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Variations on the “dilution” method for reconstituting cytochrome c oxidase into membrane vesicles
ANALYTICAL
BIOCHEMISTRY
Variations
163, loo-106 (1987)
on the “Dilution” Method for Reconstituting Oxidase into Membrane Vesicles’ JORGE RAM~REZ,
MARTHA
CALAHORRA,
AND ANTONIO
Cytochrome
c
PENA
Department of Microbiology, Institute for Cell Physiology. Universidad National Aut6noma de Mt!xico, Apartado 70-600, 04510 Mkxico, D.F., Mt!xico Received June 16, 1986 A method for the rapid incorporation of cytochrome c oxidasc into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 pg of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30°C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSCs (3)) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles. 0 1987 Academic Ress, Inc. KEY WORDS: reconstitution; cytochrome oxidase; membrane vesicles; transport; membrane potential generation.
Cytochrome c oxidase was reconstituted into liposomes several years ago by Racker (1). The procedure, still used (2), consisted of mixing liposomes with the enzyme and cholate. The detergent was then dialyzed slowly, usually overnight in a cold room, and a reconstituted preparation of the enzyme was obtained. This preparation was able to pump protons in a direction that was determined by the localization of cytochrome c. If this cytochrome was placed inside the vesicles, they pumped protons toward the inside; the opposite happened if this electron donor was placed outside the vesicles. It was also shown that this pumping of protons generated a membrane potential that could be coupled to other energy transformation systems (3). ’ This work was partially supported by a grant from the Consejo National de Ciencia y Tecnologia de M6xico.
0003-2697187 $3.00 Copyright 0 1987 by Academic Ress, Inc. All rights of reproduction in any form reserved.
Some time later, another method for reconstituting the enzyme was described; this was the “dilution” method (4) in which the enzyme was simply mixed with the liposomes and an adequate amount of cholate (0.5-0.8%), and the concentration of the detergent was decreased by diluting the mixture in the incubation medium. A reconstituted preparation of this enzyme was obtained during the study of its association with phospholipids by Eytan and Broza (5). Excellent reviews of these methods have been written by Racker (6,7). Cytochrome oxidase, incorporated into liposomes together with ATP synthetase, was found to be capable of generating an adequate electrochemical potential to drive the synthesis of ATP (3). Later, it was found (8) that cytochrome o oxidase could be incorporated into bacterial membrane vesicles and used to drive sugar transport; cy100
RECONSTITUTION
OF CYTOCHROME
tochrome c oxidase has been found also to be a very useful system for generating membrane potentials in closed membrane vesicles for different purposes (9,lO). However, it is sometimes a problem to use detergents in the reconstitution of the enzyme, especially when they have to be eliminated by long periods of dialysis. In this work, we describe a method to reconstitute the enzyme similar to that described by Eytan and Broza (5), which is actually a variation of the “dilution” method, but using no other detergent than that present during the isolation of the enzyme. The procedure consists only of substituting cholate for the detergent Tween 20 during the purification of the enzyme to produce a preparation that is incorporated instantaneously and efficiently into liposomes and other membrane preparations. MATERIALS
AND METHODS
Purification of cytochrome c oxidase. The enzyme was purified from beef heart, first following the method for preparing heavy mitochondria according to Low and Vallin (11). Submitochondrial particles were then obtained by the method of Lee and Ernster (12) and they were freed from F, (13). The extracted particles, which were essentially free of ATPase, were used for the purification of the enzyme by a procedure that is a modification of that described by Phan and Mahler for yeast (14,15) (all steps were carried out at 4°C): 1.0 g of the particles was resuspended to a concentration of 4 mg ml-’ in 1% KCl, 0.1 M potassium phosphate buffer, pH 7.4, and 2% sodium cholate. The suspension was then taken to 25% saturation with solid ammonium sulfate (144 g liter-‘). The pH was readjusted to 7.4-7.6 with 10 N NaOH, and the mixture was extracted overnight (18 h) with continuous stirring in a cold room. The following day, the mixture was centrifuged at 10,OOOg for 30 min, and the pellet was discarded. The supematant was taken to
OXIDASE
101
33% saturation with solid ammonium sulfate (47.4 g liter-‘) and it was let stand for 30 min. Then it was centrifuged at 10,OOOg for 30 min, the pellet was discarded, and the supernatant was taken to 50% saturation with solid ammonium sulfate (106.4 g liter-‘). After standing for 30 min, the mixture was centrifuged at 10,OOOg for 30 min, and the supematant was then discarded. Beyond this point, all centrifugations were carried out in the cold, at 48,OOOg, for 30 min, letting the mixtures stand for 30 min after either dissolving a pellet or changing the ammonium sulfate concentration. The procedure was the following: 1. Dissolve the pellet in 50 ml of CPA252 (50 mM potassium phosphate, pH 7.4; 1% sodium cholate; 25% saturation with ammonium sulfate). Centrifuge and discard the pellet. 2. Take to 38% saturation with solid ammonium sulfate (77.9 g liter-‘). Centrifuge and discard the supematant. 3. Dissolve the pellet in 50 ml of CPA25. Centrifuge and discard the pellet. 4. Take to 38% saturation. Centrifuge and discard the supematant. 5. Dissolve the pellet in 50 mM TPA25 (same as CPA25, but with 0.5% Tween 20 instead of cholate). Centrifuge and discard the pellet. 6. Take to 38% saturation. Centrifuge and discard the supematant. 2 Abbreviations used: CPA25, 50 mM potassium phosphate, pH 7.4, 1% sodium cholate, 25% saturation with ammonium sulfate; TPA25, 50 mM potassium phosphate, pH 7.4,0.5% Tween 20,25% saturation with ammonium sulfate; SDS, sodium dodecyl sulfate; TMPD, tetramethyl p-phenylenediamine; FCCP, p trifluoromethoxy carbonylcyanide phenylhydrazone; 1799,2: I adduct of hexafluoroacetone; Tween 20, polyoxyethylenesorbitan monolaurate; PMSF, phenylmethanesulfonyl fluoride; TPP, tetraphenylphosphonium bromide; Hepes, 4-(2-hydroxyethyl-l-piperazineethanesulfonic acid; TEA, triethanolamine; ACMA, 9-amino6-chloro-2-methoxyacridine; PC, phosphatidylcholine.
