at 170,000 g. This procedure, when applied to carotenoidless mutant Rps. spheroides, gave a pigmented “light-harvesting BChl protein” fraction at the 0.5/1.0 M-sucrose interface (LH in Plate IV) and 8 relatively non-pigmented pellet (Clayton L Wang, 1971). The LDAO-upper phase was fractionated with ammonium sulfate, most of the pigmented material becoming insoluble between 30 and 50% of saturation. This fractionation yielded relatively pure RC preparations from carotenoidless mutant Rps. epheroides, but effected little purification when applied to LDAO-upper phase from the other strains. Purity of the RC’s could be estimated from the relative o.D.‘s at 280 and 800 run; this ratio approaches 1.2 in the most exhaustively purified preparations. For the reaction centers used in this investigation the ratio was about 1.4 to 1.7. The estimated 15 to 40yb contamination was manifested by faint impurity bends in the acrylamide gel patterns. The “AUT” preparation (Loach, Sekura, Hadsell & Sterner, 1970) was made from chromatophores of wild-type Rps. *heroidea. The ohromatophores, suspended in water, were placed on a sucrose density-gradient (0.1 to 1.0 M) containing 6 M-Ure& and 1.5% Triton Xl00 at pH 11.0. After 1 hr centrifugation at about 100,000 g, all of the pigmented material was found in a layer near the top of the tube. A small amount of white material, representing 3% of the total protein, sedimented to the bottom. This substantiated Loaoh’s claim of nearly quantitative conversion of chromatophores into non-sedimenting subunits by the urea/Triton treatment at pH 11. (b)

Polyacrylamide

gel electrophoresis

Analytical polyaerylemide gels were prepared and run according to the procedure described by Laemmli (1970). The gels were cast in glass tubes of 6 mm internal diameter ; the analyzing gels were 8 cm long and contained 10% clcrylamide, 0.376 M-Tris-Cl (pH 8.8), and 0.1 O/( SDS. The stacking gels, 1 to 2 cm long, contained 3% acrylamide, O-125 M-Tris-Cl (pH 6*9), and 0.1% SDS. Samples containing 10 to 100 M protein in 0.0626 M-Tris-Cl (pH 6.9), 1.4% SDS, 3.3% 2-mercaptoethanol, 20% glycerol, and 0.002% bromophenol

100

R.

K.

CLAYTON

AND

R.

HASELKORN

blue were immersed in a boiling water bath for 60 set, unless othorwiso noted, before being layered onto the gel columns. Electrophoresis was at room temperature at 2.5 mA per gel, run until the ion front reached the end of the tube (usually 3.5 hr). The gels were fixed in 60% aqueous trichloroaoetic acid at room temperature for at least 4 hr ; usually overnight. They were then stained at 3’7°C for 90 min with 0.1% Coomassie Brilliant Blue in water with 30% trichloroacetic acid, 20% methanol, and 4% acetic acid. The gels were destained by diffusion in To/, acetic acid and 5% methanol in water. The necessary reagents and apparatus were supplied by the facilities of the Physiology Course, Marine Biological Laboratory, Woods Hole, Massachusetts, and through the kindness of Dr R. E. Stephens. We are indebted to Mr Jean-Luc Popot for help with photography. Optical densities of stained bands in the gels were scanned at 600 nm with a Gilford spectrophotometer using their linear transport accessory; the relative amounts of components were estimated from areas of bands in the resulting trace. (c)

Other

analytical

methods

Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1961) using bovine serum albumin as a standard. Optical absorption spectra and light-induced absorbancy changes, needed especially for monitoring the presence of RC’s, were measured with a home-made instrument (Bolton, Clayton L Reed, 1969). Bacteriochlorophyll was estimated roughly from absorption spectra in the near infrared, assuming an extinction coefficient of 1.3 x lo6 M-‘cm-’ for each absorption band (Clayton, 1906). Rabbit antisera against RC’s from Rp. s@eroides had been prepared by Dr R. L. Berzborn at Cornell University. We used these sera in agar-diffusion precipitation tests by the method of Ouchterlony (1949), with 0.5% Triton Xl00 present in the agar to prevent aggregation of some of the samples. More extensive serological studies by W. R. Sistrom, R. L. Benborn and R. K. Clayton will be described elsewhere.

