May 19, 1992 - procedures (Engel et al., 1980; Andrews et al., 1990). More recently these methods have also been applied for the isolation of cytochrome c ...
Eur. J. Biochem. 208,761 -767 (1992)
0FEBS 1992
Affinity purification of cytochrome c reductase from potato mitochondria Hans-Peter BRAUN and Udo K. SCHMITZ Institut fur Genbiologische Forschung Berlin GmbH, Berlin, Federal Republic of Germany (Received May 19, 1992) - EJB 92 0696
Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanurn tuberosurn L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-’. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s - l , can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individial subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional ‘core’ polypeptide which is not present in other cytochrome bel complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers
introduced by Hatefi et al. (1962), Nakajima et al. (1984) isolated cytochrome c reductase from sweet potato. They employed potassium chloride, cholate and deoxycholate for protein extraction and ammonium sulfate for protein precipitation. The procedure allowed the first analysis of the subunit composition of the complex by SDSjPAGE but did not yield an active enzyme. Further modifications of the Hatefi protocol were introduced by Degli Esposti et al. (1985) for the isolation of cytochrome c reductase from Jerusalm artichoke tuber mitochondria. The activity of the complex could be maintained through cholate solubilization, but the complex was unstable during the final precipitation step. Other methods utilize nonionic detergents for solubilization of the complex in combination with chromatographic procedures (Engel et al., 1980; Andrews et al., 1990). More recently these methods have also been applied for the isolation of cytochrome c reductase from plant mitochondria. Pfeiffer et al. (1990) used dodecyl maltoside and ion-exchange chromatography for the isolation of cytochrome c reductase from wheat germ mitochondrial membranes. The preparation was pure, but no statements on its activity were made. While the work reported here was in progress, Berry et al. (1991) used basically the same procedure to prepare, for the first time, a pure and active bel complex from a plant source. Correspondence to U . K . Schmitz, Institut fur Genbiologische In order to avoid some disadvantages of the protocols Forschung Berlin GmbH, IhnestraDe 63, W-1000 Berlin 33, Federal mentioned above, we have tried to apply to plant mitochonRepublic of Germany Abbreviation. decQ, 2,3-dimethoxy-5-decyl-6-methylbenzo-dria the very elegant and gentle method introduced by Weiss and Juchs (1978). This method uses Triton X-100 to solubilize quinone. the rather hydrophobic cytochrome e. reductase and Enzymes. Cytochrome c reductase (EC 1.6.99.3); ubiquinolcytochrome-c affinity chromatography to purify it. As a final cytochrome-c oxidoreductase (EC 1.10.2.2).
The respiratory electron-transfer chains of prokaryotes and eukaryotes differ in many respects but generally they all contain a cytochrome bel complex. The bel complex, localized in the inner mitochondrial membrane (also known as cytochrome c reductase or complex I11 of the mitochondrial respiratory chain), contains more than twice as many subunits as the b6/fcomplex localized in the thylakoid membrane or in the simple bacterial bel complex (Hauska, 1986; Yang and Trumpower, 1986). To date the role of most of the additional subunits is not clear. Studies on yeast mutants defective in the synthesis of these subunits suggest that they are essential for assembly and proper function of the complex (Crivellone et al., 1988), but direct evidence concerning their function is lacking. Even in higher eukaryotes like mammals (Schagger et al., 1985) and plants (Berry et al., 1991) the number and size of the subunits differs. Different methods have been used to purify cytochrome c reductase from mitochondria of bovine heart (Rieske et al., 1964; Engel et al., 1980), rat liver (Gellerfors et al., 1981), yeast (Siedow et al., 1978; Beattie et al., 1984) and Neurospora (Weiss and Kolb, 1979). Most of these methods had limited success for the preparation of cytochrome c reductase from plant species. Using a method similar to the one originally
762 purification step, a gel-filtration column, allowing the separation of aggregated complexes from monodisperse complexes, is employed. Isolation of cytochrome c reductase in the monodisperse state allows the determination of the molecular mass, the stoichiometry and the three-dimensional structure of the complex (Wingfield et al., 1979; Karlsson et al., 1983). Another advantage, especially relevant for plant biochemists, is the high yield obtained by this method. To date, the protocol has only been successfully applied for the preparation of cytochrome c reductase from Neurospora mitochondria. Efforts to use it for the preparation of this protein complex from yeast and bovine mitochondria failed (Weiss and Kolb, 1979). Here, we report the application and optimization of the method for the isolation of a highly pure and active cytochrome c reductase from potato tuber mitochondria.
