THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 252, No. 3, Issue of February IO, pp 943-949, 1977

Prmted in U.S.A.

Different Molecular Forms of D-Ribulose-I$-bisphosphate Carboxylase from Rhodopseudomonas sphaeroides”

(Received for publication, June 25, 1976, and in revised form, October 13, 1976)

JANET L. GIBSON AND F. ROBERT TABITA

From the Department of Microbiology, The University of Texas at Austin, Austin, Texas 78712

Ribulose-1,5-bisphosphate (Rbu-P,) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.l.Ga was sepa- rated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carbox- ylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electro- phoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P, carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight, of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme in- hibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I en- zyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensi- tive to 6-phosphogluconate.

Primary carbon dioxide fixation in photosynthetic and chemosynthetic cells is catalyzed by ribulose-1,Sbisphosphate carboxylase (3-phospho-n-glycerate carboxylase (dimerizing) EC 4.1.1.39). The discovery of this important catalyst was a direct result of the work of Calvin and colleagues who showed that the initial product of YJO, fixation by algae was 3- phosphoglyceric acid, ribulose l$bisphosphate serving as the acceptor molecule for carbon dioxide (1). In recent years, con- siderable interest has developed concerning the structure, reg- ulation, and molecular evolution of this important protein (2, 3). In all cases, the catalytic site is localized on an M, = 55,000 polypeptide (3), and it has been found that multiple copies of this subunit comprise the enzyme from Rhodospirillum rub- rum (M, = 114,000) (4), Chlorobium (M, = 350,000) (5), and several blue-green algae (M, = 450,000) (6, 7). Moreover, in many cases, a second, smaller polypeptide is found associated with the catalytic subunit. This small subunit has a molecular weight ranging from 12,000 to 20,000, depending on the source of enzyme (2, 3). The function of the small subunit in catalysis

* This investigation was supported by National Science Founda- tion Grant BMS-7410297 to F. R. T. and by a Biomedical Sciences Support Grant given to the University of Texas at Austin by the National Institutes of Health.

is uncertain; however, there are indications that this small protein might serve in some regulatory capacity (8). Certainly, recent evidence suggests that the small subunit may be a positive factor needed for the synthesis of the large, catalytic subunit (9). The small subunit has been found associated with large molecular weight carboxylases (A4, = 550,000) (3); pro- teins found in higher plants, eukaryotic algae, at least two blue-green algae (10) and several bacteria (2, 3). Presumably, eight of the large and eight of the small subunits comprise the native A4, = 550,000 protein (11).

In previous investigations, it was reported that the Rbu-P,’ carboxylase from R. rubrum could be induced over 1Bfold upon growth on reduced substrates such as butyric acid (12). Under these conditions of growth, Rbu-P, carboxylase was found to comprise from 6 to 8% of the total soluble protein (12, 13). Rhodopseudomonas sphaeroides is a related member of the Rhodospirillaceae, however, estimates of the molecular weight of the Rbu-P, carboxylase from the rhodopseudomon- ads range from 240,000 to 360,000 (14, 151, placing the carbox- ylase from Rhodopseudomonas in the intermediate size range for this protein (2, 3). In view of our ability to induce the synthesis of Rbu-P, carboxylase in R. rubrum, we sought, in this study, to ascertain whether similar induction phenomena might be operable in Rhodopseudomonas. The ability to ob- tain large masses of cells, enriched in the level of Rbu-P, carboxylase, would then make it feasible to study for the first time the molecular properties of the intermediate size enzyme from Rhodopseudomonas. This investigation reports on the purification of two apparently distinct Rbu-P, carboxylases from butyrate-induced Rps. sphaeroides.

EXPERIMENTAL PROCEDURES

Materials -The following special reagents were commercial prep- arations: tetrasodium ribulose 1,5-bisphosphate was purchased from the Sigma Chemical Co. or was prepared according to the procedure of Horecker et al. (16); ditbiothreitol, phenylmethylsulfonyl fluoride, triethanolamine, andN-tris(hydroxymethyl)methyl-2-aminoethane- sulfonic acid were all from Sigma; DEAE-cellulose was from Bio-Rad laboratories; Ultrogel AcA-22 is a product of LKB, Ine; Na,WO, (20 mCi/mmol) was from AmershamlSearle. Acrylamide (Eastman) was recrystallized from chloroform. Spinach Rbu-P, carboxylase was pur- ified according to the procedures of Paulsen and Lane (17).

Culture and Growth Conditions -A culture ofRhodopseudomonas sphaeroides 2.4.1 was kindly provided by Dr. Sam Kaplan, Depart- ment of Microbiology, The University of Illinois, Urbana, Illinois. For these studies, a spontaneous green mutant, strain 2.4.1.Ga, was used and gave the same results as the wild type. Cultures of Rps.