102
RAMiREZ,
CALAHORRA,
7. Dissolve pellet in 50 ml of TPA25. Centrifuge and discard the pellet. 8. Take to 37.5% saturation. Centrifuge and discard the supematant. 9. Dissolve the pellet with 2.0 ml of 50 IIIM potassium phosphate buffer, pH 7.4, with 0.5% Tween 20. Centrifuge and discard the pellet. 10. Measure protein concentration. Adjust to 6.8 mg ml-‘. Store in portions of 100 ~1 in the freezer at -70°C. Gel electrophoresis. Electrophoresis was run in an acrylamide-SDS gel (15%) (16) for 24 h at 12 mA in a Tris glycine-SDS buffer (17). To calculate molecular weights, standards of 66,000 to 14,000 D were run simultaneously. Enzyme properties. Cytochrome c was estimated from the absorbance ratio at 550/607 nm of the dithionite-reduced enzyme. Heme a3 content was measured according to Smith (18) from the spectrum of the dithionite-reduced enzyme ( 19), using an extinction coefficient at 605-630 nm of 20.5 mM-’ cm-’ (18). Spectrophotometric measurements. These were carried out in an SLM-Aminco DW-2c spectrophotometer, using l-cm light-path cuvettes. Reconstitution of the enzyme into liposomes. Usually, liposomes from acetonewashed soybean PC were prepared in different media by sonication for 5 to 10 min of varying amounts of phospholipid, using a bath sonicator (Bransonic 32). The enzyme was incorporated by simply mixing the enzyme with the liposomes under different conditions, as described under the individual experiments. Protein determination. Determinations were made by the method of Lowry et al. (20) with bovine serum albumin as a standard. A final concentration of 1.O% SDS was added to all samples to avoid the cloudiness produced by Tween 20. Oxygen consumption. A Clark oxygen electrode (Yellow Springs Instruments) con-
AND PEP;JA
netted to an adequate power supply and recorder was used. Fluorescence measurements. A commercial spectrofluorometer with two monochromators and a magnetic stirrer, connected to a recorder, was used. Measurement of TPP uptake. Uptake was measured by incubating the vesicles with ‘Hlabeled TPP+ and rapidly eliminating the free organic cation by filtration-centrifugation through a Sephadex G-50 fine column prepared in a disposable insulin syringe. The cation inside the vesicles was measured by scintillation counting. Preparation of vesicles from yeast plasma membrane. The procedure of Franzusoff and Cirillo (21) with minor modifications was used. Reagents. Most reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Oxonol V and DiSC3(3) were obtained from Molecular Probes (Junction City, OR). ACMA was a kind gift from Dr. P. Overath (Max Planck Institute, Tubingen, Germany). TPP was kindly donated by Drs. H. R. Kaback and N. Carrasco, Roche Institute of Molecular Biology (Nutley, NJ). RESULTS AND DISCUSSION
Table 1 shows some of the properties of the two isolated enzyme preparations. The absorbance ratios at 550/607 nm of the dithionite-reduced enzyme, as an indication of the cytochrome c content, were 0.54 and 0.33 for the two preparations (Table 1). The heme a3 contents were 12.2 and 11.2 nmol mg-‘. The specific activity is also shown in Table 1; it was approx 5000 nat g of O2 (min mg)- ’ when measured as the initial rate of oxygen consumption with the isolated enzyme. However, when the activity was measured after the enzyme was mixed at pH 7.0, as described in Fig. 4, the values were higher than 16,000 for both preparations in the uncoupled state. Similar results were obtained and both preparations produced similar degrees of coupling and similar gel-scanning
RECONSTITUTION TABLE
OF CYTOCHROME
1
PROPERTIES OF Two BEEF-HEART CYT~CHROME OXIDASE PREPARATIONS OBTAINED USING TWEEN 20 DURING THE ISXATION
Absorbance ratio at 5501607 nm
Preparation I
Preparation II
0.54
0.33
Content of heme a3 (nmol mg’)
12.2
Specific activity, nat g of oxygen (min mg)-’ Without PC Plus PC
5,042
4,808
16,545
16,724
Coupling ratio
4.5
11.24
4.3
Note. Preparations and analytical procedures were carried out as described under Materials and Methods. When PC was added for the measurement of specilic activity, liposomes were added at pH 7.0 as described in Fig. 5.
103
OXIDASE
As with other methods (5-7), the effectiveness of reconstitution depends on the lipid to protein ratio used. The data in Fig. 1 indicate that this is also the case when the enzyme was used with Tween 20. In this case, the highest coupling ratio was obtained when approximately 2 mg of PC in 100 ~1 of buffer was reconstituted with 17 pg of enzyme in 2.5 ~1 of buffer. Figure 2 shows the results of one of several similar experiments in which the amount of lipid (20 mg ml-‘) was kept constant while the concentration of the enzyme in the medium was varied. It was found, again, that 17 pg of enzyme was the optimum amount to reconstitute with 100 ~1 of the liposome suspension. Although reconstitution methods are usually carried out either in ice or in a cold
-6.0
loo-
patterns, which gave the bands that were usually identified for the enzyme (22,23), with several contaminants. Five more enzyme preparations were obtained, with similar properties, and their reconstitution properties were also similar. High purity does not appear to be essential to achieve satisfactory reconstitution by the procedure described here. Preliminary experiments showed the coupling of respiration with a preparation in which liposomes were mixed with cytochrome oxidase. FCCP or FCCP plus valinomycin produced a significant degree of stimulation of respiration, as found by varying the conditions of reconstitution. The data indicate that the Tween 20 reconstitution method is similar to the cholate dilution procedure (4) and to that reported by Eytan and Broza (5), who used Tween 80 to study the interaction of the enzyme with different phospholipids. It is important to note that these authors also used phosphatidylcholine as the main phospholipid.
