THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 20, Issue of October 25, pp. 10145-10152, 1979 Printed in U.S.A.

On the Role of Coupling Factor B in the Mitochondrial Pi-ATP Exchange Reaction*

(Received for publication, September 26, 1978, and in revised form, June 5, 1979)

Saroj Joshi, James B. Hughes,+ Fariyal Shaikh, and D. Rao Sanadig From the Department of Cell Physiology, Boston Biomedical Research Institute, Boston, Massachusetts 02114

Bovine heart mitochondrial coupling factor B (Factor B) has been prepared by a modification of the proce- dure of Lam et al. (Lam, K. W., Warshaw, J. B., and Sanadi, D. R. (1967) Arch. Biochem. Biophys. 119,477- 484). It has the highest specific activity (over 100 pmol of NAD reduced x min-’ x mg-’ of protein) compared to any related preparation. Its molecular weight under both denaturing and native conditions was approxi- mately 13,000. It appeared as a single component in polyacrylamide gel electrophoresis both in the pres- ence and absence of dodecyl sulfate. Rabbit anti-Factor B serum gave a single line in immunoelectrophoresis against partially as well as highly purified Factor B.

A preparation of energy-transducing ATPase com- plex which is partially deficient in Factor B has been isolated from particles @E-particles) derived by am- monia-EDTA extraction of heart mitochondria. The hy- drophobic membrane protein fraction (AE-Fo) derived from it by treatment with sodium bromide, recombines with mitochondrial coupling factor 1 (R) with the res- toration of oligomycin-sensitive ATPase activity. The Pi-ATP exchange activity of this AE-Fo + F1 is minimal and is stimulated over lo-fold upon addition of Factor B. Complete elimination of exchange activity of AE-Fo + F1 was achieved by prior treatment of AE-Fo with N- ethylmaleimide, although ATPase activity was retained with unaltered sensitivity to oligomycin. Addition of Factor B to this AE-F. + F1 led to restoration of Pi-ATP exchange activity and to partial inhibition of ATPase activity. The presence of Factor B in the ATPase com- plex and derivative fractions was determined by Ouch- terlony double diffusion experiments. Antigen in the ATPase complex was not detectable after the ATPase had been washed with ammonia/EDTA; concomi- tantly, the stimulation of exchange activity by Factor B increased to over B-fold. These results show that Pi- ATP exchange activity has an absolute requirement for Factor B, but binding of F1 to AE-Fo, and oligomycin sensitivity of the resulting ATPase, are independent of the factor.

One useful approach to the investigation of the mechanism of oxidative phosphorylation involves the resolution of the energy transfer components from mitochondrial membranes,

* This research was supported by Grants (GM-13641 and RR05711) from the National Institutes of Health and National Science Foun- dation Grant PCM77-09373. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Postdoctoral Trainee of the National Institutes of Health (United States Public Health Service Grant 51‘32 HL07266).

3 D. R. S. also has an appointment at the Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115.

and subsequent reconstitution of the functional catalytic sys- tems from the purified components (1). Coupling factor B (or F2),’ one such component, has been isolated and characterized in this laboratory (3-5).

While the exact function of Factor B in oxidative phospho- rylation is unknown, it has been shown that it stimulates the rate of ATP-driven NAD’ reduction by succinate, ATP-driven NAD(P)+ reduction by NADH, Pi-ATP exchange, as well as net phosphorylation coupled to either NADH or succinate oxidation in the Factor B-deficient AE particles.’ At the same time, Factor B produces up to 20% inhibition of ATPase activity in the same submitochondrial particle preparation (4). In order to obtain further insight into the function of Factor B, we have chosen for study a reaction system with the least number of components in which this coupling factor has a distinct effect; viz. the Pi-ATP exchange reaction.

Attempts at purifying the oligomycin and uncoupler-sensi- tive Pi-ATP exchange enzyme system have led to the sepa- ration of several partially purified preparations from mito- chondrial and bacterial sources (6-14). Most of these prepa- rations were isolated using detergents and were dependent on additional lipids and protein factors under special conditions for restoration of maximal exchange activity. Following a similar approach, Pi-ATP exchange activity was restored in this laboratory (9,14) to a preparation of oligomycin-sensitive ATPase (OSATPase) from beef heart mitochondria3 (15). Furthermore, it was shown for the first time that the exchange activity of the reconstituted system was enhanced over 3-fold by the addition of partially purified coupling Factor B.

Sadler et al. (8) reported on the isolation of an ATPase complex preparation with oligomycin-sensitive ATPase and Pi-ATP exchange activities. This preparation was isolated from beef heart submitochondrial particles using lysolecithin and, in our hands, showed the highest reported exchange activity (600 to 900 nmol x mir-’ X mg-‘) without supple- mentation with phospholipid or other protein factors. Using

’ Factor B has been purified lo-fold from the FS preparation of Racker et al. (2), suggesting the possibility that the two may be identical.

‘The abbreviations used are: AE particles, ammonia EDTA-ex- tracted submitochondrial particles; ETPH, electron transport particles from heavy mitochondria; F, and Fe, coupling factors 1 and 6; OSCP, oligomycin sensitivity-conferring protein; OSATPase, oligomycin- sensitive ATPase; Cl-CCP, carbonyl cyanide-m-chlorophenylhydra- zone; Mes, 2-(N-morpholino)ethanesulfonic acid, Tricine, N-[tris(hy- droxymethyl)methyl]glycine.

’ In our earlier paper (9), we had reported that the OSATPase of Tzagoloff et al. (15), reconstit.uted with F, and Factor B by the cholate dialysis procedure, had an activity of about 50 nmol x min-’ X rng-‘. We have since found that when reconstitution with F, is carried out by the cholate dilution procedure (16), the exchange activity is 200 nmol X min-’ X mg -‘, which increases in the presence of Factor B to 450 with Factor B (14). This is close to the activity seen with the ATPase complex described here or with Complex V (13).

10145

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10146 Factor B in Mitochondrial Pi-ATP Exchange

a modification of this procedure, we have been able to isolate an ATPase complex from Factor B-deficient particles. It has low exchange activity which is stimulated severalfold by added Factor B.