‘The abbreviations used are: Rbu-P,, tetrasodium ribulose 1,5- bisphosphate; Tes, N-tris(hydroxymethyl)methyl-2-aminoethane- sulfonic acid.

943

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944 R bu-P, Carboxylase in Rps. sphaeroides

sphaeroides were maintained in the synthetic malate medium of Ormerod et al. (18) as agar stabs with 0.2% ammonium sulfate as nitrogen source, supplemented with 0.1% sodium bicarbonate. Inoc- ula for large scale growth were grown in 600-ml reagent bottles of the malate media. These precultures served to inoculate 3-liter low form flasks containing 0.6% butyric acid instead of malate as the electron donor for growth. Cultures were illuminated by banks of loo-watt incandescent bulbs, above and below the culture flasks, at an inten- sity of 500 footcandles. Temperature was maintained at 32-34”. The growth conditions and apparatus were essentially as described by Stevens and Fox (19). After harvesting the cells, the cell paste was weighed, washed with 0.025 M Tris/Cl, pH 7.5, containing 1 mM EDTA, and stored at -20” until needed.

Purification of Ribulose-1 ,Sbisphosphate Carboxylase -Frozen (butyrate-grown) cell paste, 125 g, were suspended in 0.025 M Tris/Cl buffer containing 1 rnM EDTA and 5 mM 2-mercaptoethanol, pH 7.5 (Buffer A). A 1:l mass ratio of cell paste to buffer was used. The cell suspension was sonicated in 40-ml fractions for 5 min each or was passed through a French Pressure cell at 15,000 psi. Unbroken cells and debris were removed by centrifugation at 16,300 x g for 15 min and the supernatant decanted. The pellet was washed with Buffer A, centrifuged as before and the supernatants combined. The pooled supernatants were then subjected to ultracentrifugation at 105,000 x g for 60 min in a Beckman model L ultracentrifuge using a type 30 rotor to remove much of the chromatophore material.

To the high speed supernatant, 1 M MgCl, .6H,O was added to a final concentration of 50 mM. The extract was divided into lo-ml fractions and immersed in a 50” water bath for 10 min after which the tubes were immediately placed in an ice bath (12). After cooling, the heat-treated extract was centrifuged to remove denatured protein. The supernatant was then treated with streptomycin sulfate (0.75 mglmg of protein) by adding dropwise a 10% (w/v) solution with continuous stirring. The turbid suspension was allowed to stand in an ice bath for 15 min, at which time it was centrifuged and the supernatant decanted and dialyzed overnight against 10 liters of Buffer A.

The next morning the dialyzed extract was centrifuged to remove small amounts of denatured protein and then loaded onto a DEAE- cellulose column (4.2 x 41 cm) equilibrated with Buffer A. The flow rate was maintained at 60 ml/h and 20-ml fractions were collected. One liter of Buffer A was passed through the column to ensure that Rbu-P, carboxylase was adsorbed onto the cellulose, followed by 1 liter of 0.1 M NaCl in Buffer A. Protein eluted from these washes contained no carboxylase activity. A linear gradient of 0.1 M NaCl, 0.3 M NaCl in Buffer A was then passed through the column, result- ing in the elution of two distinct peaks of enzymatic activity, sepa- rated by a volume of approximately 100 ml. Active fractions from both peaks were pooled separately and concentrated by the addition of solid ammonium sulfate to 60% saturation. After standing for 30 min in an ice bath, the suspensions were centrifuged and the pellets redissolved in a small amount of Buffer A and dialyzed against the same buffer overnight to remove ammonium ion. The dialysates were then centrifuged at 27,000 x g for 15 min to remove any denatured protein. In some cases, active column fractions were concentrated by means of ultrafiltration.

The dialyzed ammonium sulfate precipitates of both Peak I and Peak II Rbu-P, carboxylase (in Z-ml aliquots) were each separately loaded onto a Ultrogel AcA-22 column (2.6 x 100 cm), equilibrated with Buffer A, and run against gravity. Flow rates were maintained at 5 ml/h at a pressure head of 15 cm, and 4-ml fractions were collected. Homogeneous Peak I Rbu-P, carboxylase was readily ob- tained by this method; however Peak II carboxylase was not pre- pared to a homogeneous state by this procedure. Subsequently, it was found that preparative gel electrophoresis could be utilized to conveniently obtain homogeneous Peak I and Peak II Rbu-P, carbox- ylase. Preparative gel electrophoresis was performed in gels (2.5 x 20 cm), using either the Tris/glycine system, with a running pH of 9.5, described by Davis (20), or the triethanolamine/Tes system described by Orr et al. (211, having a running pH of about 7.0. An apparatus was constructed to accommodate four such gels. Samples containing 5% glycerol and 20 to 30 mg of protein were applied to the gels and electrophoresed at a constant current of 15 mA/gel at 4” until the tracking dye (0.001% bromphenol blue) migrated to the bottom of the gel tube. Enzymatic activity was localized in these large gels by slicing a thin longitudinal portion of each gel followed by rapid protein staining and destaining. After washing with water, each longitudinal slice was then replaced in the gel and wherever staining indicated the presence of protein, a horizontal wedge was