.
3 \
TO-
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-----. \\
\ RATIO .‘.
n ----A
/’ eo-
- 4.0
/
o’
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Lo 12
mg of PC / yg
18
24
30
of enzyme
FIG. 1. Effect of the amount of phospholipid on the reconstitution of cytochrome oxidase into liposomes. PC (50 mg ml-‘) was sonicated in 100 mM K-phosphate, pH 7.0, for 5 min in a bath sonicator (Bransonic 32). Then, different dilutions of the liposome preparation were made with the same buffer. Seventeen micrograms of the enzyme in 2.5 pl was mixed with 100~~1 portions of the liposome suspensions containing 1.O, 2.0,3.0,4.0, and 5.0 mg ml-’ of PC. Reconstitution was carried out at 30°C. Respiration was measured by immediately adding 40 pl(6.8 pg of enzyme) of the reconstituted preparation to a medium containing 50 mM K-phosphate buffer, pH 7.0, 1.5 mM ascorbic acid-TEA, pH 7.0, 0.15 mM TMPD, 1 pM valinomycin, and 150 pg of cytochrome c. FCCP (1 PM) was added as an uncoupler. The final volume was 3.0 ml; the temperature was 30°C.
104
RAMiREZ,
CALAHORRA,
room, it was interesting to perform the reconstitution procedure at several temperatures. The data in Fig. 3 show that a marked improvement in the coupling ratio of respiration was observed when the temperature was raised to 30°C. When the temperature was raised further, a decrease in the coupling ratio was observed. Another factor that has to be considered during reconstitution is pH. Figure 4 shows that reconstitution occurred more effectively at pH 7.0-8.0, which were the highest values tested. Although coupling of respiration may indicate that the enzyme was functioning in a coupled state, its ability to generate a membrane potential was tested in two ways. The first was the measurement of the quenching of the fluorescence of a cyanine, DiSC3(3). As shown in Fig. 5, the presence of the electron-donor system, ascorbate-TMPD-cytochrome c, produced an effective quenching of fluorescence that could be inhibited or reverted by cyanide or FCCP. Table 2 shows
AND PENA
100 1
+IyM a-e-0
FCCP
l I.
/
t
/ ,’
Coupled
J
,
I 0
I
/.\ \ RATIO
‘\ ’
respirotlon
r
I
20
Temperature.
n a,
Lo
40
OC
FIG. 3. Effect of temperature on reconstitution of beef-heart cytochrome oxidase into liposomes by the Tween 20 dilution method. PC (20 mg ml-‘) was sonicated for 5 min in 100 mM K-phosphate, pH 7.0, for 5 min, using a bath sonicator. Then lOO-~1 portions of the liposomes were equilibrated to the indicated tempcratures and mixed with 17 pg of enzyme. Immediately after mixing, respiration was measured as described in Fig. 2, using 40 ~1 of the reconstituted l&some suspension. Respiration was measured at 30°C.
that these conditions produced similar results when the uptake of [3H]TPP was measured. 6 Finally, if reconstitution of cytochrome 200 . oxidase is to be used for purposes other than + 1 JM FCCP studying the functional properties of the en/ .; 160 zyme, it is interesting to test its behavior in vesicles other than liposomes. To this pur0" 120 pose, vesicles prepared essentially according 5 to the procedure of Franzusoff and Cirillo 0, a0 (2 1) were prepared from yeast. It was found that these vesicles could also incorporate the 5 40 enzyme, apparently in a functional way, since respiratory ratios of vesicles in the pres7 0I ence of 1 I.IM FCCP over those in its absence yg of enzyme/mg of PC of from 1.8 to 2.4 were obtained in different experiments. Results also showed that the FIG. 2. Effect of enzyme concentration on coupling of enzyme was capable of producing a negative respiration of cytochrome oxidase incorporated into liposomes by the Tween 20 dilution method. PC (20 mg potential inside the cell (unpublished), indiml-‘) was sonicated in 100 mM potassium phosphate, cating that it was able to pump protons topH 7.0, for 5 min, using a bath sonicator. Then, 100~~1 ward the outside of the vesicles, as expected portions of this suspension mixed with 6.8, 17,22.8,34, or 51 pg of enzyme protein at 30°C. Respiration was from previous experiments ( l-6). The data indicate the effectiveness of the measured immediately after mixing as described in Fig. I, using 40 ~1 of liposomes. modification of the dilution method for re-
1
RECONSTITUTION
120
OF CYTOCHROME
105
OXIDASE TABLE 2
-I
UPTAKEOF [‘H]TPP BYLIPOSOMESORVESICLES FROMPLASMAMEMBRANEOFYEASTWITH &TXHROME OXIDASE~NCORFWATION nmol mg-’ OfPC Complete No ascorbate NaCN FCCP
FIG. 4. Effect of pH on reconstitution of cytochrome oxidase into liposomes by the Tween 20 dilution method. PC (20 mg ml-‘) was sonicated for 5 min in 100 mM of succinate-TEA, pH 4.0 or 5.0, Mes-TEA, pH 6.0, Hepes-TEA, pH 7.0, or Tricine-TEA, pH 8.0, and was equilibrated to 30°C. Then 100~rl portions of the suspensions were mixed with 17 pg of enzyme and respiration was measured immediately, as described in Fig. 2, using 40 ~1 of liposomes.
constitution of membrane enzymes (4,5). Ey-tan and Broza also showed that the charge of the lipids is important both for the interaction and for the degree of coupling of the enzyme activity in the liposomes. In addi-
I
2 min
FIG. 5. Membrane potential, followed by the quenching of fluorescence of the cyanine DiSCs(3) in liposomes with cytochrome oxidase incorporated by the Tween 20 dilution method. Liposomes were prepared at 3O”C, as described in Fig. 4. Then 20 pl of liposome suspension was mixed with 50 mM KHzPOd, pH 7.0, in a final volume of 2.0 ml and 0.5 pM DiSCJ(3) was added, followed by substrate (0.5 mM ascorbate-TEA, pH 7.0, 0.05 mM TMPD, and 60 pg of cytochrome c) and either 1 pM FCCP or 50 pM NaCN. Fluorescence was followed at 540-580 nm.