In this communication, we also report on the preparation of a more active form of Factor B and reconstitution experiments utilizing membrane protein fractions obtained from Factor B- depleted ATPase complex. The results clearly establish that Factor B is essential for the Pi-ATP exchange reaction but not for the oligomycin-sensitive ATPase activity. In fact, the ATPase activity is partially inhibited by Factor B. Preliminary results bearing on these findings have been reported earlier4 (14).

EXPERIMENTAL PROCEDURES

Beef heart mit?chondria (17), Fti (18), OSCP (19), ATPase inhibitor (20, 21), and F1-ATPase (22) were prepared according to published methods. F, was further purified by filtration on Sepharose 4B to remove traces of low molecular weight contaminants including Factor B. Such contamination had been detected earlier with the use of anti- Factor B serum (23). For purification of F,, a l.O-ml aliquot containing 25 mg of F, protein was applied to a Sepharose 4B column (1.8 X 100 cm) equilibrated with a buffer (pH 7.5).containing 0.25 M sucrose, 10 mM Tris/SOd. 2 mM EDTA. and 4 mM ATP. The gel filtration was

. I

performed at room temperature. Active fractions had a specific activ- ity of approximately 90 pmol x min-’ x mg-‘. AE particles were prepared by ultrasonic disruption of heavy mitochondria at pH 9.0 (3).

Gel electrophoresis in the presence of sodium dodecyl sulfate was carried out either as described by Weber and Osborn (24) or essen- tially according to Swank and Munkres (25). In the latter case, 12.5% acrylamide gels (13 cm) containing 7 M urea and cross-links were introduced at a bisacrylamide to acrylamide ratio of 1:lO. The pH of the running buffer was increased to 7.4. Conventional gel electropho- resis was conducted essentially according to Davis (26) except for substituting ammonium persulfate (0.065%) for riboflavin. Both con- ventional and dodecyl sulfate-containing gels were stained and de- stained according to Weber and Osborn (24).

Protein content of the ATPase complexes and membrane proteins was determined by the biuret method (27), and of soluble proteins by the method of Lowry et al. (28), using bovine serum albumin as a standard. When dithiothreitol was present, N-ethylmaleimide was added prior to the reagents. Under these conditions, the response was linear with protein concentration, and there was little or no color in the blanks without protein. The protein content of Factor B prepa- rations after the last step of purification was further confirmed by the methods of Murphy and Kies (29) and Spector (30). Corrections for interference by dithiothreitol were introduced wherever necessary. Antiserum against Factor B was obtained by serially injecting a young female rabbit with purified Factor B (300 pg/week) which had been mixed with Freund’s adjuvant as described by Lam and Yang (31). Control and antisera were collected and purified by QAE-Sephadex chromatography (32).

Assay Methods-The binding of F1 to the membrane protein fraction (AE-Fo) and assay for ATPase activity and for restoration of oligomycin sensitivity to the ATPase activity were carried out under the conditions described by Tzagoloff et al. (33) for a similar mem- brane fraction derived from the OSATPase. The Pi-ATP exchange activity was assayed by incubating the AE . ATPase complex, or AE- F. fraction with F, or Factor B, or both, in the presence of “Pi and ATP. The details are shown in Table I. P, was determined by the method of Fiske and SubbaRow (34). Unesterified phosphate was separated from the nucleotides by extraction with isobutyl alcohol/ benzene (35).

Factor B was assayed for coupling factor activity by its stimulatory effect on the ATP-driven NAD+ reduction by succinate in AE parti- cles (3). A series of assays were performed by adding different levels of Factor B to 0.5 mg of AE particles. The specific activity was calculated from the linear part of the activation curve. As described earlier (3), specific activity is defined as the net increase in activity expressed as amol of NAD+ reduced x min-’ x mg-’ of factor protein.

’ Tenth International Congress of Biochemistry, Hamburg, July 25 to 31, 1976, Abstr. p. 197A, American Chemical Society Meeting, Miami, Fla. 1978, Abstr.

RESULTS

The data in this paper represent typical experiments based in each case on several determinations.

Preparation of Factor B-Factor B was prepared by a modification of the procedure of Lam et al. (3) as follows: 50 g of lyophilized acetone-washed mitochondrial powder was suspended in 15 volumes of 50 mM Tris/S04 buffer at pH 8.5 containing 1.0 mM dithiothreitol and homogenized in a Potter- Elvehjem homogenizer. The suspension was centrifuged at 144,000 X g for 1 h. Solid ammonium sulfate was added to the supernatant and the fraction precipitating between 30 and 50% saturation was suspended in 20 ml of 10 mM Tris/S04 buffer, pH 8.0, 1.0 mM dithiothreitol. The ammonium sulfate in the solution was removed by passage through a Sephadex G-25 column (4.3 X 23.5 cm) previously equilibrated with 10 mu Tris/SO, buffer, pH 8.0,l.O mu dithiothreitol. The eluates from two batches of 50 g of mitochondrial powder were combined and applied to a DEAE-cellulose column (3.6 x 10 cm) equilibrated with the same buffer. The column was washed with 10 mu buffer and 1.0 mM dithiothreitol until the absorbance at 280 nm was below 0.1 absorbance unit and then with 75 to 100 ml of 50 mu buffer and 1.0 mM dithiothreitol. Factor B activity began to appear immediately after a pink band, and the buffer was increased to 125 mu in order to facilitate Factor B elution in a small volume. Fractions con- taining Factor B activity were pooled and the protein was collected by precipitation with solid ammonium sulfate to 60% saturation. The precipitate was suspended in 5 ml of 2 mM

Tris/S04 buffer, pH 7.5, 1.0 mu dithiothreitol, 0.1 mM EDTA, and 2% glycerol. Removal of ammonium sulfate and CM- cellulose chromatography were carried out as described by Lam et al. (3) except that 20 mM Tris/SO., buffer, pH 7.5, and 1.0 mM dithiothreitol instead of 10 mM buffer was used for elution of coupling factor activity. The active fractions from the CM-cellulose column were pooled and lyophilized to 1 to 2 ml prior to subsequent purification. Addition of glycerol minimized loss of activity during lyophilization and subse- quent purification. Concentration of Factor B by ultrafitra- tion through a DiafIo membrane (Amicon Corp.) or precipi- tation by ammonium sulfate resulted in significant loss of protein and activity.