cut from the rest of the gel and placed into a vessel containing 25 rnM TrisKl, 1 rnM EDTA buffer (pH 7.5 at 25”). The acrylamide wedge was then macerated in this buffer to elute the protein. The elution process was repeated twice with fresh buffer to elute virtually all the protein in the acrylamide wedge. Excess acrylamide in the eluate was removed by centrifugation and dialysis. Assays were performed on each of these eluates as described.

Preparation ofAntisera to Rps. sphaeroides Peak I Rbu-P, Carbox- ylase-Electrophoretically pure Rbu-P, carboxylase in 0.01 M potas- sium phosphate, pH. 7.5, purified from the first DEAE-cellulose activity peak, was mixed with an equal amount of Freund’s complete adjuvant. This mixture, containing 100 pg of enzyme was injected subcutaneously into the shoulder of a female white New Zealand rabbit. Ten days later, the same amount of enzyme/adjuvant mix- ture was injected. Two weeks after the second injection the rabbit was bled from the ear, and the blood was allowed to clot for several hours at room temperature. The clot was removed by centrifugation and the supernatant was treated by the dropwise addition of 25% sodium sulfate to a final concentration of 18% saturation. The cloudy suspension was centrifuged and the pellet was dissolved in physio- logical saline to about one-fifth the original volume and dialyzed overnight against 0.02 M Tris/Cl, pH 7.5. This dialyzed antisera was stored frozen at -20”. Control sera were obtained prior to injection of antigen and treated similarly.

lmmunodiffusion -1mmunodiffusion was carried out in 1% aga- rose gels dissolved in 50 mM Tris/Cl (pH 7.4) as previously described (12).

Electrophoresis - Polyacrylamide disc-gel electrophoresis was used throughout the purification procedure to monitor homogeneity. Samples were loaded onto 7.5 or 6.0% acrylamide gels and electro- phoresed at pH 9.5 using the Buchler anionic gel system (121, as originally described by Davis (20) or at pH 7.0 using the triethanola- mine/Tes system (211. A current of 1.25 mA/gel was used until the tracking dye (0.001% bromphenol blue) could be seen entering the gels, at which time the current was increased to 2.5 mA/gel and maintained until the tracking dye was approximately 1 cm from the end of the gels. The gels were stained in 1% Amido Schwarz in 7.5% acetic acid for 30 min and then destained in 7.5% acetic acid in a diffusion destainer. Sodium dodecyl sulfate gel electrophoresis was carried out according to the procedure of Weber et al. (22) using 10% gels. Standards used included bovine serum albumin, catalase, lyso- zyme, ovalbumin (monomer and dimer), lactic dehydrogenase, and cytochrome c, plus spinach leaf and Rhodospirillum rubrum Rbu-P, carboxylase. The gels were run at 9 mA/gel for approximately 5 h until the tracking dye was 1 to 2 cm from the end of the gel. The gels were measured and then stained in Coomassie blue (4) for approxi- mately 2 h and then destained according to the procedure of Fair- banks et al. (23).

Enzyme Assay-The assay for Rbu-P, carboxylase was as previ- ously described (4, 7, 13). For the pH studies, assays were performed using the standard procedure except that the incubation mixtures contained the following buffering species: imidazole at pH 6.6, 6.8, 7.0,7.2, 7.4, andTrisatpH7.0, 7.2,7.4, 7.6,7.8,8.0,8.2, and8.6. All adjustments in pH were made with HCl.

Protein was estimated according to the procedure of Lowry et al. (24).

Polyacrylamide Gel Assay for Rbu-P, Carbonylase - In order to locate enzymatic activity on polyacrylamide gels, an in situ poly- acrylamide gel assay for Rbu-P, carboxylase activity was developed. For these experiments, the sample to be analyzed was electropho- resed on two replicate gels, one of which was stained for protein and then destained in the usual manner. The duplicate gel was sliced into 2-mm slices, each of which was placed into a test tube containing the constituents of the Rbu-P, carboxylase assay in a total volume of 0.2 ml. Rbu-P,, 50 ~1, was then added and the assay was allowed to proceed for 30 to 90 min in order to detect slight amounts of activity. At the appropriate time, the assay was terminated by the addition of 100 ~1 of propionic acid and acid-stable radioactivity was determined by liquid scintillation spectrometry using a ZOO-p.1 aliquot of the terminated reaction mixture.