48 12 15 12.4
Note. Liposomes were prepared as described in Fig. 5. The suspension (20 ~1) was mixed with 50 mM potassium phosphate buffer, pH 7.0, 50 jhM TMPD, 60 pg of cytochrome c, and other indicated additions in a final volume of 300 ~1 at 30°C. After 2 min, 2 pM TPP was added, and after 3 more min, the mixture was passed by centrifugation at 3500 rpm for 2 min through a column of 1 ml of Sephadex G-50 fine in a disposable insulin syringe, from which excess liquid had been eliminated by suction. The liquid coming out of the column was measured to correct results for dilution; an aliquot of it was placed on a filter paper, dried, and counted in a scintillaton counter.
tion, as expected for almost any reconstitution or biological system, results depended on a series of factors, such as pH, temperature, protein to lipid ratio, and salt concentration. From the results obtained, a general method for reconstitution of this enzyme is the following: Liposomes from acetonewashed soybean PC are prepared in 100 mM Tricine-TEA buffer, pH 7.0-8.0, by sonicating 20 mg ml-’ for 5 to 10 min in a bath sonicator (Bransonic 32 or similar). Then 100 ~1 of the liposome suspension (or vesicles) is mixed with 17 pg of the enzyme in 2.5 ~1 at 30°C. However, other systems may require their own conditions. Another important factor is that the procedure seems to work, at least in the system tested, with vesicles prepared from liposomes of phosphatidylcholine and plasma membranes from yeast. The results of respiration measurements indicate some degree of coupling, and it was found that the enzyme incorporated into these vesicles was capable of generating an electrochemical potential, with
106
RAMiREZ,
CALAHORRA,
an electric component, that was negative inside. This finding is in agreement with the expected functioning of the enzyme, pump ing protons to the outside when cytochrome c was added to the outside. This electrochemical potential was also found to be useful for driving ion transport into the vesicles (unpublished).
1. 2.
3.
4. 5. 6. 7.
8.
AND PENA
9. Driessen, A. J. M., De Vrij, J. M., and Konings, W. N. (1985) Proc. Natl. Acad. Sci. USA82, 7555-7559. 10. Hirata, H., Sone, N., Yoshida, M., and Kagawa, Y.
(1977)J. Supramol.Struct.6,77-84. 11. Low, H.. and Vallin, J. (1963) B&him. Bioohys. _
Acta69,361-364.
12. Lee, C. P., and Emster, L. (1967) in Methodsin Enzymology (Estabrook, R. W., and Pullman, M. E., Eds.), Vol. 10, pp. 543-548, Academic Press, New York. 13. Tuena de G6mez-Puyou, M., and Gomez-Puyou, REFERENCES A. (1977) Arch.Biochem. Biophys.182,82-86. 14. Phan, S. H., and Mahler, H. R. (1976) J. Biol. Racker, E. (1972) J. Membr.Biol. 10,221-235. Chem.251,257-269. Hinkle, P. C. (1979) in Methods in Enzymology 15. Phan, S. H., and Mahler, H. R. (1976) J. Biol. (Fleischer, S., and Packer, L., Eds.), Vol. 55, pp. Chem.251,270-276. 748-75 1, Academic Press, New York. 16. Fuller, S. D., Darley-Usmar, V. M., and Capaldi, HinWe, P. C. (1976) in Mitochondria, Bioenergetics, R. A. (198 1) Biochemistry 20,7046-7053. Biogenesis and Membrane Structure (Packer, L., 17. Laemli, V. K. (1970) Nature (London) 227, and G6mez-Puyou, A., Eds.), pp. 183-192, Aca680-685. demic Press, New York. 18. Smith, L. (1978) in Methods in Enzymology Racker, E., Chien, T. F., and Kandrach, A. (1975) (Fleischer, S., and Packer, L., Eds.), Vol. 53, pp. FEBSLett. 57, 14-16. 202-2 13, Academic Press, New York. Eytan, G. D., and Broza, R. (1978) J. Biol. Chem. 19. Rieske, S. (1967) in Methods in Enzymology (Esta253,3196-3202. brook, R. W., and Pullman, M. E., Eds.), Vol. 10, Racker, E. (1979) in Methods in Enzymology pp. 488-493, Academic Press, New York. (Fleischer, S., and Packer, L., Eds.), Vol. 55, pp. 20. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and 699-7 11, Academic Press, New York. Randall, R. (195 1) J. Biol. Chem.193,265-275. Racker, E. (1985) Reconstitutions of Transporters, 21. Franzusoff, A., and Cirillo, V. P. (1983) J. Biol. Chem.258,3608-3614. Receptors, and Pathological States, Academic 22. Azzi, A, (1980) Biochim. Biophys. Acta 594, Press, Orlando, FL. 231-252. Matsushita, K., Patel, L., Gennis, R. B., and Kaback, H. R. (1983) Proc.Natl.Acad.Sci.USA80, 23. Downer, N. W., Robinson, N. C., and Capaldi, R. A. (1976) Biochemistry l&2930-2936. 4889-4893.