For further purification, an ahquot containing 10 to 20 mg of Factor B (specific activity, 3 to 6) was applied to a Sephadex G-75 (40 mesh) column (2.5 X 45 cm) which had been equili- brated with 50 mM Tris/SO1 or 10 mM KH2P04 buffer, pH 7.5, containing 0.1 mu EDTA, 1.0 mM dithiothreitol, and 2.0% glycerol. A protein peak constituting 80% of the total protein and less than 5% of coupling factor activity appeared in the earlier fractions. Coupling factor activity was separated from the bulk of the protein as shown in Fig. 1. This step resulted in a lo- to 12-fold purification with 80 to 90% recovery of activity units as compared to the starting material maintained at 0” to 5°C. In over 30 such preparations, the specific activity of the pooled fractions has been in the range of 100 to 125 pm01 X min-’ X mg-‘. The overall purification from the acetone powder extract was roughly 2000-fold.

The order of the last two steps of purification could be reversed and the final product was essentially similar in spe- cific activity and gel pattern on dodecyl sulfate gels. However, purification of Factor B by CM-cellulose chromatography preceding the Sephadex filtration step was preferred since more protein could be processed in a given time without loss of resolution.

After the final step of purification, the activity in the pooled fractions containing 10 to 20 pg of protein X I&’ is stable on ice for 3 days but is lost on freezing. However, it may be stabilized by adding bovine serum albumin to 1 mg x ml-’ or

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Factor B in Mitochondrial Pi-ATP Exchange 10147

ELUTION VOLUME, ml

FIG. 1. Sephadex chromatography of Factor B. Twenty-one milligrams of Factor B protein after the CM-cellulose step are con- centrated and applied to a Sephadex G-75 column, which had been equilibrated with 50 mM, pH 7.5, Tris/SO, buffer containing 1 mM

dithiothreitol, 1 mM EDTA, and 2% glycerol. For estimation of molecular weight of Factor B, 5-mg samples of bovine serum albumin (BSA), carbonic anhydrase, and cytochrome (Crt C), along with 5 mg of Factor B were applied to the Sephadex column. The arrows indicate the elution volumes for the respective standard proteins.

increasing the glycerol concentration to 20% prior to freezing in liquid nitrogen. Under these conditions, the preparation may be repeatedly thawed and frozen without measurable loss of activity.

Properties of Factor B-The molecular weight of Factor B, estimated by the Sephadex retention method using bovine serum albumin, carbonic anhydrase and horse heart cyto- chrome c, approximated 13,000 (Fig. 1, inset). It appeared as a single band following electrophoresis under nondenaturing conditions on 7% acrylamide gels at pH 8.5 (26) or in gels containing dodecyl sulfate processed according to Weber and Osborn (24) or Swank and Munkres (25). The average mono- mer molecular weight of Factor B estimated from three ex- periments involving gel electrophoresis containing dodecyl sulfate was 13,000 in Weber and Osborn’s system, and from two experiments in the Swank and Munkres system, it was 15,000. In the latter system, horse heart myoglobin peptides (M, = 2,500 to 16,949) were used as standards. In conventional gel electrophoresis and subsequent staining with Coomassie Brilliant Blue R-250 under the Weber-Osbom (24) conditions, the Factor B band stained very poorly as described earlier (3). For example, 15 pg of the factor was barely visible while 1 pg of bovine serum albumin was brightly stained. The dye bind- ing was greater in the presence of dodecyl sulfate and has been quantitated for Factor B, ATPase inhibitor, and bovine serum albumin by measuring the area under the respective peaks after staining, and scanning the gels (Fig. 2). While the staining intensity of all three proteins was linear with protein concentration, bovine serum albumin and ATPase inhibitor stained 3 to 5 times more intensely than Factor B on a microgram basis. We have found that at least 10 pg of protein are required under routine conditions for detection of Factor B on gels containing dodecyl sulfate. Detection of low levels of Factor B (1 to 5 pg) was facilitated by labeling with fluorescamine (36) prior to gel electrophoresis. Purity of Fac- tor B preparation was further confiied by the observation that application of 15 pg of fluorescamine-labeled Factor B to acrylamide gels with dodecyl sulfate (Weber and Osbom’s (24) system) yielded a single fluorescent band which had the same relative mobility as unlabeled Factor B.

In the Swank and Munkres gel system (25), Factor B was clearly distinguishable from other low molecular weight com- ponents of the ATPase complex such as oligomycin sensitiv-

h Rm=0.45

ATPase Inhibitor

R,=0.18 OSCP

4PJ TO

FIG. 2. Densitometric traces (AcaO nm) of ATPase inhibitor, Factor B, and OSCP. The samples were electrophoresed on 12.5% acrylamide gels in the presence of sodium dodecyl sulfate as described by Swank and Munkres (25). Other details are given under “Experi- mental Procedures.” R,,, refers to mobility of the protein relative to tracking dye (TD).

ity-conferring protein (OSCP), coupling factor 6 (Fs) and the 6 and E subunits of F1. The ATPase inhibitor protein, which has been reported to have a molecular weight of 10,500 (20), migrated with an average apparent molecular weight of 5,000 (two samples) and thus was easily resolved from Factor B. Whether the presence of urea in the Swank and Munkres (25) procedure results in dissociation of the inhibitor into smaller subunits, or the anomalous behavior of the inhibitor is an artifact of this particular gel system, is not clear at present. However, deviation from linearity has been observed with other proteins in this system (25).

Coupling factor activity of the preparation is lost upon exposure to high temperatures (2 min at 75°C) (see also Ref. 3). Incubation of the factor withp-chloromercuriphenylsulfon- ate or with N-ethyhnaleimide results in loss of the activity suggesting thiol involvement. These results are consistent with the previous observations of Lam et al. (4).