4
A
Absorbance
> 510
550
530
570
610
590
630
Wavelength
Inml
650
B
4 Absorbance
MATERIALS AND METHODS Preparation and subfractionation of potato mitochondria
Mitochondria from different potato varieties (Hansa, Bintje, Jaerla, Ukama, Marfona) were prepared following a modified version of the procedure of Boutry et al. (1984) and Douce (1985). The method includes purification of mitochondria on discontinous Percoll gradients. After several washes in a buffer containing 0.4 M mannitol, 10 mM Tricine, pH 7.2, 1 mM EGTA and 0.2 mM phenylmethylsulfonyl fluoride, the mitochondria were resuspended at a concentration of 20 mg protein/ml and stored at - 80 "C. Preparation and solubilization of mitochondrial membranes was performed according to Linke and Weiss (1986) with some modifications. About 3 g wet mitochondria were lysed in hypotonic 0.2 M sodium phosphate, pH 7.2, by sonication with a Sonifier B 12 (Branson) for 40 s (four intervals) at 4°C. 'Silicone antifoam emulsion M 30' (Serva) was added to avoid shearing forces generated by foaming. Unbroken mitochondria were pelleted by centrifugation for 5 min at 5000 x g; the supernatant was subjected to ultracentrifugation (90 min at 150000 x g), yielding a pellet of mitochondria1 membranes free of cytochrome c. The membrane fraction, containing about 150 mg protein, was resuspended by sonication (two intervals of 10 s) in 4 ml water. Membrane proteins were solubilized by successively adding 10% Triton X-100 up to a final concentration of 3.3%. To remove membrane fragments and lipids, the suspension was further centrifuged for 10 min at 60000 x g. Purification of cytochrome c reductase
The purification of cytochrome c reductase was carried out at 4 "C and included affinity chromatography, gel-filtration chromatography and an ultrafiltration step (Linke and Weiss, 1986). For affinity chromatography, cytochrome c from horse heart (Sigma, type 111) was coupled to CNBr-activated Sepharose 4B (Pharmacia) as described by Weiss and Juchs (1978). 8 ml cytochrome-c- Sepharose were equilibrated in a small column (1.0 cm x 10 cm) at a flow rate of 5 ml/h with elution buffer containing 20 mM Tris/acetate, pH 7.0, 0.04% Triton, 5% sucrose and 0.2 mM phenylmethylsulfonyl fluoride. After applying the solubilized membrane proteins to the column, it was washed at the same flow rate with elution buffer. Cytochrome c reductase was eluted with a linear gradient (50 ml, 5 ml/h) of 20-200 mM Tris/acetate, pH 7.0, in elution buffer supplemented with 2 mM ascorbate, which reduces cytochrome c, thereby decreasing its affinity for cytochrome cl. Fractions containing cytochrome b and
I
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t
,
,
530
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,
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,
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610
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I
I
t Wavelength
[nml
650
C
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I 510
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Fig. 1. Dithionite-reduced-minus-air-oxidized difference spectra of cytochromes during purification of potato cytochrome c reductase. (A) Whole mitochondria.(B) Mitochondria1 membranes. (C) Cytochrome bcl peak fractions after affinity chromatography of the complex. All spectra were taken at room temperature and in the presence of Triton x-100.