RESULTS

Purification of Rbu-P, Carboxylase -The key to obtaining homogeneous Rhodopseudomonas sphaeroides Rbu-P, carbox- ylase is to obtain cells derepressed for the synthesis of this protein. It was found that photoheterotrophic growth with

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Rbu-P, Carboxylase in Rps. sphaeroides 945

butyric acid as the electron donor resulted in a significant expression of Rbu-P, carboxylase activity, with over a 1Zfold elevation in specific activity when compared to growth with malate, the usual growth substrate for the Rhodospirillaceae. Moreover, under these conditions of growth, 5 to 6 g (wet weight) of derepressed cells/liter could be obtained, providing a ready source of enzyme. Significant amounts of protein and extraneous pigment could be removed from the crude high speed supernatant fraction by the magnesium-heat treatment (4, 15). Surprisingly chromatography on DEAE-cellulose re- sulted in the elution of two distinct peaks of Rbu-P, carboxyl- ase activity, well separated from the bulk soluble protein and pigments present in the crude extracts (Fig. 1). Two activity peaks were obtained when extracts were obtained from cells harvested throughout the growth phase in the presence or absence of phenylmethylsulfonyl fluoride, a protease inhibi- tor, and with extracts that had not been treated prior to DEAE-cellulose chromatography. Thus, enzyme from each peak was pooled separately and concentrated. Enzyme from the first activity peak emerging after DEAE-cellulose chroma- tography is hereby referred to as Peak I Rbu-P, carboxylase; Peak II Rbu-P, carboxylase refers to the second peak of activ- ity.

Samples of the purified Peak I and Peak II carboxylases, obtained from the DEAE-cellulose column, were analyzed by electrophoresis and subsequent assays in polyacrylamide gel slices. When electrophoresed at pH 9.5, the Peak I preparation showed one major active species, with a slight indication of activity toward the top of the gel (Fig. 2). Conversely, Peak II carboxylase was resolved into five distinct active species upon electrophoresis at this pH (Fig. 2). Moreover, Rhodospirillum rubrum Rbu-P, carboxylase, which has a molecular weight of 114,000 (12) co-migrates with the first active species of Peak II Rps. sphaeroides Rbu-P, carboxylase. Thus, we estimate that the 5 active Peak II carboxylase species have molecular weights of 114,000, 240,000,360,000, 500,000, and 700,000. That is, each active species of Peak II appears to be a multiple of the smallest active M, = 114,000 species. A sample of the crude extract was also electrophoresed and assayed in gel slices (Fig. 2), showing the same basic pattern of activity obtained for Peak I and Peak II. With regard to the crude extract, it is

FIG. 1. DEAE-cellulose chromatography of Rbu-P, carboxylase from Rhodopseudomonas sphaeroides. (O), absorbance at 280 nm. Rbu-P, carboxylase activity (A) is expressed as disintegrations/min of [‘Vlbicarbonate (‘VO,) fixed during a 5-min assay/ZO-pl aliquot from each fraction of 20 ml in the standard assay. TEM, Buffer A.

noteworthy that significant carboxylase activity is associated with the top of the gel, perhaps indicating the formation of higher states of aggregation or enzyme that is associated with residual chromatophore membrane material (Fig. 2).

By contrast to the results obtained at pH 9.5 (Fig. 2), when the same preparations of crude extract Peak I and Peak II carboxylases were electrophoresed at pH 7.0 and assayed by the gel slice method, an entirely different pattern was ob- tained (Fig. 3). The crude extract showed two major activity peaks with only a slight indication of large molecular weight active aggregates (Fig. 3). Peak I exhibited one major active

Since Number Slice Number

Peak II i

20 30 40

FIG. 2 (left). Polyacrylamide gel electrophoresis at pH 9.5. Sam- ples were applied in duplicate to 6.0% aerylamide gels in a 20-p] volume containing 5% sucrose. Top, high speed (100,000 x g) super- natant fraction (75 pg of protein); middle, Peak I DEAE-cellulose 60% ammonium sulfate precipitate fraction (55 pg of protein); bot- ton, Peak II DEAE-cellulose 60% ammonium sulfate precipitate fraction (45 pg of protein). Following electrophoresis in the Tris/ glycine system, one gel was sliced into 2-mm slices and assayed for Rbu-P, (R&V’) carboxylase activity as described in the text. Assay mixtures contained 20 rnM MgZ+, 20 rnM HY!O,-, 0.8 rnM Rbu-P,, 10 rnM dithiothreitol, in 64 mM Tris/Cl (pH 7.2) in a final reaction volume of 200 ~1. The reaction was incubated at 30” for 90 min and then terminated by addition of 100 ~1 of propionic acid. Acid stable radioactivity was determined with a 200-pl aliquot.