3. Results (8) Reaction center proteins of

Rhodopseudomonas

spheroides

R26

Reaction centers from carotenoidless mutant Rps. spheroides, subjected to SDS/ acrylamide gel electrophoresis as described, exhibited three components as shown in Plate I. We designate these RC,, RC!, and RC, in order of decreesing molecular weight. Sharpness of these b&rids was not affected by removal of the chromophores, BChl and bacteriopheophytin, before dissociation of the RC’s in SDS. The result with samples that had not been boiled was essentially the same as that with 1 minute of boiling (the faintness of the bands in gel (a) of Plate I is spurious ; we had inadvertently used too little material). Boiling longer than 1 minute caused a marked loss of RC, and RC,, while the origin (top) of the gel showed the accretion of non-moving material. RC, was not affected by boiling. Following a remark by W. Hagins, we noted that prolonged boiling caused solutions of SDS to become flocculent. Perhaps RC,, and RC, have a special atlinity for SDS and are bound by the flocculent material derived from it. Two of the gels shown in Plate I contained proteins of known molecular weights: bovine serum albumin (68,000), deoxyribonuolease I (31,000), trypain (23,500), and cytochrome c (12,499). The positions of these marker proteins were plotted semilogarithmically as shown in Figure 1; the positions of the RC proteins then corresponded to molecular weights of 27,22 and 19 kdaltons, in fair agreement with the results of Feher et al. (1971). Densitometer traces of these gels revealed the relative amounts of Coomassie Brilliant Blue stain bound to each component, If we assume

(b)

0

I

(Cl

(d)

(e)

(f)

2

5

IO

0

(h)

I

5

I AUT

2

PLATE I. Analysis of proteins of reaction centers prepared from csrotenoidless mutant Rps. plw~~ides R26 by electrophoresis on polyaorylamide gels containing SDS. Gels (a) to (h) illustrate the effects of boiling on the distribution of RC proteins. Gel (c) contains bovine serum albumin and cytochrome e as markers. Samplea were prepared as described in Materials and Methods and boiled for the times, in minutes, indicated on the Plate. The sample in (i) was incubated 1 hr in 6 M-urea, 1.5% Triton Xl00 at pH 11 before boiling for 1 min in SDS. Gel (j) contains, in descending order: bovine serum albumin, a trace contaminant, pancreatic DNase I, RC,, trypsin, RCb, RC,, and cytochrorne c. The symbols to the right of gel (j) are explained in the legend to Plate III.

(4 PLATE illuminated

II.

Fluorescence from the rear;

of the RC, in (b) from

lb) band stained the side.

with

Coomassie

Brillient

Blue.

In (R) the gel

E

su

C

LC

U

RC

LH

~'LATE III. Analysis of proteins in fractions prepared from Rps. spheroides R26, as described in Materials and Methods. Samples were boiled 2.5 min before electrophoresis. Bands identified as the RC subunits are indicated by a chevron ( <). A dot to the right of a band indicates strong fluorescence of Coomassie Brilliant Blue; a dash indicates non-fluorescence. E, crude extract of broken cells; SU, supernatant after first high-speed centrifugation (soluble proteins, no chromatophores) : C, chromatophores; LC, light chromatophores; U, LDAO-upper phase; RC, reaction centers; LH. light-harvesting BChl protein (see Materials and Methods).

E PLATE IV. are designated Triton Xl00

su

C

LC

U

Analysis of proteins in fractions prepared from wild-type Rps. as given in the legend to Plate III; AUT is a preparation at pH 11 as described in the text. Samples were boiled 1 min.

AU1

8pheroides. made with

Fractions urea and

V. Analysis of proteins PM8 and from wild-type were added to some of the samples subunits should be sought. Symbols PLATE

spheroides

in

fractions prepared from non-photosynthetic mutant Bps. spheroides grown aerobically in darkness. Purified RC’s from PM8, as indicated, to mark the places where RC’ protein are identified in the legend for Plate III.

Rps.