cytochrome c1 were pooled and concentrated by ultrafiltration using Diaflo XM 300 filters (Amicon). The concentrate was passed through an ultrogel AcA 34 column (1.0 cm x 50 cm). The protein complex was eluted with 40 mM Tris/acetate, pH 7.0, 0.04% Triton X-100 and 0.2 mM phenylmethylsulfonyl fluoride at a flow rate of 3 ml/h. The purity of the fractions was monitored spectrophotometrically and by SDS/ PAGE after each chromatography step. Fractions containing cytochrome b and cytochrome c1 but no traceable amounts of cytochrome c oxidase were pooled, frozen in liquid nitrogen and stored at -80°C. Protein analysis by SDS/PAGE and immunoblotting
Protein concentrations were determined according to Bradford (1976). Proteins were fractionated in 14% SDS/ polyacrylamide gels (Laemmli, 1970). The proteins were either stained with Coomassie Blue R 250 (Serva) or blotted onto nitrocellulose membranes (Schleicher & Schull). Blots were incubated with antibodies directed against individual subunits of cytochrome c reductase from potato, yeast and Neurospora as previously described (Braun et al., 1992). Antibodies against the core proteins from potato were raised in rabbits using about 50 pg polypeptides separated by SDS/PAGE. Antibodies directed against cytochrome b and cytochrome c1 from yeast were kind gifts of Prof. G. Schatz, Basel; antibodies
763 69
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30
~ } c o r eP r o t e i n s
-
-
21 5
-
14.3
-
c
Cyt b c y t c,
+
FeS
-1
s m a l l SUs
c
t
Fig. 2. Electrophoreticand spectral analysis of fractions eluted from the cytochrome c affinity column. The upper part shows a Coomassie-stained SDS/polyacrylamide gel; a specific set of mitochondria1 membrane proteins are eluted from the column. A graphical illustration of the cytochrome-b content in the fractions loaded is shown underneath the gel. The cytochrome-h content was determined spectrophotometrically in the presence of dithionite at 557 nm using an absorption coefficient of 27 in accordance with Weiss and Ziganke (1974) and Weiss and Juchs (1978). Fractions 1-6 contain 16 nmol cytochrome b and fractions 14- 19 32 nmol cytochrome b. The dashed line indicates the Tris/acetate gradient. The arrows point to the subunits of potato cytochrome c reductase. The distortions at the bottom of the gel (lanes 1-6) are due to Triton X-100jSDS interactions. The numbers on the left indicate the size of molecular-mass standard proteins in kDa. SU, subunit.
against the 'core' proteins and the FeS protein of cytochrome c reductase from Neurospora were kindly supplied by Prof. H. Weiss, Diisseldorf. Visualization of immunopositive bands was performed with biotinylated anti-(rabbit IgG)serum, avidin and biotinylated horseradish peroxidase (Vector Laboratories) as recommended by the supplier. Reconstitution of cytochrome c reductase in phospholipid vesicles and activity measurements
Cytochrome c reductase was reconstituted in phospholipid membranes according to Zweck et al. (1989). A solution containing 17% phosphatidylcholine (Sigma) in 21 mM Mops, pH 6.8, 42 mM KzS04 and 145 mM lithium cholate was sonicated five times for 10 s at 4°C. Equal volumes of this solution and purified cytochrome c reductase (about 2 pM) in 40 mM Tris/acetate, pH 7.0 and 0.04% Triton X-100 were combined and diluted with 100 volumes of a buffer containing 50 mM Mops, pH 6.8, and 100 mM K2S04. Quinol: ferricytochrome c reductase activity was assayed in the presence of 40 pM KCN, 40 pM cytochrome c from horse heart (Sigma, type III), 2 pM Valinomycin (Fluka) and 100 pM Decylchinol, which was a kind gift of Prof. H. Weiss, Diisseldorf. The decrease of the ferricytochrome-c concentration was determined at 25°C with a dual-wavelength photometer (Sigma 11, Biochem) at 550nm and 580nm using the absorption coefficient 20mM-' cm-' for cytochrome c (Linke and Weiss, 1986). The inhibitors, antimycin and myxothiazol (Boehringer), were added to a final concentration of 2 pM.
The duroquinol/2,3-dimethoxy-5-decyl-6-methylbenzoquinone (decQ) transhydrogenation reaction of cytochrome c reductase reconstituted into phospholipid vesicles was analyzed in a buffer containing 100 pM duroquinol (Sigma). 5 pM decQ and 2 pM myxothiazol. The reduction of decQ is monitored spectrophotometrically using wavelengths 285 nm and 320 nm, which are isobestic for duroquinol and duroquinone, respectively (Boveris et al., 1971). A dual-wavelength photometer, a spectral band width of 3 nm and the molar absorbance coefficient 9.1 mM-' cm-' were used as described by Zweck et al. (1989). The non-enzymatic turnover rate was determined in the presence of 2 pM antimycin and subtracted from the total turnover rate.