FIG. 3 (right). Polyacrylamide gel electrophoresis at pH 7.0. Top, high speed supernatant fraction (75 pg of protein); middle, Peak I DEAE-cellulose 60% ammonium sulfate precipitate fraction (55 Kg of protein); bottom, Peak II DEAE-cellulose 60% ammonium sulfate precipitate fraction (45 pg of protein). All conditions are the same as those described in Fig. 2, except that gels were prepared and electro- phoresed at pH 7.0 as described in the text. Gels were polymerized using 6.0% acrylamide. R&P, ributose 1,5bisphosphate.

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946 Rbu-P, Carboxylase in Rps. sphaeroides

species (Fig. 3) similar to the results obtained after electropho- resis of pH 9.5 (Fig. 2). Peak II, however, after electrophoresis at pH 7.0, was resolved into a single major active species, corresponding to the second major activity peak obtained with the crude extract (Fig. 3). It is likely that the minor peak of activity found associated with Peak II represents contaminat- ing Peak I enzyme.

The same Peak I and Peak II carboxylase preparations used in the electrophoresis experiments were chromatographed on an Ultrogel AcA-22 column at pH 7.5. Clearly, only one major active species was found associated with each preparation at pH 7.5 (Fig. 4). Moreover, as a result of gel filtration, Peak I carboxylase was purified to homogeneity; Peak II carboxylase was substantially purified, but not to a homogeneous state. From the elution volume of each enzyme and subsequent comparison to the elution volume of several standards of known molecular weight, it was found that Peak I Rbu-P, carboxylase co-elutes with spinach Rbu-Pp carboxylase and thus has a molecular weight of approximately 550,000. Peak II Rbu-PZ carboxylase has an apparent molecular weight of 360,000 by this criterion.

It was subsequently found that good yields of homogeneous Peak I and Peak II Rbu-P, carboxylase could be obtained after preparative polyacrylamide gel electrophoresis, at pH 7.0, of the concentrated pooled DEAE-cellulose eluates. Electropho- retograms of homogeneous Peak I and Peak II carboxylase are shown in Fig. 5, along with the corresponding activity profiles. The homogeneous Peak I enzyme co-migrates with purified spinach carboxylase, again indicative that the Peak I enzyme has a molecular weight of about 550,000. In each case, one major peak of activity is associated with a single stained protein (Fig. 51, with some slight minor activity for the puri- fied Peak II enzyme further down the gel, indicating that our assay is more sensitive than the usual protein staining proce- dures. The purified Peak II enzyme readily dissociates into at least four active species at pH 9.5, giving a rather diffuse staining pattern when contrasted to electrophoresis at pH 7.0 (Fig. 6). Occasionally, the presence of a very high molecular weight active species is seen after electrophoresis at pH 9.5 when large amounts of purified Peak II enzyme are electropho- resed.

43

40 16.0 % e

f -

I 2.0 3.0 12.0

OS 20

0.6 1.5

0.4 I.0

0.2 0.5

20 40 60 SO 100 120 140

Ve (ml) FIG. 4. Gel filtration of Peak I and Peak II Rhodopseudomonas

sphaeroides Rbu-P, carboxylase. Concentrated Peak I and Peak II Rbu-P, carboxylase (2 ml) were chromatographed on a column of Ultrogel equilibrated with Buffer A at pH 7.5 and eluted against gravity. Ve is the elution volume.

Table I summarizes the purification of Peak I and Peak II Rbu-P, carboxylase from Rps. sphaeroides. Both carboxylases are highly stable for at least 3 months when stored sterile at 2 at about 2 to 3 mglml in Buffer A. The enzymes obtained were of high specific activity and represent major proteins of the total soluble protein of extracts from derepressed Rps. sphae- roides. Indeed it can be calculated that, together, Peak I and Peak II Rbu-P, carboxylase represent 12 to 16% of the total soluble protein.