BIOCHEMISTRY
Variations
163, loo-106 (1987)
on the “Dilution” Method for Reconstituting Oxidase into Membrane Vesicles’ JORGE RAM~REZ,
MARTHA
CALAHORRA,
AND ANTONIO
Cytochrome
c
PENA
Department of Microbiology, Institute for Cell Physiology. Universidad National Aut6noma de Mt!xico, Apartado 70-600, 04510 Mkxico, D.F., Mt!xico Received June 16, 1986 A method for the rapid incorporation of cytochrome c oxidasc into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 pg of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30°C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSCs (3)) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles. 0 1987 Academic Ress, Inc. KEY WORDS: reconstitution; cytochrome oxidase; membrane vesicles; transport; membrane potential generation.
Cytochrome c oxidase was reconstituted into liposomes several years ago by Racker (1). The procedure, still used (2), consisted of mixing liposomes with the enzyme and cholate. The detergent was then dialyzed slowly, usually overnight in a cold room, and a reconstituted preparation of the enzyme was obtained. This preparation was able to pump protons in a direction that was determined by the localization of cytochrome c. If this cytochrome was placed inside the vesicles, they pumped protons toward the inside; the opposite happened if this electron donor was placed outside the vesicles. It was also shown that this pumping of protons generated a membrane potential that could be coupled to other energy transformation systems (3). ’ This work was partially supported by a grant from the Consejo National de Ciencia y Tecnologia de M6xico.
0003-2697187 $3.00 Copyright 0 1987 by Academic Ress, Inc. All rights of reproduction in any form reserved.
Some time later, another method for reconstituting the enzyme was described; this was the “dilution” method (4) in which the enzyme was simply mixed with the liposomes and an adequate amount of cholate (0.5-0.8%), and the concentration of the detergent was decreased by diluting the mixture in the incubation medium. A reconstituted preparation of this enzyme was obtained during the study of its association with phospholipids by Eytan and Broza (5). Excellent reviews of these methods have been written by Racker (6,7). Cytochrome oxidase, incorporated into liposomes together with ATP synthetase, was found to be capable of generating an adequate electrochemical potential to drive the synthesis of ATP (3). Later, it was found (8) that cytochrome o oxidase could be incorporated into bacterial membrane vesicles and used to drive sugar transport; cy100
RECONSTITUTION
OF CYTOCHROME
tochrome c oxidase has been found also to be a very useful system for generating membrane potentials in closed membrane vesicles for different purposes (9,lO). However, it is sometimes a problem to use detergents in the reconstitution of the enzyme, especially when they have to be eliminated by long periods of dialysis. In this work, we describe a method to reconstitute the enzyme similar to that described by Eytan and Broza (5), which is actually a variation of the “dilution” method, but using no other detergent than that present during the isolation of the enzyme. The procedure consists only of substituting cholate for the detergent Tween 20 during the purification of the enzyme to produce a preparation that is incorporated instantaneously and efficiently into liposomes and other membrane preparations. MATERIALS
AND METHODS
Purification of cytochrome c oxidase. The enzyme was purified from beef heart, first following the method for preparing heavy mitochondria according to Low and Vallin (11). Submitochondrial particles were then obtained by the method of Lee and Ernster (12) and they were freed from F, (13). The extracted particles, which were essentially free of ATPase, were used for the purification of the enzyme by a procedure that is a modification of that described by Phan and Mahler for yeast (14,15) (all steps were carried out at 4°C): 1.0 g of the particles was resuspended to a concentration of 4 mg ml-’ in 1% KCl, 0.1 M potassium phosphate buffer, pH 7.4, and 2% sodium cholate. The suspension was then taken to 25% saturation with solid ammonium sulfate (144 g liter-‘). The pH was readjusted to 7.4-7.6 with 10 N NaOH, and the mixture was extracted overnight (18 h) with continuous stirring in a cold room. The following day, the mixture was centrifuged at 10,OOOg for 30 min, and the pellet was discarded. The supematant was taken to
OXIDASE
101
33% saturation with solid ammonium sulfate (47.4 g liter-‘) and it was let stand for 30 min. Then it was centrifuged at 10,OOOg for 30 min, the pellet was discarded, and the supernatant was taken to 50% saturation with solid ammonium sulfate (106.4 g liter-‘). After standing for 30 min, the mixture was centrifuged at 10,OOOg for 30 min, and the supematant was then discarded. Beyond this point, all centrifugations were carried out in the cold, at 48,OOOg, for 30 min, letting the mixtures stand for 30 min after either dissolving a pellet or changing the ammonium sulfate concentration. The procedure was the following: 1. Dissolve the pellet in 50 ml of CPA252 (50 mM potassium phosphate, pH 7.4; 1% sodium cholate; 25% saturation with ammonium sulfate). Centrifuge and discard the pellet. 2. Take to 38% saturation with solid ammonium sulfate (77.9 g liter-‘). Centrifuge and discard the supematant. 3. Dissolve the pellet in 50 ml of CPA25. Centrifuge and discard the pellet. 4. Take to 38% saturation. Centrifuge and discard the supematant. 5. Dissolve the pellet in 50 mM TPA25 (same as CPA25, but with 0.5% Tween 20 instead of cholate). Centrifuge and discard the pellet. 6. Take to 38% saturation. Centrifuge and discard the supematant. 2 Abbreviations used: CPA25, 50 mM potassium phosphate, pH 7.4, 1% sodium cholate, 25% saturation with ammonium sulfate; TPA25, 50 mM potassium phosphate, pH 7.4,0.5% Tween 20,25% saturation with ammonium sulfate; SDS, sodium dodecyl sulfate; TMPD, tetramethyl p-phenylenediamine; FCCP, p trifluoromethoxy carbonylcyanide phenylhydrazone; 1799,2: I adduct of hexafluoroacetone; Tween 20, polyoxyethylenesorbitan monolaurate; PMSF, phenylmethanesulfonyl fluoride; TPP, tetraphenylphosphonium bromide; Hepes, 4-(2-hydroxyethyl-l-piperazineethanesulfonic acid; TEA, triethanolamine; ACMA, 9-amino6-chloro-2-methoxyacridine; PC, phosphatidylcholine.