RESULTS A prerequisite for the isolation of cytochrome c reductase by affinity chromatography is the preparation of mitochondrial membranes free of cytochrome c. This was achieved by sonication of isolated mitochondria in a hypotonic sodium phosphate buffer and subsequent differential centrifugation as described in Materials and Methods. The spectral properties of the membrane fraction were monitored at different wavelengths (Fig. 1B). Loss of the maximum at 550 nm, which is present in whole mitochondria (Fig. 1A), indicated that the membrane fraction was essentially free of undisrupted mitochondria or loosely bound cytochrome c. The CI bands at 600 nm and 557 nm indicated that cytochrome c oxidase and
764 cytochrome c reductase were present in the membrane fraction. Different concentrations of Triton X-100 were tested for the solubilization of cytochrome c reductase. While a final concentration of 5% is used for the solubilization of this complex from Neurospora mitochondrial membranes (Linke and Weiss 1986), we achieved best results with respect to integrity and yield of potato cytochrome c reductase at 3.3% Triton X-100. After separation from membrane fragments and lipids, the solubilized complex was immediately pumped onto a cytochrome-c- Sepharose column (see Materials and Methods) and eluted by employing a salt gradient (20 200 mM Tris/acetate, pH 7.0, and 2 mM ascorbate). Fig. 2 shows an analysis of the resulting fractions by SDS/PAGE and the spectrophotometric determination of the cytochromeb content. The majority of the total cytochrome b bound specifically to the column and was eluted at 100 mM Tris/ acetate as a sharp peak together with a distinct subset of polypeptides (fractions 14- 20). The presence of cytochrome b in the earlier fractions may be due to partial disintegration of the complex in the presence of Triton X-100 or may result from other cytochrome-b sources as discussed previously (for review see von Jagow and Sebald, 1980). The amount of cytochrome b found in the peak fractions and the flow through was exactly reproducible during several different preparations if the same amount of Triton X-100 was used for solubilization. The greater part of mitochondrial proteins did not bind to the cytochrome-c column and was collected in fractions 16 before the salt gradient was started. The elution of proteins in fractions 7 - 13 is probably due to the weak anion-exchange properties of the cytochrome c coupled covalently to the Sepharose. Potentially, cytochrome c oxidase might also bind to the column, but under our conditions no cytochrome a (a band at 600 nm) was detectable in fractions 7 - 22 (Fig. 1C). The dithionite-reduced spectra of cytochromes in fractions 14-20 show the absorbance extrema typical for the mitochondrial cytochrome bel complexes. As already reported by Degli Esposti et al. (1985) and Berry et al. (1991) the CI band of the plant complex has a rather broad and symmetrical shape and a maximum at 557 nm. There are shoulders at 552 nm and 562 nm; these values correspond to the a-band maxima of cytochrome c1 and cytochrome b reported for other organisms. Our measurements of the bands of the complex show two maxima at 525 nm and 530 nm and a shoulder at 537 nm. The peak fractions of the affinity-chromatography step were concentrated by ultrafiltration using filters with an exclusion limit of 300 kDa. As contaminating proteins most likely have a smaller size, the ultrafiltration is also an effective purification step. The concentrate was passed through a gelfiltration column and the resulting fractions were analyzed by SDSjPAGE and spectrophotometric measurements (Fig. 3). A symmetric cytochrome-b peak in fractions 21 - 28 correlates with the appearance of a distinct set of protein bands already present in the peak fractions of the cytochrome-c affinity column. Aggregated proteins, including oligomeric cytochrome c reductase complexes, elute in the void volume (fractions 13 - 18), while incomplete cytochrome c reductase and Triton X-100 micelles are found in later fractions (fractions 30 - 50, not shown). Fractions 21 - 28 contain cytochrome c reductase in a monodisperse state. The purification protocol is summarized in Table 1. Cytochrome c reductase was purified about 38-fold from isolated mitochondrial protein. As mitochondria only form a minor part of potato tubers, the total
A 69
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Core Proteins
t
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Fig. 3. Fractions of the gel-filtration column analyzed by SDS/PAGE and by spectrophotometric determination of the cytochrome-6 content (see Fig. 2). The Coomassie-stained polyacrylamide gel (A) shows a distinct set of proteins eluting from the column (fractions 21 -28). The identity of individual subunits was determined immunologically (see Fig. 5). (B) Graphical illustration of the cytochrome-b content in the fractions analyzed. The numbers on the left indicate the size (kDa) of a molecular-mass standard. SU, subunit.