Subunit Structure - Electrophoretic studies of purified Peak I and Peak II carboxylase in gels polymerized from 4.0 to 7.5% acrylamide concentration using the techniques of Hedrick and Smith (25) indicated that the two carboxylases were neither size nor charge isomeric proteins. Thus, we sought to deter- mine the quaternary structure of each protein. After denatur- ation of the proteins in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (22), the carboxylases were electropho- resed in 10% sodium dodecyl sulfate gels (Fig. 7). Both en- zymes contained one large polypeptide that migrated similar to the large subunit of spinach Rbu-P, carboxylase. However, Peak I carboxylase was also composed of a small polypeptide (Fig. 7). No evidence for the presence of a small subunit was obtained for Peak II Rbu-P, carboxylase (Fig. 7). The molecu- lar weight for the large subunit of Peak I carboxylase was found to be about 52,000 (Fig. 8); the small subunit of Peak I carboxylase exhibits a molecular weight of 11,000. Moreover, both subunits of Peak I carboxylase co-electrophoresed with the two subunits from spinach Rbu-P, carboxylase (Fig. 8). The polypeptide obtained from dissociation of Peak II carbox-

Slice Number

FIG. 5. Polyacrylamide gel electrophoresis of homogeneous Peak I (29 pg) and Peak II (26 pg) Rhodopseudomonas sphaeroides Rbu-P, (RuBP) carboxylase at pH 7.0. Conditions of electrophoresis, stain- ing and assay are as described in the text and in the legend to Fig. 2. Gels were polymerized using 7.5% acrylamide.

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Rbu-P, Carboxylase in Rps. sphaeroides 947

4” z 0 2.0-

r, ::

0 1.5 - L l.5 1

k-k l.O-

G Dye 0.5- Front

+ I * I I, -La..&.,

IO 20 30 40

TABLE I

Isolation summary of ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides

Fraction Total Total BC- A.,: protein tivity A*60

say:: Yield ity”

mg units unit.71 %

mg 100,000 x g superna- 4845 1788 0.79 0.369 100

tant Mg*+-heat supernatant 2280 1631 0.70 0.716 91 Streptomycin superna- 2065 1524 0.96 0.738 85

tant Peak I DEAE-cellulose 178 306 1.28 1.72 17

eluates (pooled) Peak II DEAE-cellulose 139 207 1.49 1.49 12

eluatos (pooled) Preparative gel electro- 14.5 43 1.60 2.98 2.1

phoresis, Peak I Preparative gel electro- 14.5 33.3 1.70 2.30 2.0

phoresis, Peak II

a Prior to DEAE-cellulose chromatography, assays were per- formed at pH 7.2. Subsequent to DEAE-cellulose chromatography, Peak I carboxylase was assayed at pH 8.0 and Peak II carboxylase at pH 7.2. All assay tubes contained 10 mM dithiothreitol.

Slice Number

FIG. 6. Polyacrylamide gel electrophoresis of homogeneous Peak II Rhodopseudomonas sphaeroides Rbu-PZ (RUM’) carboxylase at pH 7.0 and pH 9.5. Homogeneous Peak II carboxylase (25 pgl was analyzed on polyacrylamide gels using the triethanolamine/Tes (pH 7.01 electrophoresis system (upper) and Trislglycine system at pH 9.5 (lower). Conditions of electrophoresis, staining, and assay are as described in the text and in the legend to Fig. 2. Gels were polymer- ized using 6.0% acrylamide.

ylase was shown, in similar experiments, to have a molecular weight ranging from 52,000 to 55,000.

Immunological Studies -By use of the double diffusion Ouchterlony technique, one precipitin band was obtained against antisera directed against the purified enzyme of Peak I when either purified Peak I carboxylase or crude extracts were used in the precipitin reaction. Interestingly, however, no cross-reactivity towards the anti-Peak I carboxylase was ob- served with preparations of enzyme from Peak II in these experiments. Significantly, antibodies to Peak I carboxylase inhibited the Peak I enzyme but had no effect on Peak II carboxylase activity (Fig. 9). These data further distinguish between the two carboxylases obtained from Rps. sphaeroides.

Catalytic Properties -To further delineate the properties of Peak I and Peak II Rbu-P, carboxylases, the effect of pH on catalysis was determined. Peak I carboxylase exhibits optimal activity from pH 7.8 to 8.0, while the Peak II enzyme has a fairly sharp optimum at pH 7.2 (Fig. 10). Furthermore, the Peak I enzyme is markedly sensitive to low concentrations of 6-phosphogluconic acid, while the Peak II enzyme is relatively insensitive to this ligand (Table II). Results were comparable at pH 7.2 and 8.0, with the Peak II enzyme showing slight inhibition (8 to 13%) at 1.2 mM phosphogluconate (Table II).

DISCUSSION

In this investigation, we have found that Rbu-Pz carboxyl- ase from Rps. sphaeroides is induced upon growth on butyric acid. This finding is consistent with prior results obtained

0 Rps. sphaeroides

0.2 0.4 0.6 0.8

%l

FIG. 7 (left). Sodium dodecyl sulfate polyacrylamide gel electro- phoretogram of Peak I (right) and Peak II (left) Rbu-P, carboxylase from Rhodopseudomonas sphaeroides. Aliquots of each enzyme were treated and subjected to electrophoresis as described in the text. The tracking dye position was near the bottom of the gel.