102
RAMiREZ,
CALAHORRA,
7. Dissolve pellet in 50 ml of TPA25. Centrifuge and discard the pellet. 8. Take to 37.5% saturation. Centrifuge and discard the supematant. 9. Dissolve the pellet with 2.0 ml of 50 IIIM potassium phosphate buffer, pH 7.4, with 0.5% Tween 20. Centrifuge and discard the pellet. 10. Measure protein concentration. Adjust to 6.8 mg ml-‘. Store in portions of 100 ~1 in the freezer at -70°C. Gel electrophoresis. Electrophoresis was run in an acrylamide-SDS gel (15%) (16) for 24 h at 12 mA in a Tris glycine-SDS buffer (17). To calculate molecular weights, standards of 66,000 to 14,000 D were run simultaneously. Enzyme properties. Cytochrome c was estimated from the absorbance ratio at 550/607 nm of the dithionite-reduced enzyme. Heme a3 content was measured according to Smith (18) from the spectrum of the dithionite-reduced enzyme ( 19), using an extinction coefficient at 605-630 nm of 20.5 mM-’ cm-’ (18). Spectrophotometric measurements. These were carried out in an SLM-Aminco DW-2c spectrophotometer, using l-cm light-path cuvettes. Reconstitution of the enzyme into liposomes. Usually, liposomes from acetonewashed soybean PC were prepared in different media by sonication for 5 to 10 min of varying amounts of phospholipid, using a bath sonicator (Bransonic 32). The enzyme was incorporated by simply mixing the enzyme with the liposomes under different conditions, as described under the individual experiments. Protein determination. Determinations were made by the method of Lowry et al. (20) with bovine serum albumin as a standard. A final concentration of 1.O% SDS was added to all samples to avoid the cloudiness produced by Tween 20. Oxygen consumption. A Clark oxygen electrode (Yellow Springs Instruments) con-
AND PEP;JA
netted to an adequate power supply and recorder was used. Fluorescence measurements. A commercial spectrofluorometer with two monochromators and a magnetic stirrer, connected to a recorder, was used. Measurement of TPP uptake. Uptake was measured by incubating the vesicles with ‘Hlabeled TPP+ and rapidly eliminating the free organic cation by filtration-centrifugation through a Sephadex G-50 fine column prepared in a disposable insulin syringe. The cation inside the vesicles was measured by scintillation counting. Preparation of vesicles from yeast plasma membrane. The procedure of Franzusoff and Cirillo (21) with minor modifications was used. Reagents. Most reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Oxonol V and DiSC3(3) were obtained from Molecular Probes (Junction City, OR). ACMA was a kind gift from Dr. P. Overath (Max Planck Institute, Tubingen, Germany). TPP was kindly donated by Drs. H. R. Kaback and N. Carrasco, Roche Institute of Molecular Biology (Nutley, NJ). RESULTS AND DISCUSSION
Table 1 shows some of the properties of the two isolated enzyme preparations. The absorbance ratios at 550/607 nm of the dithionite-reduced enzyme, as an indication of the cytochrome c content, were 0.54 and 0.33 for the two preparations (Table 1). The heme a3 contents were 12.2 and 11.2 nmol mg-‘. The specific activity is also shown in Table 1; it was approx 5000 nat g of O2 (min mg)- ’ when measured as the initial rate of oxygen consumption with the isolated enzyme. However, when the activity was measured after the enzyme was mixed at pH 7.0, as described in Fig. 4, the values were higher than 16,000 for both preparations in the uncoupled state. Similar results were obtained and both preparations produced similar degrees of coupling and similar gel-scanning
RECONSTITUTION TABLE
OF CYTOCHROME
1
PROPERTIES OF Two BEEF-HEART CYT~CHROME OXIDASE PREPARATIONS OBTAINED USING TWEEN 20 DURING THE ISXATION
Absorbance ratio at 5501607 nm
Preparation I
Preparation II
0.54
0.33
Content of heme a3 (nmol mg’)
12.2
Specific activity, nat g of oxygen (min mg)-’ Without PC Plus PC
5,042
4,808
16,545
16,724
Coupling ratio
4.5
11.24
4.3
Note. Preparations and analytical procedures were carried out as described under Materials and Methods. When PC was added for the measurement of specilic activity, liposomes were added at pH 7.0 as described in Fig. 5.
103
OXIDASE
As with other methods (5-7), the effectiveness of reconstitution depends on the lipid to protein ratio used. The data in Fig. 1 indicate that this is also the case when the enzyme was used with Tween 20. In this case, the highest coupling ratio was obtained when approximately 2 mg of PC in 100 ~1 of buffer was reconstituted with 17 pg of enzyme in 2.5 ~1 of buffer. Figure 2 shows the results of one of several similar experiments in which the amount of lipid (20 mg ml-‘) was kept constant while the concentration of the enzyme in the medium was varied. It was found, again, that 17 pg of enzyme was the optimum amount to reconstitute with 100 ~1 of the liposome suspension. Although reconstitution methods are usually carried out either in ice or in a cold
-6.0
loo-
patterns, which gave the bands that were usually identified for the enzyme (22,23), with several contaminants. Five more enzyme preparations were obtained, with similar properties, and their reconstitution properties were also similar. High purity does not appear to be essential to achieve satisfactory reconstitution by the procedure described here. Preliminary experiments showed the coupling of respiration with a preparation in which liposomes were mixed with cytochrome oxidase. FCCP or FCCP plus valinomycin produced a significant degree of stimulation of respiration, as found by varying the conditions of reconstitution. The data indicate that the Tween 20 reconstitution method is similar to the cholate dilution procedure (4) and to that reported by Eytan and Broza (5), who used Tween 80 to study the interaction of the enzyme with different phospholipids. It is important to note that these authors also used phosphatidylcholine as the main phospholipid.
