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Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 218, No. 2, October 15, pp. 525430, 1982
Influence
of Membrane Fluidity on Transport Mediated Ubiquinones through Phospholipid Vesicles
FRANCISCO Departamento
J. ARANDA
Znterfmultatiuo
JUAN
AND
de Bioquimim,
Facultad
Received
January
25, 1982,
C. GGMEZ-FERNANDEZ’
de Medicinn,
Espinardo,
Murcia,
by
Uniuersidad
de Murcia,
Apartado
61,
Spain
and in revised
form
June
6, 1982
Coenzymes Q1,, and Q3 are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ,,, and CoQQ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.
Coenzyme Q10 (ubiquinone, UQ)’ is a component of the electron transport chain which appears to be involved in the transfer of electrons between respiratory complex I (or II) and respiratory complex III in the inner mitochondrial membrane (see (l), for a review). CoQ has been implied in the mechanism of vectorial proton translocation coupled to electron transport in mitochondria (2). However, the exact localization of ubiquinones in the membrane and the molecular mechanisms of electron and proton transport are still a matter of discussion. Hauska and co-workers (3), using ferricyanide trapped inside quinone-containing lipid vesicles, adding dithionite externally have measured the rate of ferricyanide reduction. Their conclusions were that quinones with side chains longer than those of Qz were more efficient in electron and proton translocation. They suggested that the quinones with longer isoprenyl 1 To whom all correspondence should ’ Abbreviations used: Q, ubiquinone; mitoylphosphatidylcholine.
be addressed. DPPC, dipal-
525
side chains formed aggregates within the phospholipid bilayer. NMR studies recently have shown that ubiquinones in lipid vesicles exhibit some degree of local molecular motion, and that CoQs with longer side chains tend to have a deeper localization in the lipid bilayer. At the same time the ubiquinol rings are closer to the membrane surface than the ubiquinone rings. On the other hand, monolayer experiments have shown that ubiquinones (n = 3 or greater) are immiscible with the phospholipid, being squeezed out of the monolayer (5,6). More recently it has been shown by fluorescence and differential scanning calorimetry experiments (7, 8) that CoQlo does not perturb the phospholipid phase transition, therefore suggesting that its localization should not be within the bilayer but rather between both monolayers where the terminal methyl residues of the hydrocarbon chains are located. In this paper we study the effect of lipid fluidity on the ubiquinone ability to transport reducing equivalents across phospholipid bilayers. Some of these results have 0003.9861/82/120525-06$02.00/O Copyright AI1 rights
Q 1982 by Academic Preen. Inc. of reproduction in any form reserved.
526
ARANDA
AND
GGMEZ-FERNANDEZ
I FIG. 1. Traces for the reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine vesicles. The different experiments were done with vesicles containing the Co& indicated and without CoQ in the control, and at the temperatures shown.
been published form (9). MATERIALS
already
AND
in preliminary
METHODS
Coenzyme Q10 and coenzyme Qs were the kind gifts of Dr. Weber and Dr. Jenni, Hoffmann-La Roche, Basel. Dipalmitoylphosphatidylcholine (DPPC) was from Fluka, Buchs. Egg yolk lecithin from Sigma Chemical Company (Poole, Dorset, U. K.). Phospholipid vesicles containing ferricyanide were prepared essentially as described by Futami and co-workers (3). Briefly, phospholipids and CoQs were mixed in chloroform. A phospholipid:CoQ ratio of 5O:l (w/w) was used in these experiments. After evaporation to dryness under Nz in the dark, the last traces of solvent were removed in a vacuum dessicator. Small unilamellar vesicles were formed by sonicating the dry lipids in a 0.2 M ferricyanide solution, buffered at pH 8.0 with 50 mM tricine/NaOH, 0.3 M KCl. Sonication was performed under Nz. Temperature was kept between 50 and 60°C during the sonication. The external ferricyanide was removed on a 50 X 1.5~cm Sephadex G-50 column, equilibrated and eluted with the same buffer. Absorbance of the eluted fractions was measured at 550 nm, and fractions diluted less than twofold with respect to the unfractionated sample were collected. Reduction of trapped ferricyanide by external 20 mM dithionite, in the same buffer used for sonication, was studied by a stopped flow technique; a dual wavelength Aminco DW2a spectrophotometer equipped with an Aminco Morrow stopped flow accessory was used. Syringe A contained the lipid vesicle suspension and syringe B the dithionite solution. Data were ac-