FIG. 8 (right). Estimation of molecular weight of sodium dodecyl sulfate-dissociated Rhodopseudomonas sphaeroides Rbu-P, carbox- ylase. Experimental details are described in the text. The molecular weight of the subunits of the Rps. sphaeroides enzyme was deter- mined with a calibration curve obtained from the following standard proteins of known molecular weight: 1, cytochrome c; 2, y-globulin; 3, lactate dehydrogenase; 4, ovalbumin monomer; 5, catalase; 6, ovalbumin dimer.

with the physiologically related organism R. rubrum (4). With the ability to obtain large masses of bacteria rich in Rbu-PI carboxylase, it became feasible to develop a procedure for purifying this enzyme from Rhodopseudomonas. Discrepant reports are found in the literature with regard to the molecu- lar weight of the rhodopseudomonad enzyme (14,151, however, these investigations were performed with preparations of un-

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948 Rbu-P, Carboxylase in Rps. sphaeroides

I 0 1 I , 100 M

6o- Peak II 60 -

40 -

20 - I I 1 I I IO 20 30 40 60

Antisera bl)

FIG. 9. Antibody titration of Peak I and Peak II Rhodopseudomo- nas sphaerordes Rbu-P, carboxylase activity. Aliquots of partially purified antisera to Peak I Rbu-P, carboxylase (0) and control sera (0) were incubated with homogeneous Peak I and Peak II Rbu-P, carboxylase for 30 min at 30’, at pH 7.2, and sybseqwmtly assayed as described in the text.

I r I6 0 Peai3 I

4.0 -

L 1 I I A 6.6 7.0 7.4 7.8 6.2 6.6

PH

FIG. 10. Effect of pH on the activity of Rbu-P, carboxylase from Rhodopseudomonas sphaeroides. The standard reaction mixture containing Tris/Cl or imidazole/Cl at the desired pH was used. The Iv@*+ concentration in this experiment was 4 mM.

TABLE II

Effect of 6-phosphogluconate on Peak I and Peak II Rbu-P, carboxylase from Rhodopseudomonas sphaeroides

Enzymes were premcubated for 5 min at 30” in the presence of Mg2+, H1’CO,-, and 6-phosphogluconate in 0.064 M Tris/Cl prior to initiation of the reaction by addition of Rbu-Ps. The final concentra- tion of each assay component (in millimolarityl was as follows: Me;*+ (101, H”CO,- (201, Rbu-P, (0.8). The reaction was terminated atier 5 min by addition of 0.1 ml of propionic acid. Purified Peak I enzyme (1.6 ng) having a specific activity of 2.9, and 1.4 ng of purified Peak II enzyme, having a specific activity of 2.3, were used.

Per cent Activity 6.Phosphogluco-

nate pH 1.2 pH 8.0

Peak I Peak II Peak I Peak II

ITLM

0.0 100 100 100 100 0.2 68 97 66 100 0.4 58 95 57 108 0.6 49 94 54 101 0.8 45 90 50 97 1.2 35 87 36 92

known purity (14, 15). During the purification of Rbu-P, car- boxylase, we found that two peaks of carboxylase activity were eluted from columns of DEAE-cellulose. Both activities were eluted shortly after the salt gradient was applied to the col- umn. Subsequent purification of each activity peak resulted in homogeneous Peak I and Peak II Rps. sphaeroides Rbu-P, carboxylase. The Peak I carboxylase has an approximate mo- lecular weight of 550,000, based on its co-elution during molec- ular sieve chromatography with spinach leaf Rbu-Pt carboxyl- ase and the co-migration of the Peak I enzyme with the spinach carboxylase on polyacrylamide gels. Furthermore, dissociation and subsequent sodium dodecyl sulfate polyacryl- amide gel electrophoresis of the Peak I enzyme revealed two dissimilar polypeptides: a large subunit of 52,000 daltons and a small subunit of 11,000 daltons. Each of the Peak I Rps. sphaeroides Rbu-P, carboxylase subunits co-electrophoreses with the corresponding subunits obtained after dissociating the spinach leaf enzyme, again reinforcing the thought that the Peak I carboxylase exhibits a molecular weight that ap- proaches 550,000. Thus, the Peak I Rps. sphaeroides Rbu-P, carboxylase is probably composed of eight large and eight small subunits, similar to the spinach enzyme (11). Certainly, further experimentation is needed to rigorously determine the molecular weight and actual quaternary structure of the Rps. sphaeroides Peak I Rbu-P, carboxylase.