Double diffusion experiments with Factor B antiserum showed a single precipitin line against Factor B fractions at all stages of purification. ETPH also gave a single line showing that Factor B preparations after the final Sephadex filtration have a single antigen. Immunoelectrophoresis experiments involving Factor B and antiserum were performed at pH 8.8 and, again, a single precipitin line was obtained as in the studies of Lam and Yang (31), indicating that the antibody was monovalent. I f there are traces of other proteins in the antigen preparation, they do not give rise to detectable anti- bodies under our experimental conditions.

AE. ATPase Complex-The preparation used in the pres- ent investigation is designated as AE. ATPase since it is derived from Factor B-depleted AE particles. The AE a ATP- ase was prepared by the procedure of Sadler et al. (a), modi- fied as follows. AE particles (10 mg/ml) were incubated in a buffer containing 0.25 M sucrose. 10 mM Tris/Cl, pH 7.8,0.1% lysolecithin, and 2.6 mM CaClz at 0°C. After 30 min, an equal volume of K/Mes buffer, pH 6.5, was added and the incubation was continued for an additional 20 min. The extracted mate- rial was collected by centrifugation as described earlier by Sadler et al. (8). The modified procedure yielded an ATPase complex with an exchange activity of 230 to 250 nmol x min-’ X mg-‘, which was stimulated 2- to 3-fold (580 to 690 nmol X min-’ x mg-‘) by the addition of Factor B. No protein was

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10148 Factor B in Mitochondrial Pi-ATP Exchange

sedimented in the absence of CaCL in the extraction medium. The yield of AE. ATPase complex was 1 to 2% of the

particle protein. Higher yields could be obtained by increasing lysolecithin concentration during extraction of AE particles, but the preparations thus obtained had lower specific activity in the Pi-ATP exchange reaction and showed less stimulation by Factor B.

Subsequent washing of the AE’ ATPase (10 mg/ml) in a buffer containing 10 mu Tris/acetate, pH 8.6, and 0.6 mM EDTA decreased the basal exchange activity further (80 to 160 nmol X min-’ X mg-I). This led to greater stimulation (4- to 6-fold) by the added factor. The extraction procedure apparently resulted in solubilization of Factor B from the ATPase complex rather than inactivation of the coupling factorper se since the wash liquid, separated by centrifugation at 100,000 X g for 1 h, showed a precipitin line with antiserum to Factor B when tested on double diffusion plates. The effectiveness of EDTA and alkaline conditions in separating Factor B from AE. ATPase is consistent with our previous experience concerning the optimal conditions for extracting Factor B from lyophilized mitochondrial acetone powder (3). It is interesting, however, that washing the ATPase complex derived from ETPn with ammonia/EDTA buffer did not cause a significant increase in Factor B dependency.

It has been reported earlier that oligomycin stimulates the energy-linked activities of AE particles at low levels and inhibits at higher levels (3, 4). A similar effect was observed with the washed AE . ATPase complex. The stimulation was optimal at 50 ng of oligomycin/mg of ATPase protein. The maximal stimulation with oligomycin was less than 2-fold, while that with Factor B was generally &fold.

Membrane Protein Fraction (AE-FO) from AE. ATPase Complex-The AE. ATPase complex preparations were ex- tracted with 3.5 M NaBr as described by Tzagoloff et al. (33). A soluble (AE-NaBr extract) and a membrane protein (AE- F,,) fraction were obtained. Two-thirds of the total protein, including all the subunits of Fr-ATPase and some of the higher molecular weight polypeptides was obtained in the extract, and nearly one-third remained in the membrane fraction. The results are similar to those obtained with OS- ATPase (33). In dodecyl sulfate gel electrophoresis, AE-Fo appeared to be similar to the membrane fractions reported by Capaldi (37) and Serrano et al. (12) in terms of number of polypeptide bands. Bands corresponding to F, subunits were not seen, which is consistent with the complete absence of ATPase activity in AE-Fo. However, oligomycin-sensitive ATPase activity could be restored by the addition of F,- ATPase to the AE-Fo fraction. The ATPase activity of the reconstituted AE e ATPase complex increased linearly as a function of added F1 up to 200 pg of FJmg of AE-Fo. Resto- ration of maximal activity required the addition of 400 to 600 pg (1.0 to 1.65 nm01)~ of FJmg of AE-Fo, which is consistent with published values for similar reconstitution using OSAT- Pase (33). Up to levels of 50 pg of Fl/mg of AE-Fo, all of the added F, remained bound to the membrane pellet. In experi- ments using higher levels of F1, it was necessary to centrifuge Fo-F1 suspensions to remove the unbound Fr.

The reconstituted ATPase activity was inhibited over 90% by 1.0 pg of oligomycin at all the levels of F, that were tested. Addition of OSCP occasionally produced up to 10% increase in ATPase activity, but did not increase oligomycin sensitivity, suggesting that the OSCP present in the membrane protein fraction may be adequate for oligomycin sensitivity.

’ The conversion of activity units to moles of protein is based on a molecular weight of 847,000 for F1-ATPase (38) and 13,000 for Factor B (present work).

Effect of Factor B on the Pi-ATP Exchange Activity of AE-F,,--Concomitant with the appearance of oligomycin-sen- sitive ATPase activity in AE-Fo supplemented with Fr, P,- ATP exchange activity also appeared, but with a low rate of 10 to 20 nmol X min-’ X mg-’ of AE-Fo (Table I).

An 8- to lo-fold stimulation of activity was generally ob- served on further addition of Factor B. The reconstituted activities were between 150 to 200 nmol x min-’ x mg-’ of AE-Fo. The stimulation was specific and was not observed if Factor B was replaced by OSCP, Fe, additional Fr, or a combination of these. Table I also shows that Factor B which had been inactivated with N-ethylmaleimide produces no increase in Pi-ATP exchange activity. This is consistent with previous data showing involvement of a functionally active -SH in Factor B.

The exchange activity of AE-Fo reconstituted with F1 was not stimulated by low or high levels of oligomycin as in the case of AE . ATPase.

Restoration of maximal Pi-ATP exchange activity to AE-F.