quired and digitally stored by means of an Aminco DASAR unit which allows the transfer of the curves to the spectrophotometer recorder. Reduction was monitored at 420 with the reference at 600 nm. The high excess of dithionite should make the reaction of pseudo-first order as studied in (3). Initial rates were obtained by a numerical procedure described in (10). Phospholipid concentration was measured as inorganic phosphate by the Bartlett procedure (11). Ubiquinone-10 concentration was assayed spectrophotometrically at 275 nm, comparing the oxidized and the reduced states, using an extinction coefficient of 14.6 M-‘-cm-’ (12). Ubiquinone-3 was assayed in petroleum ether at 270 nm, using an extinction coefficient of 12.6 M-i *cm-i (13). Millipore immersible CX-10 ultrafilters were used to investigate the possible leakiness of the vesicles for the trapped ferricyanide. These filters have a nominal exclusion molecular weight of 10,000 for proteins. After filtration through the Sephadex column to eliminate the nontrapped ferricyanide, aliquots of the lipid vesicle suspension were ultrafiltered at 20°C and at 50-55”C, i.e., below and above of the phase transition of the pure DPPC. The sample and the ultrafiltration system were introduced in a temperature-controlled cupboard in order to carry out the experiments at 5055V. The first 4 ml of ultratiltrate were ignored in all the measurements, in order to avoid dilution with the water retained in the system. The fifth milliliter was used for ferricyanide assay using a 1.04 X 10’ M-l. cm-i extinction coefficient at 420 nm. Comparisons were made with a control sample treated with 2% Triton X-100 to dissolve the lipid vesicles. To exclude any possibility of specific retention of free ferricyanide by the filter another control, consisting of a lipidfree ferricyanide solution, was run through the ultrafiltration system. It was found that free ferricyanide is not retained at all by the filter. The total amount of trapped ferricyanide for both dipalmitoylphosphatidylcholine and egg yolk lecithin vesicles containing Co&,0 was calculated in a way similar to that described in (14). DPPC/CoQis vesicles contained 0.35 liter/m01 DPPC and those of egg yolk lecithin/CoQ,, 0.50 liter/m01 phospholipid. These values are in the range of others described in the literature for small unilamellar vesicles, contrasting with the notably higher values of up to approximately 1.8 liters/m01 of phospholipid, found for multilamellar vesicles (15).
RESULTS
AND
DISCUSSION
Figure 1 shows the spectrophotometric traces for the reduction of trapped ferricyanide, by externally added dithionite. They were obtained with CoQlo and CoQs reconstituted in dipalmitoylphosphatidyl-
MEMBRANE
FLUIDITY
AND
UBIQUINONE
choline vesicles, at temperatures both above and below the main T, transition temperature of the pure phospholipid (41’C). Controls consisting of vesicles including ferricyanide but not Co& were also studied. One of the controls, at a temperature above T,, is shown in Fig. 1. It can be seen that no ferricyanide reduction takes place in the control case, in the time scale of the experiment. Different reduction patterns are found at temperatures above and below T,. Similar experiments, but using CoQlo reconstituted with egg yolk lecithin, are shown in Fig. 2. In this case the reduction of trapped ferricyanide by externally added dithionite is almost unaffected by the changes in temperature; this phospholipid does not show any thermotropic phase transition in the temperature range under study. We conclude that the physical state of the membrane is important in determining the rate of UQmediated ferricyanide reduction. In order to confirm that the observed initial ferricyanide reduction does not correspond to ferricyanide which has leaked out of the vesicles, ultrafiltration experiments were carried out using Millipore ultrafilters. Vesicle suspensions were filtered at different times after their passage through the Sephadex column, as described under Methods. Less than 3% of the total ferricyanide leaks out of the DPPC/CoQi,-, vesicles after 6 h at either 20 or 50°C; 6 h was the maximum period of time during which these vesicles were used after Sephadex filtration. Identical results are obtained for egg yolk lecithin/CoQ,, vesicles at 20°C; however, some more ferricyanide leaks after 6 hours at 5O”C, but always less than 7%. We deduce that this small leak is not important enough to influence our conclusions. Other arguments can be presented against the possibility of significant leakage of ferricyanide out of the vesicles. One of them is based on the comparison of rate constants. The lz value for the reduction of ferricyanide by dithionite without the membrane barrier was estimated from measurements with vesicle suspensions in the presence of 2% Triton X-100 to be higher than 300 s-’ (3). However, the UQ-
MEMBRANE
TRANSPORT
527
DlthlcdF I !------““‘ro’~Ls’c
FIG. 2. Traces for the reduction of ferricyanide trapped in egg yolk vesicles, including Co&,, or without CoQ (control) and at the temperatures shown.