purifiction factor is much higher. The yield was about 60 nmol cytochrome b/c starting mitochondrial protein. The molecular mass of the complex was calculated from its properties during gel filtration. The complex migrates close to thyroglobulin, a protein of 669 kDa (Fig. 4). This value is higher than that of Neurospora cytochrome c reductase, which has a molecular mass of 550 kDa. As phospholipids and Triton X-100 are bound to the complex, the exact molecular mass is about 100 kDa smaller (Weiss and Kolb, 1979). After subtracting this value, the size of the potato enzyme is still about twice the minimal molecular mass calculated by summing up the apparent molecular masses of individual subunits. This indicates that, as in other organisms, cytochrome c reductase from potato occurs as a dimer. To analyze the integrity of the purified protein complex we tested quinol-cytochrome-c oxidoreductase activity. The activity was assayed in a dual-wavelength photometer using 5 pM ubiquinol analogue as electron donor and 6 pM oxidized cytochrome c from horse heart mitochondria as electron acceptor (see Materials and Methods). The protein complex was integrated into phospholipid vesicles and measurements were carried out in the presence of KCN to inhibit potential contaminations by cytochrome c oxidase. The turnover number of potato tuber cytochrome c reductase was 50 s- l/dimer, which is slightly less than the value reported for Neurospora (Weiss and Wingfield, 1979; Linke and Weiss, 1986). Upon addition of antimycin or myxothiazol, more than 90% of the activity was inhibited.
765 Table 1. Purification of cytochrome reductase from potato. The difference absorbance coefficient for cytochrome b was taken from Weiss and Ziganke (1974). n. d., not determined. Source of enzyme
Volume
Mitochondria Mitochondria1 membranes Triton-X-100-solubilized membranes Protein complex after affinity chromatography Protein complex after ultrafiltration Protein complex after gel filtration
t
Protein
cytochrome b
cytochrome b/Protein
ml
mg
nmol
nmol/mg
%
16 4 7 10 0.5 4
300 n. d. 110 5.1 n.d. 2.1
67 n.d. 58 30 n.d. 16
0.2 n.d. 0.5 5.9 n.d. 7.6
100 n. d. 89 45 n.d. 24
Table 2. Polypeptide composition and molecular masses of ubiquinolcytochrome-c oxidoreductasefrom bovine (Schagger et al., 1986), yeast (Beattie et al., 1984), Neurospora (Karlsson et al., 1983) and potato.
molecular mass (lo6kDa) Thyroglobulin (669 kDa)
.-\
Yield of
Polypeptide
Molecular mass of enzyme from bovine heart
I
: :
Cytochrome Reductase
4:23
5
Neurospora cram
potato tuber
49 39 30 29 22 13 11 10 9
50 45
55 53 51 35 33 25 14 12 11 10
kDa
1 4
Saccharomycetes cerevisiae
time 6
7
[hl
Fig. 4. Determination of the molecular mass of cytochrome c reductase from potato. The complex elutes from an AcA 34 gel-filtration column shortly after thyroglobulin.