ANALYSIS

OF

PHOTOSYNTHETIC

MEMBRANES

E D&mce

I” gel L mm)

FIG. 1. Determination of the molecular weights of the RC protein oomponenta from Rpa. ap~roidee R26. The semi-logarithmic plot of molecular weight wef8Bzu) band position wae established for the four marker proteins shown, and the RC components were located on the line so determined. The apparent molecular weights are RC, = 27 kdaltons, RCb = 22 kdaltons, and RC, = 19 kdaltons. Markers and RC proteins were run on the same 10% gel, aa in Plate I, gel (j).

that the amount of stain is proportional to the mass of protein, the samples boiIed 1 minute or less showed a 1: 1: 1 molecular ratio for RC,, RC$,, and RC,. This corresponds to a minimum weight of 68 kdeltons for the RC prtrticle. These numerical conclusions can be drawn only tentatively, because the estimation of molecular weight by electrophoresis is based on the behavior of hydrophilic proteins of known molecular weight. RC’s are hydrophobic; 65% of their amino acids are non-polar (Feher, 1971), and a detergent is needed to maintain solubility in water. We do not know how these factors affect the mobility of the SDS/protein complex in polyacrylamide. Also we cannot be sure that dye is bound in proportion to the mass of these proteins. Therefore, the foregoing estimates of molecular weights and ratios of the RC proteins are only provisional. (b) Fluorescence of Coomassie Brilliant

Blue

Following a chance observation at sunset, we saw that the dye adsorbed to the proteins in acrylamide gels exhibited a pink fluorescence on some bands and not on others. The effect was most striking when viewed by strong illumination from the side. It is illustrated in Plate II for the RC subunits: Coomassie Brilliant Blue is fluorescent on RC, but not on RC, or RC,. The multiple bands from complex materials such as chromatophores could be classified on the basis of their fluorescence. In several cases this property provided the basis for establishing non-identity of proteins having similar mobilities. The distinctive pattern of fluorescent RC, and non-fluorescent RC, and RC, made this triad easy to recognize even in a complicated band system.

102

R. .K. CLAYTOS

AND

R. 11,1RELKORPI‘

It will be useful, of course, if t,he fluorescenceof this dye proves to be a reliable indicator of some general property, such as hydrophobicity of the protein to which the stain is bound. The structure of CoomassieBrilliant Blue is given by Fazekas de St. Groth, Webster & Datyner (1963). (c) Analyses

of fractions

from Rhodopseudomonasspheroides

Chromatophores and other fractions from Rps. spheroides yielded numerous protein components as shown in Plates III and IV for the blue/green mutant strain R26 and the wild type, respectively. Chromatophores showed the typical RC proteins and also gave dense, non-fluorescent bands near the center and the leading edge of the gel, corresponding to molecular weights 46 and 11 kdaltons. The 46-kdalton component was evident only if the samplehad been boiled 1 minute or longer; boiling appeared to release this protein from larger complexes. The II-kdalton component might be the sameas the “band 15” protein described by Fraker $ Kaplan (1971). In agreement with Fraker & Kaplan’s contention that this protein bearsmost of the light-harvesting pigment, we saw that with wild-type Rps. spheroides the obvious color of carotenoids moved with the 11-kdalton component in the gels. The 46-and 11-kdalton proteins of chromatophores are conspicuousin fractions E, C, LC, LH, and AUT (Plates II and IV). The sample for AUT was made by treating chromatophores with urea and Triton Xl00 at pH 11, as described by Loach et al. (1970). Consistent with Loach’s claim that the AUT treatment converts chromatophores quantitatively into smaller particles, we find the same spectrum of proteins in AUT as in C. The relatively washed-out appearance of the AUT gel probably resulted from the presenceof Triton Xl00 in the sample. Note the high proroportionof the 46- and 11-kdalton proteins and the absence of RC proteins, in fraction LH (Plate III). LH is a pigmented membrane-protein fraction, a byproduct in the purification of RC’s (seeMaterials and Methods). In the membrane-free fraction SU, the sharp band near the leading edge(bottom) can be ascribed to cytochromes of the c type, which can be isolated from this fraction. The characteristic RC proteins are evident in fractions E, C, LC, U, RC, and AUT. The samplesof Plate III had been boiled 2.5 minutes, so RC, and RC, are relatively faint (compare the effect of boiling as shown in Plate I). With prolonged boiling of chromatophores in the presenceof SDS, the disappearanceof RC, and RC, unveiled smaller amounts of other proteins in nearly (but not exactly) the samepositions on the gels. These other proteins seemeddistinct from RC, and RC, becausethey were not attenuated by boiling and CoomassieBrilliant Blue on them was fluorescent. The progressiveenrichment of the RC proteins in fractions C, U, and RC, representing successivestagesin the purification of RC’s, can be seenin Plate III. The foregoing preparations were assayed for protein and BChl, and the ratios of protein components in the gelswere estimated from band areasin densitometer traces. Because RC, was not altered by boiling, the total amount of RC protein was based on the area of the RC, band. The results of these analyses are given in Table 1. In both carotenoidless and wild-type Rps. spheroides,the RC protein accounted for 20 to 30% of the total protein in chromatophores and light chromatophores. This is consistent with the result of a serological experiment performed by W. R. Sistrom (personal communication), which showed that a certain amount of RC antiserum could precipitate either 40 pg of RC’s or approximately 200 pg of chromatophores from Rps. spheroides.