TABLE I

Specificity of Factor B in restoring P,-ATP exchange activity to AE-F,,.

The AE-F. fraction (0.2 mg) was preincubated with F1 and other components as indicated above in a final volume of 0.5 ml containing 100 prnol of Tricine/KOH (pH 8.0) buffer, 0.5 pmol of MgCb, 1 pmol of dithiothreitol, and 2.5 mg of bovine serum albumin for 10 min at 23°C. The reaction was initiated by adding a 0.5 ml aliquot containing 10 pmol of 32P, (2 to 3 x 10” cpm), 15 pmol of ATP, 5 pmol of ADP, and 20 pmol of MgCl*. After 15 min at 38°C the reaction was terminated by the addition of 0.5 ml of 20% trichloroacetic acid. Factor B in Line 5 was preincubated with 0.1 mM N-ethylmaleimide (indicated as NEM) at room temperature. After 15 min, 5 mM dithi- othreitol was added and the sample was transferred to ice.

Additions Exchange

FI Other nmol X min-’ X

m&T mg-’ AE-Fo

0.2 None 14

0.1 None 14 0.1 Dithiothreitol, 5 mm 15

0.1 Factor B, 1 unit 147 0.1 Factor B (NEM) 12 0.1 OSCP, 2 pg 15 0.1 OSCP, 4 /Jg 13 0.1 Fs, 2 pg 12 0.1 F6r 10 pg 15

5

F x

-L

c 200 x z E c & loo e 0.28 m-d F

r” 2

w 1.0 2.0 0 2.5 5 7.5 IO

5, nmol x mg-1 AE-F, Factor B, nmol x mg-I AE-Fe

FIG. 3. Effect of Factor B on Pi-ATP exchange activity of AE-F. reconstituted with F1. In A, AE-FU (0.25 mg) was incubated with ([?---o) or without (M) Factor B (0.62 nmol) and indi- cated levels of F, for 10 min at 23°C in the presence of 100 pmol of Tricine buffer (pH 8.0). 0.5 pmol of MgCb, 1 pmol of dithiothreitol, and 2.5 mg of bovine serum albumin in 0.5-ml volume. The reconsti- tuted complex was then assayed as described in the legend to Table I. In B, AE-F,, was incubated with two different levels of F, (0.28 nmol X mg-’ of AE-Fo, M, 1.65 nmol x rng-’ of AE-F,,, M) and indicated levels of Factor B and assayed as described in the legend to Table I.

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Factor B in Mitochondrial P,-ATP Exchange 10149

required approximately the same levels of F1 (about 1.5 nmol/ mg of AE-Fo) whether additional Factor B was present or not (Fig. 3A). When AE-F. was titrated with Factor B at two widely varying levels of F1, about 2 nmol of Factor B were required to obtain maximal activity (Fig. 3B). The level of Factor B or F, required for restoration of maximal activity thus appeared to be independent of the level of the other factor present.

Fig. 3A also shows that at subsaturating levels of F1 (up to 1 nmol x mg-’ of AE-Fo), the stimulation of exchange provided by Factor B was relatively greater than at saturating levels, leading to a sigmoid curve. A similar observation with more distinct sigmoidicity was made with the membrane protein fraction derived from ETPn (14). The membrane fraction in those experiments showed only 2-fold stimulation by Factor B in contrast to approximately IO-fold effect seen with AE-Fo. The shapes of the curves in Figs. 3A and 4 were reproducible.

Binding of Fl and Factor B to AE-Fo--In order to further test whether Factor B and F1 bind independently to AE-Fo, the latter was incubated with either FL or Factor B, and then subjected to centrifugation to remove any unbound coupling factor. The pellets were assayed for exchange activity after adding the missing coupling factor(s). It is seen from Table II that AE-Fo, which was preincubated with F1 (Line 2) regains full activity with the addition of Factor B alone. Similarly, AE-F0 preincubated’with Factor B (Line 3) shows maximal activity with F1 alone. Addition of more Factor B or F1 during the subsequent assay, over and above what is already bound to the AE-Fo, does not cause any further increase in activity (Column 4), indicating that binding of F1-ATPase and Factor B to the AE-F. is mutually independent and complete. These results are consistent with the data in Fig. 3.

The reconstituted Pi-ATP exchange activity is inhibited by carbonyl cyanide-m-chlorophenylhydrazone, 2,4-dinitrophe- nol, and a combination (and not singly) of valinomycin and Nigericin + K’ (data not shown). The sensitivities were sim- ilar to those of an intact ATPase complex made from heavy bovine heart mitochontial particles (14).

Effect of Factor B on the ATPase Activity of AE-F. + F,- The ATPase activity of the reconstituted complex was par- tially inhibited (from 3.4 to 2.7 pmol X min-’ x mg-‘) by Factor B (Fig. 4). The inhibited activity was linear with time for the duration of the assay. The inhibition is greater at F1 levels that are below saturation, leading to sigmoidicity of the curve (see also Ref. 14), as in the stimulation of exchange activity in Fig. 3A. It is unlikely that the inhibition is caused by displacement of bound F1 by Factor B, since unbound F1 would be insensitive to oligomycin, and experiments showed that the sensitivity was unaltered by the presence of Factor

TABLE II

Binding of F, and Factor B to AE-F. AE-F, (0.25 mg) was reconstituted with 100 /.tg of F, or 10 pg of

Factor B, or both, as described in the legend to Table I, centrifuged to remove unbound components, and then suspended in assay buffer. An additional 100 pg of F, or 10 pg of Factor B, or both, were supplemented wherever indicated in the table. The exchange activity was assayed as described in the legend to Table I.