A quinol/quinone transhydrogenation reaction which has been investigated in Neurospora (Zweck et al., 1989) is also traceable in cytochrome c reductase from potato mitochondria. After reconstitution into lipid membranes, the enzyme catalyzes electron transfer from duroquinol to decQ. The turnover number of this reaction is 16 s-'; the activity is insensitive towards myxothiazol but antimycin sensitive. As detailed in Zweck et al. (1989), the reaction is supposed to occur in a ping-pong mode with duroquinol first reducing cytochrome c reductase, then making way for the electron-accepting decQ to react. The transhydrogenation reaction of potato cytochrome c reductase may be interpreted in support of the model of a proton-motive ubiquinone cycle in plant mitochondria (Mitchell, 1976). As revealed by SDS/PAGE (Fig. 3), cytochrome c reductase from potato mitochondria consists of ten major bands with molecular masses 55 kDa, 53 kDa, 51 kDa, 35 kDa, 33 kDa, 25 kDa, 14 kDa, 12 kDa, 1 1 kDa and 10 kDa. In contrast, the complex from Neurospora (see Table 2) comprises nine subunits which, apart from the two largest polypeptides, have similar molecular masses to the potato enzyme. There is less conformity with the polypeptide patterns of the bc, complexes from yeast and bovine heart mitochondria. In addition to the ten major bands of potato cytochrome c reductase, two substoichiometric polypeptides of 50 kDa and 42 kDa co-elute with the enzyme complex (see Fig. 3). They could potentially be fragments of one of the high-molecularmass polypeptides or represent proteins which are associated with the protein complex from potato. The polypeptide patterns of cytochrome c reductase from different potato varieties (see Materials and Methods) are identical (not shown).
Core 1 Core 2 Core 3 Cytochrome b Cytochrome c1 FeS Small subunits
49 47 44 28 25 13 11 9 8
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35 31 25 14 12 11 8
The identity of individual subunits of the complex was determined by immunoblotting and amino-terminal sequencing of some of the polypeptides. Antibodies directed against the five largest subunits of the complex from fungi cross-react with individual bands of our preparation (Fig. 5). The 55 kDa, 53 kDa and 51 kDa bands are recognized by antibodies directed against subunits I and I1 of the Neurospora complex. The fact that both antibodies against the core proteins recognize the same potato polypeptides is not surprising as the largest subunits of the Neurospora enzyme belong to the same protein family (Schulte et al., 1989). Our data suggest that the three largest polypeptides of potato cytochrome c reductase belong to this protein family as well. The three redox-center-carrying proteins (cytochrome b, cytochrome c1 and the Rieske-FeS protein) are obviously well conserved between fungi and potato ; they specifically cross-react with antibodies directed against the yeast cytochromes and the FeS protein from Neurospora. The electroimmunoblots shown in Fig. 5 C - E suggest that the 35-kDa band of potato cytochrome c reductase represents cytochrome b, the 33-kDa band cytochrome c1 and the 25-kDa band of the Rieske-FeS protein. These results have been corroborated by amino-terminal sequencing of the polypeptides (not shown). The first 20 amino acids of the 33 kDa polypeptide are identical with a peptide
766 22 23 24 25 26 27
22 23 24 25 26 27
46 -
30 -
215-
E D Fig. 5. Immunological analysis of the polypeptides present in the peak fractions (22 - 27) eluted from the gel-filtration column. Equal volumes C
from each fraction were subjected to SDS/PAGE, blotted onto nitrocellulose and incubated with antibodies directed against subunit I (A), I1 (B), cytochrome b (C), cytochrome c1 (D) and the FeS protein (E) of cytochrome c reductase from fungi. The numbers on the left indicate the size (kDa) of a molecular-mass standard.
encoded by one of the cytochrome-cl genes from potato (Braun et al., 1992). The amino-terminal sequence of the 35kDa polypeptide corresponds to the sequence encoded by the cytochrome-6 gene which forms part of the mitochondrial genome (Zanlungo et al., 1991; Braun and Schmitz, unpublished results). As the antibodies directed against the core proteins from Neurospora cross-reacted with all three high-molecular-mass subunits of cytochrome c reductase from potato, we attempted to exclude the possibility that two of the three bands might be isoformes of the same polypeptide. Antibodies against the 53 kDa and the 51 kDa polypeptide were rasied in rabbits and tested with potato cytochrome c reductase. As shown in Fig. 6, the antibodies exlcusively react with the polypeptides used for immunization but not with any other core protein. This indicates the presence of three immunologically distinct ‘core’ proteins in cytochrome c reductase from potato. DISCUSSION A recent report (Berry et al., 1991) and this paper describe for the first time a method for the purification of an active and complete cytochrome c reductase complex from a plant source. The cytochrome c affinity purification (Weiss and Juchs, 1978; Linke and Weiss, 1986) applied here is based on the biological activity of the complex and yields a highly pure and active enzyme. A transhydrogenase reaction of cytochrome c reductase, which to date has not been analysed in plants, could be demonstrated using the affinity-purified enzyme from potato. In contrast to other methods, the final gel-filtration step of the procedure leads to a monodisperse enzyme, thus allowing determination of its molecular mass. Our results suggest a size of more than 600 kDa for potato cytochrome c reductase, indicating that it most likely occurs
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Fig. 6. Immunoblot of cytochrome c reductase fractionated by SDS/ PAGE after incubation with antibodies against the 51-kDa protein (A) and the 53-kDa protein (B). While the pre-immune sera (PI) did not react with any band, the immune sera (I) specificallyrecognized single core proteins. The numbers on the left indicate the molecular mass (kDa). The slight background on all blots is due to cross-reaction of the blotted proteins with the secondary antibody which was used to develop the immune reaction.