ANALYSIS

OF

PHOTOSYNTHETIC

MEMBRANES

103

TAFJLE 1 BacteriochlorophyU

and reaction center protein infractions

prepared from

Rhoaopf3eua0m0n8s spheroiaes mg BChl/lOO protein

Preptwration Cerotenoidless mutant Chromatophores Light chromatophores LDAO-upper phase RC preparetion

Rpa.

Wild-type Rpa. ~p?mr&des: Chrometophores Light chromatophores LDAO-upper phase

spheroides

mg

RC protein (% of total protein)

mg BChl/lOO RC protein

mg

: 3.4 3.9 0.8 2.0

26 30 40 80

13 13 2.0 2.5

4.5 5.6 6.7

23 21 28

20 27 24

LDAO-upper

phase (fraction U) contained relatively more RC protein than did This fraction from wild-type Rps. spheroides also contained an abundance of the 11-kdalton component, but was relatively free of proteins of higher molecular weight. In carotenoidless mutant Rps. spheroides we note a dramatic decrease in the ratio of BChl to RC protein (last column in Table 1) when passing from chromatophores to LDAO-upper phase and to RC’s. This reflects the separation of light-harvesting pigments from the RC’s; the BChl in LDAO-upper phase and in RC’s w&s almost exclusively PSOO and P870. In wild-type Rps. spheroides, however, the ratio of BChl to RC protein remained fairly constant in the different fractions and the ratio of BChl to total protein was greater in LDAO-upper phase than in chromatophores. The absorption spectrum of LDAO-upper phase from wild-type cells was like that of light chromatophores; it reflected the preponderance of light-harvesting BChl. The LDAO-upper phase from wild-type Rps. spheroides wits therefore enriched for RC protein, for the 11-kdalton component, and for light-harvesting BChl relative to total chromatophore protein. It follows that the light-harvesting pigment is bound preferentially to RC protein, or to the Il-kdalton component, or to both?. The fractions from wild-type Rps. spheroides contained P870 in roughly the amount expected from the content of RC protein; they presumably contained the other RC chromophores as well. chromatophores.

(d) Non-photosynthetic mutant Rhodopseudomonas spheroides PM8 The non-photosynthetic mutant strain PM8 of Rps. spheroides was grown aerobically in darkness, with wild type grown in the same way as a control. Gels made with preparations from these cultures are shown in Plate V. We could not &d any RC protein in any fraction from PMS, whereas the characteristic RC components were conspicuous in preparations from the aerobically grown wild-type cells. The absence of RC protein in PM8 was confirmed by serological tests: extracts of PM8 did not contain any material that was precipitated by antibody specific toward RC’s. Also the PM8 extracts, when added to antisera, did not lessen the potency of these sera in precipitating RC’s. The lack of photosynthetic activity in this mutant can thus be linked to a failure to make RC protein, -f See Note

added

in proof.