Addition during assay

FI + None F, Fa$or Factor

B

Exchange (nmd x min-’ x mg-’ AE-F,,J

AE-Fo, centrifuged 0 22 0 133 AE-Fo + F,, centrifuged 21 28 151 146 AE-Fe + Factor B, centrifuged 0 125 0 135 AE-Fo, not centrifuged 0 21 0 167

(2.5 nmol x mg-I AE-Fo)

G 0.5 1.0 1.3

F, , nmol x mg-I AE-F,

FIG. 4. Effect of Factor B on the ATPase activity of AE-Fo reconstituted with FL-ATPase. AE-Fo (1 mg) was reconstituted with indicated levels of FI as described by Tzagoloff et al. (33). The protein pellets collected after centrifugation of the reconstituted complex were washed twice with sucrose/T&acetate buffer (pH 7.5) containing 1 mrvr dithiothreitol. Particles were then incubated with (M) or without (M) Factor B for 10 min at 23°C and suitable aliquots were assayed for ATPase activity as described by Tzagoloff et al. (33).

B. In one experiment containing 0.28 nmol of F1 x mg-’ of AE-Fo, addition of 0.5 pg of oligomycin lowered the reconsti- tuted ATPase activity from 0.75 to 0.08 pmol x mini’ x mg-’ of AE-Fo in the absence of Factor B, and from 0.45 to 0.05 pmol x mm-’ x mg-’ in the presence of Factor B.

Since the inhibition of ATPase activity by Factor B was low, it was necessary to exclude the presence of contaminating ATPase inhibitor (21). This was shown by the following criteria. (a) The inhibitor is clearly separated from Factor B in gels containing dodecyl sulfate processed according to Swank and Munkres. No inhibitor, or any other protein band for that matter, was seen in our Factor B preparations. (b) The inhibitory effect due to addition of Factor B was observed only with the AE. ATPase complex, not with the soluble ATPase, while the ATPase inhibitor inhibits both. (c) The ATPase inhibitor protein is heat-stable (20), but the effects of Factor B, both the stimulation of exchange and ATPase inhibition, were lost on heating at 75°C for 2 min (Table III). (d) Similarly, it is known that the inhibitor protein is resistant to the action of -SH inhibitors (see also Ref. 22) while Factor B is sensitive. Table III shows that the effects of Factor B on ATPase and exchange activities of AE . ATPase complex are lost on treatment with N-ethyhnaleimide but the inhibitor retains its activity. (e) Factor B effects, namely, inhibition of ATPase activity and stimulation of exchange activity, are reversed by the uncoupler carbonyl cyanide-m-chlorophen- ylhydrazone (Cl-CCP) (Table III), while the inhibition caused by ATPase inhibitor is still observed in the presence of un- coupler. (f) The inhibition of ATPase levels off (Fig. 4) when the added Factor B reaches the level that provides maximal stimulation of exchange activity. I f the inhibition were due to a contaminant, inhibition may be expected to increase further.

The inhibition of the ATPase activity of the reconstituted AE. ATPase complex by Factor B is similar to that seen earlier with AE particles (3). The inhibition was low (15 to 20%) since these particles have a nearly full complement of F1. Greater inhibition with the reconstituted AE.ATPase is noted only at suboptimal levels of F1. It may be noted that You and Hatefi (39) did not observe similar inhibition with their Factor B-like protein.

Effect of --SH Reagents on Reconstitution of AE-F. with F, and Factor B-It is seen in Table IV that after preincu- bation with N-ethyhnaleimide, AE-Fo still bound F1 and the resulting ATPase activity was oligomycin-sensitive. However, the exchange activity was totally lost and was not restored by the addition of excess dithiothreitol. Factor B restored roughly

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10150 Factor B in Mitochondrial P,-ATP Exchange

TABLE III Effect of Factor B on the ATPase and exchange activities of AE.

A TPase For measuring P,-ATP exchange activity, the AE.ATPase (0.2 mg)

was preincubated with Factor B or ATPase inhibitor, or both, as indicated in the table, in a final volume of 0.5 ml containing Tricine/ KOH, MgCh, dithiothreitol, and bovine serum albumin and activity was assayed as indicated in the legend to Table I. For treatment with N-ethylmaleimide, Factor B and ATPase inhibitor were incubated in the presence of 100 PM N-ethybnaleimide at room temperature for 15 min foIIowed by addition of dithiothreitol. In Experiment 2, P,-ATP exchange activity of the AE. ATPase was measured by preincubating 0.2 mg of AE.ATPase with Factor B or ATPase inhibitor in a final volume of 0.5 ml containing 50 pmol of Tris/SO, buffer (pH 7.5) and 0.5 pmol of MgCIz for 10 mm at 23°C. Cl-CCP was then added wherever indicated in the table, followed by additional incubation for 2 min at 38°C. The reaction was then initiated by adding ATP, ADP, Mg*‘, and P,, and activity was assayed as indicated in the legend to Table I. The ATPase activity was assayed by preincubating AE. ATPase (106 to 200 pg) with Factor B or ATPase inhibitor, or both, as indicated in the table, in a final volume of 0.9 ml containing 50 pmol of Tris/SO, buffer (pH 7.5) and 5 pmol of MgCL for 2 min at 38% This was followed by addition of Cl-CCP as indicated and further incubation for 2 more min at 38°C. Activity was initiated by the addition of 6 pm01 of ATP and measured for a period of 4 min at 38°C. The reaction was terminated by the addition of 0.5 ml of 20% trichloroacetic acid. After brief centrifugation, the supernatant was analyzed for inorganic phosphate.

Additions to AE. ATPaee Exchange ATPase

Experiment 1 None + Factor B, 10 erg + Factor B (75°C 2 min) + Factor B, N-ethyImaIeimide-

treated + ATPase inhibitor, 10 gg + ATPase inhibitor, 10 ag + Fac-

tor B, 10 erg + ATPase inhibitor, N-ethyhnal-

eimide-treated Experiment 2

None + Cl-CCP, IaM + Factor B, 2 cc~ + Factor B, 2 I.rg + Cl-CCP 1 PM + ATPase inhibitor, 5 s + ATPase inhibitor, 5 M + Cl-

CCP. 1 UM

nmol X nun-’ pm01 X mm-’ X x nag-’ w-’

78 4.6 409 3.5

79 4.8 73 4.2

51 1.0 245 1.0

57 1.2

33” 4.1 0.96 4.6

251” 3.0 29 4.8

1.3 1.4

“The exchange activity in this preparation when assayed in the presence of 5 ~01 of dithiothreitol and 2.5 mg of bovine serum albumin was 77 nmol x mm-’ x mg-’ and was stimulated to 437 nmol upon addition of Factor B.