as a dimer. Assuming that similar amounts of Triton X-100 and phospholipids as in Neurospora (Weiss, 1987) are associated with the complex, potato cytochrome c reductase is about 100 kDa larger than the fungal enzyme. This may be due to the association of an additional ‘core’ protein with the potato complex. While bacterial and chloroplast cytochrome be, (b& complexes only contain three or four polypeptides (Hauska, 1986; Trumpower, 1990), mitochondrial bel complexes have
767 5-7 additional subunits which do not carry redox centers. The complex from bovine heart mitochondria is composed of eleven subunits (Schagger et al., 1986), the five largest of which are similar to subunits I - V of fungal cytochrome bcl complexes (Beattie et al., 1984; Linke and Weiss, 1986). The few publications specifying the number of polypeptides in plant mitochondria1 cytochrome bcl complexes have different conclusions; between eight (Nakajima et al., 1984) and ten subunits (Berry et al., 1991) have been reported. Our data indicate that the complex from potato mitochondria contains ten subunits. Interestingly, the potato enzyme seems to have three core proteins, as revealed by an immunological analysis of individual bands. In contrast, the complexes from fungi and mammals contain only two core proteins. There are different possibilities to explain the occurrence of the three core proteins. Potentially, one of the three bands may result from partial proteolysis. Considering that we applied a very gentle isolation procedure which reproducibly gave identical protein patterns, this explanation does not seem to be very likely. Alternatively, two of the three bands may be isoforms of the same protein, as proposed by Berry et al. (1991). However, antibodies directed against the 53-kDa and 51-kDa subunits of the potato complex exclusively recognize the proteins used for immunization and do not cross-react with other subunits. These data suggest that three distinct ‘core’ proteins form part of potato cytochrome c reductase. Recently, Schulte et al. (1989) have shown that subunit I of cytochrome c reductase from Neurospora is identical with the processing enhancing protein of the mitochondrial-processing peptidase. This enzyme is composed of two subunits, the processing enhancing protein and the catalytically active matrix-processing peptidase, which form a soluble complex in yeast. It would be tempting to speculate that, in potato, both components form part of cytochrome c reductase. We are currently investigating this issue by direct protein sequencing of the three largest subunits of the complex. To find out whether isoforms occur in different parts of potato plants or in other species, it would also be worthwhile to isolate cytochrome c reductase from different tissues and developmental stages as well as from other plants. We already applied the isolation protocol successfully to Oenothera mitochondria. Thus, the affnity-purification protocol seems to be a gentle, simple and effective method for the isolation of cytochrome c reductase from plants and may help to answer some of the above mentioned questions. We arc very grateful to Prof. H. Weiss, M. Bodicker and M. Peters, Diisseldorf, for instructing us on the cytochrome c affinitypurification procedure and cy tochrome-c-reductase activity measurements in their laboratory. We are also indebted to them for supplying the synthetic quinol and antibodies directed against individual subunits of the enzyme. We wish to thank Udo Herz, Berlin, who helped us to make antibodies against subunits of cytochrome c reductase from potato. Thanks are also due to Prof. G. Schatz, Basel, for sending us antibodies directed against cytochrome b and cytochrome el from yeast and to Prof. A. Brennicke, Berlin, for constant support and encouragement. This work was supported by the Deutsche Forschungsgemeinschaft, grant Schm 69812-2.
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