104

R. K. CLAYTON

AND

R. HASELKORN

The distribution of proteins in aerobically grown wild-type Rps. spheroidesundoubtedly differed in many small ways from that in cells grown photosynthetically; differences can be gleaned by studying the appropriate gels in Plates IV and V. We are not yet in a position to evaluate such differences. (e) Other photosynthetic bacteria We prepared sonic extracts, chromatophores and LDAO-upper phase fractions from R. rubrum and from Rps. capsuluta, palustris, gelatinosaand viridis. The preparations from R. rubrum and Rps. capsulata and palustris showed patterns suggesting the presence of RC proteins: a strongly fluorescent band above two strikingly nonfluorescent ones, in the neighborhood of 15 to 30 kdaltons. Such patterns were not evident in the preparations from Rps. gelatinosa and viridis. All of thesephotosynthetic bacteria showed a major chromatophore component of apparent molecular weight between 10 and 15 kdaltons, and the color of light-harvesting pigments moved in the corresponding region of the gels. Finally, none of the materials from speciesother than Rps. spheroidesshowed any precipitin cross-reaction with antibody specific toward RC’s from Rps. spheroides. 4. Discussion Neither the light-harvesting BChl protein fraction from carotenoidlessmutant Rps. spheroidesR26 nor the pigmented material from the non-photosynthetic mutant PM8 contained the proteins characteristic of RC’s. This showsthat the light-harvesting BChl can be bound to other proteins in the photosynthetic tissue, probably to the 11-kdalton protein. RC’s can be isolated from chromatophores of carotenoidless mutant Rps. spheroidesby a combination of exposure to lauryl dimethyl amine oxide, centrifugation, and fractionation with ammonium sulfate. This method fails when applied to cbromatophores of the wild type; the RC’s are accompanied persistently by the light-harvesting pigments and the 11-kdalton protein. Non-photosynthetic mutant Rps. spheroidesPM8 appears to lack all three components, RC,, RC,, and RC,. Furthermore, we could find no material in PM8 that shares antigenic determinants with RC’s. When PM8 was originally isolated by Sistrom it reverted to wild-type photosynthetic activity with a frequency of about 10e7 (Sistrom, 1966), suggestingthat the original PM8 was a point mutation. After culture for many years, the strain PM8 used in this work appears not to revert. This stability could be due to the accretion of more point mutations, or to a deletion. Against this background, the absenceof the three RC proteins can be explained in several ways consistent with current models of molecular genetics. Lacking an experimental genetic system for Rps. spheroides,we are not yet in a position to make such speculations fruitful. This work was supported by U.S. Atomic Energy Commission contract no. AT(30-l)3759 awarded to one of us (R. K. C.) and by a National Science Foundation grant GB17514 to the other author (R. H.). The latter is grateful to A. G. Szent-GyBrgyi for the invitation to Woods Hole that made this collaboration possible. REFERENCES Bolton, J. R., Clayton, R. K. & Reed, D. W. (1969). Photo&em. Photobiol. 9, 209. Clayton, R. K. (1966). Photo&em. Photobiol. 5, 669. Clayton, R. K. (19710). Light and Living Matter, vol. 2, chapter 1. New York : McGraw

Hill,

ANALYSIS

OF PHOTOSYNTHETIC

MEMBRANES

105

Clayton, R. K. (19715). Adwanc. Chem. Phys. 19, 353. Clayton, R. K. & Smith, C. (1960). Biochewa. Biophys. Re.9. Comm. 3, 143. Clayton, R. K. & Wang, R. T. (1971). Methde in Enzymol. 23, 696. Cohen-Bazire, G., Sistrom, W. R. & Starrier, R. Y. (1957). J. Cell. Camp. Physiol. 49, 25. Eimhjellen, K. E., Aasmundrud, 0. & Jensen, A. (1963). Biochem. Biophys. Res. Comm. 10, 232. Fazekas de St. Groth, S., Webster, R. G. & Datyner, -4. (1963). Biochim. biophys. Acta, 71, 377. Feher, G. (1971). Photo&m. Photobiol. 14, 373. Feher, G., Okamura, M. Y., Raymond, J. A. & Steiner, L. A. (1971). Biophys. J. 11, 38a. Fraker, P. J. & Kaplan, S. (1971). J. Boct. 108, 465. Laemmli, U. K. (1970). Nature, 227, 680. Loach, P. A., Sekura, D. L., Hadsell, R. M. & Sterner, A. (1970). Biochemistry, 9, 724. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265. Ouchterlony, 0. (1949). Acta Path. MicrobioE. Scund. 26, 507. Sistrom, W. R. (1966). Photochem. PhotobioZ. 5, 845. Sistrom, W. R. & Clayton, R. K. (1964). Biochim. biophys. Acta, 88, 61. Worden, P. B. 8r Sistrom, W. R. (1964). J. Cell BioZ. 23, 135.

Note added in proof: One of us (R. K. C.) has now found that reaction centers, free of light harvesting bacteriochlorophyll, can be precipitated from suspensions of detergenttreated chromatophores of wild-type Rps. spheroides by adding anti-reaction center serum. Furthermore the 46 kdalton protein is found mainly in a relatively non-pigmented fraction associated with cell-wall fragments, whereas the 11 kdalton protein is found mainly in pigmented fractions from Rps. epheroides.