Polyacrylamide gel assays performed on samples of Peak II Rps. sphaeroides Rbu-P, carboxylase electrophoresed at pH 9.5 and at pH 7.0 yielded surprising results. Multiple activities are observed from gels electrophoresed at pH 9.5 which are resolved into one major activity at pH 7.0, corresponding to an estimated molecular weight of about 360,000. Indeed this ob- servation has allowed us to isolate the Peak II enzyme to a state of apparent electrophoretic homogeneity by preparative polyacrylamide gel electrophoresis at pH 7.0. The dissociation and aggregation of Peak II Rps. sphaeroides Rbu-Pp carboxyl- ase at high pH is reminiscent of the alkaline pH-dependent dissociation of Escherichia coli adenine phosphoribosyltrans- ferase into multiple active species originally observed by Hochstadt-Ozer and Stadtman (26).

The subunit structure of the Peak II Rbu-P, carboxylase was found to be entirely different from the Peak I carboxylase in that we have no evidence for the presence of the small subunit upon dissociation of the Peak II enzyme. Perhaps the apparent dissociation and aggregation of the Peak II carboxylase at alkaline pH is somehow mediated by the absence of the second polypeptide. Certainly, the Peak II enzyme can be considered to be a less stable protein by these criteria.

Plots of protein migration at different concentrations of acrylamide for Peak I and Peak II Rps. sphaeroides Rbu-P, carboxylases (data not shown), gave presumptive evidence that these were proteins of different size and charge. These data, combined with the apparent differences in quaternary structure, clearly indicated that one protein is not an aggre- gate of the other. Moreover, no immunological cross-reactivity was observed when antibodies elicited against the Peak I carboxylase were reacted with purified Peak II carboxylase or with any of the dissociated or aggregated species of Peak II carboxylase. Indeed, antibodies to Peak I carboxylase did not inhibit Peak II carboxylase activity.

Peak I and Peak II Rbu-P, carboxylase promise to show considerable differences in catalytic properties. Our data, at this point, certainly show differences in the response to pH, with the Peak I enzyme showing a rather broad optimum and maximum activity between pH 7.8 and 8.0. Conversely, the

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Rbu-P, Carboxylase in Rps. sphaeroides 949

Peak II enzyme shows a rather sharp optimum at pH 7.2. Recently, it was found by Tabita and McFadden (27) that small and intermediate size Rbu-P, carboxylases are insensi- tive to 6-phosphogluconic acid. However, the large molecular weight carboxylases were markedly affected by this ligand (27) with phosphogluconate acting as a competitive inhibitor with respect to ribulose 1,5-bisphosphate (27, 28). The physiological implications of phosphogluconate regulation of CO, fixation are well described (27, 29). Our findings that phosphogluco- nate inhibits only the Peak I enzyme are perfectly consistent with the initial investigations of Tabita and McFadden (27). Thus, it is apparent that Peak I and Peak II Rps. sphaeroides Rbu-P, carboxylase show marked differences in the topogra- phy of the active site which may be reflective of the quater- nary structure.

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In conclusion, we have isolated two major and distinctive molecular forms of Rbu-P, carboxylase from extracts of Rps. sphaeroides. One of these enzymes structurally resembles the plant enzyme. Previously, it was demonstrated that the small subunits of the plant enzyme may be buried within the enzyme structure, with the large subunits exposed at the surface of the protein (30). Thus, antisera to isolated small subunits react only weakly with the native enzyme and have no effect on catalytic activity. Moreover, enzyme activity is inhibited only by antisera to isolated large subunits and antibodies to the native enzyme and not by antisera to the small subunits (30). In this investigation, we find that antibodies to the Peak I Rps. sphaeroides carboxylase, which structurally resembles the typical high molecular weight two-subunit type of carbox- ylase (2, 3), does not react with the Peak II enzyme which is composed only of large subunits. This may indicate that the Peak I and Peak II enzymes are quite unrelated. However, we remain cautious in interpreting the limited immunological data thus far obtained. Further experiments using antisera directed against isolated large subunits of Peak I carboxylase and antisera to the Peak II enzyme are warranted. Certainly tryptic or chymotryptic digests of the isolated large subunits of Peak I and Peak II enzymes should be examined to further discern the relationship of the Peak I and Peak II proteins. In any case, the ability to isolate two structurally distinct molec- ular forms of Rbu-P, carboxylase from Rps. sphaeroides pre- sents an unusual opportunity to study how function is related to structure.

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Acknowledgment - We thank Jack Myers for allowing us to use his facilities for growing large batches of bacteria.

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J L Gibson and F R TabitaRhodopseudomonas sphaeroides.

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from

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