mediated ferricyanide reduction in intact lipid vesicles has k values of l-2 s-l as calculated from initial rates (see below). Furthermore, the possibility of ubiquinone having a detergent-like effect increasing membrane permeability to dithionite or ferricyanide was also discussed and excluded by those authors, on the basis of experiments involving analog compounds, that do not catalyze the reaction. This is the case, among others, of l-nonprenyl2,3,4,5-tetramethoxy-6-methyl benzene which is the dimethyl ether of QHz-9. Furthermore, it has been shown for other structural analogs of ubiquinones, such as tocopherols, that they do not increase, but rather decrease membrane permeability (16, 17). The stopped-flow traces of both CoQlo and CoQg (Figs. 1 and 2) show complex patterns with a fast absorbance decay followed by a phase of slower ferricyanide reduction. It is interesting to note that for CoQi,, and CoQB reconstituted in DPPC vesicles above T,, the initial rapid phase accounts for the reduction of most of the ferricyanide. However, the second, slow, phase makes an important contribution to that reduction below T,. The dependence
528
ARANDA
TIME
FIG. 3. Pseudo-first-order reduction of ferricyanide phatidylcholine vesicles, temperatures shown.
AND
GGMEZ-FERNANDEZ
I s~cardsl
plots of the traces for the trapped in dipalmitoylphosmediated by CoQlo, at the
of this behavior on temperature will be discussed later. But first, attention will be paid to the order of the reaction which is important in order to understand its mechanism. An excess of dithionite, 10 mM, is used in this reaction (3). Therefore, if the reaction is of second order it should be made of pseudo-first order for Co&, due to the excess dithionite and a straight line should be found in a semilogarithmic plot of the stopped-flow traces. Figure 3 shows such a plot and a straight line is not found and therefore the reaction is of an order higher than two. This might suggest that not all the CoQ molecules in the membrane react in the same way. In turn, this diversity could be due to a heterogeneous distribution of CoQ in the membrane (3). Given this complex kinetics, rate constant calculations are difficult. Consequently initial rates are used in order to
-A
l/T x 16~
FIG. 4. Arrhenius plots of initial rates of reduction of ferricyanide trapped in lipid vesicles containing CoQrs. Filled circles correspond to egg yolk phosphatidylcholine vesicles and open circles to dipalmitoylphosphatidylcholine vesicles.