50% of the exchange activity in these experiments compared to the control that was not exposed to N-ethylmaleimide. These results show an absolute dependence on this coupling factor for the reaction. Excess F, or bovine serum albumin failed to replace Factor B in the exchange reaction. This is the first demonstration of total dependence on Factor B for exchange activity in a suitably treated membrane preparation. The observation that the oligomycin sensitivity of the recon- stituted ATPase remained unaltered under these conditions indicates that Factor B is not involved in binding F1 to the membrane or in rendering the ATPase sensitive to oligomycin (Table IV).

Immunological Evidence for the Participation of Factor B in the P,-ATP Exchange-In order to detect the presence of Factor B in AE particles and derivative preparations, Ouch- terlony double diffusion experiments using antiserum to Fac- tor B were carried out (Fig. 5). Single precipitin lines were obtained against AE particles, unwashed AE. ATPase com-

plex, and its NaBr extract. No precipitin lines were obtained against AE. ATPase complex that had been washed with Tris/ acetate/EDTA buffer, pH 8.8, or AE-F. made from it. These preparations showed over 5-fold stimulation of exchange ac- tivity by added Factor B. Thus, there is a direct correlation between loss of a component that gives a precipitin reaction with anti-Factor B serum and loss of Pi-ATP exchange activ- ity. The activity is restored specifically by Factor B, the antigen. The above data also show that the dependence of the AE particles on added Factor B for activity is actually due to removal of Factor B and not to some structural change in the membrane system.

Examination of the precipitin lines on other double diffusion plates where Factor B was placed adjacent to AE particles and AE. ATPase indicated that the same immunoreactive component was present in all of them.

Apparently, Factor B diffuses from the membranous struc-

TABLE IV Effects of oligomycin and Factor B

AE-F, (0.75 mg) was preincubated at room temperature in a total volume of 0.1 ml containing 0.25 M sucrose and 10 mu Tria/acetate buffer, pH 7.5. Ten mihimolars of N-ethyhnaleimide was present where it is indicated by NEM. After 30 min, 10 pmol of dithiothreitol were added. Ahquota were withdrawn and coupling factors were added where indicated to measure P,-ATP exchange or oligomycin- sensitive ATPase activity as in Table I. For restoration of OSATPase activity, 100 pg of AE-F. was reconstituted with a limiting amount of Fi (2.5 pg) and 0.5 unita of Factor B. For the exchange assay, 0.25 mg of the AE-F. was reconstituted with 100 H of F, and 1.0 unit of Factor B. No centrifugation is necessary since unbound FI does not catalyze exchange. Assays were carried out as described under “Experimental Procedures.”

Exchange ATPaw ~_ _

AE-Fo AE-Fo + F, AE-Fo (NEM) + F,

-Factor B +Factor B -Oligomy- +Oligomy- tin tin nmol X mm”’ X mg-’ pm01 x mitt-’ x mg

0 0 0 0 32 148 0.60 0.13 0 74 0.61 0.16

FIG. 5. Immunodiffusion studies with Factor B antiserum. Precoated immunodiffusion plates containing 0.9% agar, borate/saline (0.9% NaCl solution) buffer pH 8.5, and 0.01% merthiolate was ob- tained from Miles Laboratories. Two to three milligrams of crude antiserum were placed in the central weII and 800 pg of the AE- particles (AE-Part), AE. ATPase complex, wash from the AE. ATP- ase, AE-F,,, or 100 pg of Factor B (Fact B) at the CM-cellulose step, were applied to the peripheral webs as indicated on the plate (see Table I and text for the exchange activity of some of these prepara- tions). The immunodiffusion plates were covered and placed at 2’ to 4°C in a humid environment.

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Factor B in Mitochondrial Pi-ATP Exchange 10151

tures and reacts with the antiserum as a soluble protein. The slightly alkaline condition present during immunodiffusion experiments may promote separation of Factor B from the membranes.

Single precipitin lines were seen in immunoelectrophoresis also when the antiserum was used against purified Factor B. These results would show that the antibody was monovalent. Also, if there is more than one form of Factor B in the particles, they all react similarly under the conditions for double diffusion.

On the basis of these results, it may be expected that the undepleted ATPase complex should contain Factor B. In fact, we can detect Factor B if high levels of the protein (say 50 pg) are applied to polyacrylamide gels, electrophoresed in the presence of dodecyl sulfate and stained with Coomassie Blue overnight.6 The poor staining property of Factor B requires these conditions. These samples of ATPase complex showed less than 10% increase in Pi-ATP exchange activity on addition of Factor B, and evidently had a nearly full complement of endogenous Factor. B.

DISCUSSION

Factor B-The purity of the preparation described here can be judged from several criteria: polyacrylamide gel elec- trophoresis both in the presence (in two systems) and absence of dodecyl sulfate and immunoelectrophoresis using antiserum to Factor B. No impurity was detected even using the sensitive protein detection method involving labeling with fluoresca- mine.

The preparation of Factor B obtained by Lam et al. (3) showed a minimum molecular weight of 14,600 by amino acid composition and a sedimentation coefficient of 3.11 to 3.39 S. The results suggested that the preparation was a dimer of 29,200 daltons (3). The molecular weight of the present prep- aration is about one-half that value by gel electrophoresis in dodecyl sulfate as well as Sephadex filtration, indicating that it could be a monomer. The principal difference in isolation procedure that may have led to the dissociation may be the presence of dithiothreitol right from the beginning of the purification. Factor B has -SH groups and shows activation by -SH compounds if it is isolated in the presence of EDTA instead of dithiothreitol (3). Thus, the Factor B preparation of Lam et al. (3) might have been stabilized as a dimer by disulfide formation.