.
3 \
TO-
I’ I
-----. \\
\ RATIO .‘.
n ----A
/’ eo-
- 4.0
/
o’
6
Lo 12
mg of PC / yg
18
24
30
of enzyme
FIG. 1. Effect of the amount of phospholipid on the reconstitution of cytochrome oxidase into liposomes. PC (50 mg ml-‘) was sonicated in 100 mM K-phosphate, pH 7.0, for 5 min in a bath sonicator (Bransonic 32). Then, different dilutions of the liposome preparation were made with the same buffer. Seventeen micrograms of the enzyme in 2.5 pl was mixed with 100~~1 portions of the liposome suspensions containing 1.O, 2.0,3.0,4.0, and 5.0 mg ml-’ of PC. Reconstitution was carried out at 30°C. Respiration was measured by immediately adding 40 pl(6.8 pg of enzyme) of the reconstituted preparation to a medium containing 50 mM K-phosphate buffer, pH 7.0, 1.5 mM ascorbic acid-TEA, pH 7.0, 0.15 mM TMPD, 1 pM valinomycin, and 150 pg of cytochrome c. FCCP (1 PM) was added as an uncoupler. The final volume was 3.0 ml; the temperature was 30°C.
104
RAMiREZ,
CALAHORRA,
room, it was interesting to perform the reconstitution procedure at several temperatures. The data in Fig. 3 show that a marked improvement in the coupling ratio of respiration was observed when the temperature was raised to 30°C. When the temperature was raised further, a decrease in the coupling ratio was observed. Another factor that has to be considered during reconstitution is pH. Figure 4 shows that reconstitution occurred more effectively at pH 7.0-8.0, which were the highest values tested. Although coupling of respiration may indicate that the enzyme was functioning in a coupled state, its ability to generate a membrane potential was tested in two ways. The first was the measurement of the quenching of the fluorescence of a cyanine, DiSC3(3). As shown in Fig. 5, the presence of the electron-donor system, ascorbate-TMPD-cytochrome c, produced an effective quenching of fluorescence that could be inhibited or reverted by cyanide or FCCP. Table 2 shows
AND PENA
100 1
+IyM a-e-0
FCCP
l I.
/
t
/ ,’
Coupled
J
,
I 0
I
/.\ \ RATIO
‘\ ’
respirotlon
r
I
20
Temperature.
n a,
Lo
40
OC
FIG. 3. Effect of temperature on reconstitution of beef-heart cytochrome oxidase into liposomes by the Tween 20 dilution method. PC (20 mg ml-‘) was sonicated for 5 min in 100 mM K-phosphate, pH 7.0, for 5 min, using a bath sonicator. Then lOO-~1 portions of the liposomes were equilibrated to the indicated tempcratures and mixed with 17 pg of enzyme. Immediately after mixing, respiration was measured as described in Fig. 2, using 40 ~1 of the reconstituted l&some suspension. Respiration was measured at 30°C.
that these conditions produced similar results when the uptake of [3H]TPP was measured. 6 Finally, if reconstitution of cytochrome 200 . oxidase is to be used for purposes other than + 1 JM FCCP studying the functional properties of the en/ .; 160 zyme, it is interesting to test its behavior in vesicles other than liposomes. To this pur0" 120 pose, vesicles prepared essentially according 5 to the procedure of Franzusoff and Cirillo 0, a0 (2 1) were prepared from yeast. It was found that these vesicles could also incorporate the 5 40 enzyme, apparently in a functional way, since respiratory ratios of vesicles in the pres7 0I ence of 1 I.IM FCCP over those in its absence yg of enzyme/mg of PC of from 1.8 to 2.4 were obtained in different experiments. Results also showed that the FIG. 2. Effect of enzyme concentration on coupling of enzyme was capable of producing a negative respiration of cytochrome oxidase incorporated into liposomes by the Tween 20 dilution method. PC (20 mg potential inside the cell (unpublished), indiml-‘) was sonicated in 100 mM potassium phosphate, cating that it was able to pump protons topH 7.0, for 5 min, using a bath sonicator. Then, 100~~1 ward the outside of the vesicles, as expected portions of this suspension mixed with 6.8, 17,22.8,34, or 51 pg of enzyme protein at 30°C. Respiration was from previous experiments ( l-6). The data indicate the effectiveness of the measured immediately after mixing as described in Fig. I, using 40 ~1 of liposomes. modification of the dilution method for re-
1
RECONSTITUTION
120
OF CYTOCHROME
105
OXIDASE TABLE 2
-I
UPTAKEOF [‘H]TPP BYLIPOSOMESORVESICLES FROMPLASMAMEMBRANEOFYEASTWITH &TXHROME OXIDASE~NCORFWATION nmol mg-’ OfPC Complete No ascorbate NaCN FCCP
FIG. 4. Effect of pH on reconstitution of cytochrome oxidase into liposomes by the Tween 20 dilution method. PC (20 mg ml-‘) was sonicated for 5 min in 100 mM of succinate-TEA, pH 4.0 or 5.0, Mes-TEA, pH 6.0, Hepes-TEA, pH 7.0, or Tricine-TEA, pH 8.0, and was equilibrated to 30°C. Then 100~rl portions of the suspensions were mixed with 17 pg of enzyme and respiration was measured immediately, as described in Fig. 2, using 40 ~1 of liposomes.
constitution of membrane enzymes (4,5). Ey-tan and Broza also showed that the charge of the lipids is important both for the interaction and for the degree of coupling of the enzyme activity in the liposomes. In addi-
I
2 min
FIG. 5. Membrane potential, followed by the quenching of fluorescence of the cyanine DiSCs(3) in liposomes with cytochrome oxidase incorporated by the Tween 20 dilution method. Liposomes were prepared at 3O”C, as described in Fig. 4. Then 20 pl of liposome suspension was mixed with 50 mM KHzPOd, pH 7.0, in a final volume of 2.0 ml and 0.5 pM DiSCJ(3) was added, followed by substrate (0.5 mM ascorbate-TEA, pH 7.0, 0.05 mM TMPD, and 60 pg of cytochrome c) and either 1 pM FCCP or 50 pM NaCN. Fluorescence was followed at 540-580 nm.