The specific activity of the preparation of Lam et al. (3) was approximately 2.0. Other partially purified preparations of higher specific activity were encountered by Schuurmanns- Stekhoven et al. (40), Shankaran et al. (41) and Racker et al. (2). Since then, You and Hatefi (39) reported a highly purified Factor B-like protein of specific activity around 12. The prep- arations described here have a lo-fold higher specific activity. The assay conditions used in the different laboratories using the same type of submitochondrial particle are very similar and may not account for the large differences in activity, although it cannot be ruled out.

The properties of Factor B and You and Hatefi’s Factor B- like protein are similar in (a) ability to restore the activity of AE particles in several energy-linked reactions, (b) heat sen- sitivity (both are inactivated at 75’C in a few minutes). (c) sensitivity to -SH inhibitors, and (d) antigenicity. By private communication, Dr. Hatefi has informed us that the antiserum supplied by us cross-reacted in the Ouchterlony precipitin reaction with their Factor B-like protein. The preparation also differs in some ways. (a) Factor B binds to CM-cellulose,

” S. Joshi, J. B. Hughes, F. Shaikh, and D. R. Sanadi, unpublished data.

which is a routine procedure in its isolation. The Factor B- like activity of You and Hatefi does not bind this cation exchange resin (39). (b) Our preparation has about 10 times higher specific activity. (c) In conventional gel electrophoresis, Factor B is stained poorly compared to bovine serum albumin (3) while there appears to be no significant anomaly in dye binding with the Factor B-like protein (39), although the same staining procedure (24) was used in both laboratories. In the presence of dodecyl sulfate, staining of Factor B is distinctly more intense, but still less than that observed with bovine serum albumin or the ATPase inhibitor. (d) Factor B partially inhibited the ATPase activity of AE particles (3) and of the Factor B-depleted ATPase complex (Fig. 4). You and Hatefi (39) report lack of such effect. (e) The molecular weights of Factor B and Factor B-like protein, determined by gel filtra- tion, are close, but it is not certain they are identical. In these determinations using Sephadex G-75, we have found that the elution volume of Factor D is less than that of horse heart cytochrome c, yielding a molecular weight of 13,000. You and Hatefi’s (39) data show that the elution volume of their Factor B-like protein is slightly greater than that of cytochrome c. Unless those experiments are carried out in the same labora- tory, it is difficult to place too much significance on this difference. However, the possibility must be kept open that the Factor B-like protein may have been derived from Factor B in view of the differences in charge (shown by differences in binding to CM-cellulose), specific activity, and elution volume relative to cytochrome c. Alternatively, Factor B may be the active species in the Factor B-like preparation.

On the Role of Factor B-The data in Fig. 3 and Table II indicate that the binding sites on AE-F. for F, and Factor B are distinct and independent of each other. Even in the total absence of Factor B, the F1 bound to AE-F. is functional since the ATPase activity is inhibited by oligomycin (Table IV). It is clear from these data that Factor B cannot be a subunit of F1 and membrane-bound F1 can have an activity independent of Factor B. It is not known, however, whether F1 ever acts independently of Factor B in intact mitochondria.

In earlier work, we had demonstrated that the Pi-ATP exchange reaction is inhibited by -SH inhibitors, but the oligomycin-sensitive ATPase is insensitive and suggested in- volvement of Factor B in the exchange but not in the ATPase activity (5). The present findings (Table IV) fully substantiate this conclusion.

It is seen in Figs. 3A and 4 that the Pi-ATP exchange and ATPase activity curves become sigmoidal as a function of increasing F,. Our preferred explanation for the sigmoidicity assumes that two types of F1 binding sites exist in AE-Fo, some capable of coupling and others incapable of coupling. These sites may exist in different assemblies of subunits constituting the ATPase complex. Melnick et al. (42) have similarly considered the possibility of F1 existing in submito- chondrial particles in partly energy-coupled and partly en- ergy-uncoupled states, in order to explain the differential inhibitory effects of adenylyl imidodiphosphate (AMP-PNP) in mitochondrial and in submitochondrial particles. The data are consistent with the hypothesis that Factor B binds pref- erentially at the sites that have retained the capacity for coupled reactions. The remaining sites may have been irre- versibly damaged as a result of structural disorganization or loss of an essential component. The reversal of the Factor B- mediated partial ATPase inhibition by the uncoupler (Table III) strongly favors the above explanation.

The results described here establish for the first time that coupling factor B is a component of the Pi-ATP exchange system, but is not required for the binding of F1 to AE-Fo or

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10152 Factor B in Mitochondrial Pi-ATP Exchange

16. for the restoration of oligomycin sensitivity. Concomitant with the restoration of the exchange activity, Factor B also reduces the ATPase activity as expected from the change from an uncoupled, energy-wasting reaction to a coupled reaction. This change may occur by two possible mechanisms. One, as suggested by Racker (43), Factor B might block a H’ leak in the membrane. An alternative is the possibility that Factor B might participate in the generation of the H+ gradient from ATP (see Ref. 5). In either case, an increase in the steady state level of the H’ gradient and associated membrane po- tential (44) would result, leading to restoration of the coupled Pi-ATP exchange activity.

Racker, E., Chien, T. F., and Kandrach, A. (1975) FEBSLett. 57, 14-18

17. Joshi, S., and Sanadi, D. R. (1979) Methods Enzymol. SBF, 3&4- 391

18. 19. 20.

Fessenden-Raden, J. M. (1972) J. Biol. Chem. 247.2351-2357 Senior, A. E. (1971) Bioenergetics 2, 141-150 Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238,

3762-3769 21.

22.

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Kanner, B. I., Serrano, R., Kandrach, M. A., and Racker, E. (1976) Biochem. Biophys. Res. Commun. 69, 1050-1056

Ho&man, L. L., and Racker, E. (1970) J. Biol. Chem. 245,1336- 1344

Sanadi, D. R. (1971) in Probes of Structure and Function of Macromolecules and Membranes (Chance, B., Lee, C. P., and Blasie, J. K., ed) Vol. 1, pp. 449-452, Academic Press, New York Acknowledgment-We appreciate the excellent technical assist-

ance of Ronald Keough.

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S Joshi, J B Hughes, F Shaikh and D R Sanadireaction.

On the role of coupling factor B in the mitochondrial Pi-ATP exchange

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