48 12 15 12.4
Note. Liposomes were prepared as described in Fig. 5. The suspension (20 ~1) was mixed with 50 mM potassium phosphate buffer, pH 7.0, 50 jhM TMPD, 60 pg of cytochrome c, and other indicated additions in a final volume of 300 ~1 at 30°C. After 2 min, 2 pM TPP was added, and after 3 more min, the mixture was passed by centrifugation at 3500 rpm for 2 min through a column of 1 ml of Sephadex G-50 fine in a disposable insulin syringe, from which excess liquid had been eliminated by suction. The liquid coming out of the column was measured to correct results for dilution; an aliquot of it was placed on a filter paper, dried, and counted in a scintillaton counter.
tion, as expected for almost any reconstitution or biological system, results depended on a series of factors, such as pH, temperature, protein to lipid ratio, and salt concentration. From the results obtained, a general method for reconstitution of this enzyme is the following: Liposomes from acetonewashed soybean PC are prepared in 100 mM Tricine-TEA buffer, pH 7.0-8.0, by sonicating 20 mg ml-’ for 5 to 10 min in a bath sonicator (Bransonic 32 or similar). Then 100 ~1 of the liposome suspension (or vesicles) is mixed with 17 pg of the enzyme in 2.5 ~1 at 30°C. However, other systems may require their own conditions. Another important factor is that the procedure seems to work, at least in the system tested, with vesicles prepared from liposomes of phosphatidylcholine and plasma membranes from yeast. The results of respiration measurements indicate some degree of coupling, and it was found that the enzyme incorporated into these vesicles was capable of generating an electrochemical potential, with
106
RAMiREZ,
CALAHORRA,
an electric component, that was negative inside. This finding is in agreement with the expected functioning of the enzyme, pump ing protons to the outside when cytochrome c was added to the outside. This electrochemical potential was also found to be useful for driving ion transport into the vesicles (unpublished).
1. 2.
3.
4. 5. 6. 7.
8.
AND PENA
9. Driessen, A. J. M., De Vrij, J. M., and Konings, W. N. (1985) Proc. Natl. Acad. Sci. USA82, 7555-7559. 10. Hirata, H., Sone, N., Yoshida, M., and Kagawa, Y.
(1977)J. Supramol.Struct.6,77-84. 11. Low, H.. and Vallin, J. (1963) B&him. Bioohys. _
Acta69,361-364.
12. Lee, C. P., and Emster, L. (1967) in Methodsin Enzymology (Estabrook, R. W., and Pullman, M. E., Eds.), Vol. 10, pp. 543-548, Academic Press, New York. 13. Tuena de G6mez-Puyou, M., and Gomez-Puyou, REFERENCES A. (1977) Arch.Biochem. Biophys.182,82-86. 14. Phan, S. H., and Mahler, H. R. (1976) J. Biol. Racker, E. (1972) J. Membr.Biol. 10,221-235. Chem.251,257-269. Hinkle, P. C. (1979) in Methods in Enzymology 15. Phan, S. H., and Mahler, H. R. (1976) J. Biol. (Fleischer, S., and Packer, L., Eds.), Vol. 55, pp. Chem.251,270-276. 748-75 1, Academic Press, New York. 16. Fuller, S. D., Darley-Usmar, V. M., and Capaldi, HinWe, P. C. (1976) in Mitochondria, Bioenergetics, R. A. (198 1) Biochemistry 20,7046-7053. Biogenesis and Membrane Structure (Packer, L., 17. Laemli, V. K. (1970) Nature (London) 227, and G6mez-Puyou, A., Eds.), pp. 183-192, Aca680-685. demic Press, New York. 18. Smith, L. (1978) in Methods in Enzymology Racker, E., Chien, T. F., and Kandrach, A. (1975) (Fleischer, S., and Packer, L., Eds.), Vol. 53, pp. FEBSLett. 57, 14-16. 202-2 13, Academic Press, New York. Eytan, G. D., and Broza, R. (1978) J. Biol. Chem. 19. Rieske, S. (1967) in Methods in Enzymology (Esta253,3196-3202. brook, R. W., and Pullman, M. E., Eds.), Vol. 10, Racker, E. (1979) in Methods in Enzymology pp. 488-493, Academic Press, New York. (Fleischer, S., and Packer, L., Eds.), Vol. 55, pp. 20. Lowry, 0. H., Rosebrough, N. J., Fat-r, A. L., and 699-7 11, Academic Press, New York. Randall, R. (195 1) J. Biol. Chem.193,265-275. Racker, E. (1985) Reconstitutions of Transporters, 21. Franzusoff, A., and Cirillo, V. P. (1983) J. Biol. Chem.258,3608-3614. Receptors, and Pathological States, Academic 22. Azzi, A, (1980) Biochim. Biophys. Acta 594, Press, Orlando, FL. 231-252. Matsushita, K., Patel, L., Gennis, R. B., and Kaback, H. R. (1983) Proc.Natl.Acad.Sci.USA80, 23. Downer, N. W., Robinson, N. C., and Capaldi, R. A. (1976) Biochemistry l&2930-2936. 4889-4893.