study the effect of temperature on the reaction. Arrhenius plots of initial rates of ferricyanide reduction in CoQlO-dipalmitoylphosphatidylcholine, and CoQ,,,-egg yolk lecithin are shown in Fig. 4. Sharp changes in CoQlo functionality can be seen in the first case, near the main T, phase transition of the pure dipalmitoylphosphatidylcholine. No sharp change, i.e., discontinuity, can be detected in the second case, with CoQJegg yolk lecithin vesicles, where no phospholipid phase transition is observed in the range of temperatures under study. Similar results are observed in the case of CoQ, reconstituted with dipalmitoyllecithin (Fig. 5). Below T,, similar rates are observed for Co&, and CoQlo in DPPC membranes (approx. 45 Adz0 U/min) but, above T,, they are higher for CoQ,,, (approx. 145 A42,, U/min) than for CoQB (approx. 100 A420 U/min) . In general, our results show the same behavior for CoQQ and for CoQi,, with respect to the influence of membrane fluidity on their transport ability. However, in agreement with previous authors (3), we find that CoQlo is more efficient than CoQg in this transport. This agrees as well with what was found in mitochondria reconstituted with ubiquinones where long side chains were found to be necessary for adequate functionality (17). It was already mentioned that the patterns of the stopped-flow traces were quite different above and below the phase transition both for CoQlo and CoQg reconstituted in DPPC (Fig. 1). The decrease in absorbance corresponding to the initial (rapid) phase is plotted against temperature in Fig. 6. The form of measuring this decrease is defined in the insert of Fig. 6. Results obtained with CoQlo reconstituted in both DPPC and egg yolk lecithin vesicles, are used for these plots. Figure 6 shows that, below T,, the amount of trapped ferricyanide which is reduced during the first (rapid) phase, is only slightly increased when the temperature is raised. However, near T,, a sharp increase is observed. From this temperature up, the increase is again small. In the
MEMBRANE
FLUIDITY
AND
UBIQUINONE
FIG. 5. Arrhenius plot of initial rates of reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine vesicles containing CoQs.
case of CoQlo reconstituted in egg yolk lecithin vesicles the temperature dependence is small in all cases, as expected. There is a striking similarity between the behavior of CoQlo in fluid and rigid DPPC membranes (Fig. 1) and that of ubiquinones with long and short isoprenyl chains (see Fig. 1, Ref. (3)). Looking at the ferricyanide reduction patterns, CoQS (long isoprenyl chain) in a fluid membrane can be compared to CoQlo in DPPC above T,, and a first (rapid) phase with a large ferricyanide reduction followed by a second (slower) phase, are observed. On the other hand, CoQp (short isoprenyl chain) also in a fluid membrane, can be compared to CoQlo in DPPC membranes below T,, and a first (rapid) phase of ferricyanide reduction is also observed; however, the amount of ferricyanide reduced in this phase is lower than in the previous cases and in consequence most of the ferricyanide is reduced here in the second (slower) phase. By making this comparison we do not necessarily mean that the same explanation can be applied to both experiments. In any case it seems that at least two different mechanisms are implied in the reduction of ferricyanide included in ubiquinonecontaining vesicles by externally added dithionite. The first of them would imply a rapid reaction with a high order for ubiquinone and the second a reaction of pseudofirst order (see Fig. 3 in this paper and Fig. 6 of Ref. (3)). Although the available data do not allow a molecular explanation for all the events described above, it is clear that the localization of CoQs in the membrane must be an important clue to it. Recent studies (7,
MEMBRANE
529
TRANSPORT
8) using CoQ reconstituted in liposomes, have suggested that most of the CoQlo would be situated between both monolayers in the region where the terminal methyl groups of the fatty acids are also found. This would be true, at least for the type of ubiquinone:phospholipid ratio used in our experiments. According to the suggestion made in Fig. 3 of Ref. (8), CoQlo molecules will be more heterogeneously distributed and more freely mobile above than below T,; with the membrane in the rigid condition the CoQlo molecules would be predominately sandwiched between the gel phase monolayers. It is interesting to relate these suggestions with the NMR observations (4), that quinones with long isoprenyl chains could form ubiquinone-rich phases, rather than be uniformly distributed in fluid phospholipid bilayers. The inability of short chain quinones to build such ubiquinone-rich domains could be an explanation for their lower transport efficiencies (3, 4, 18) and perhaps for the transport mechanism somewhat different to that of long-chain quinones (Q3 or longer). Another interesting question is why CoQ behaves differently in fluid and rigid membranes. Reconstitution in membranes with fully saturated phospholipids undergoing phase transitions at physiological
a2-
I
20
.
30 lemperatws
40
.
.
50
(‘C )
FIG. 6. Plot of the decrease in A4% during the first (rapid) phase of reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine and egg yolk lecithin vesicles containing Co&i,,, at different temperatures. The magnitude of the decrease in this first phase is calculated as indicated in the insert. l , CoQ&DPPC; 0, CoQ&egg yolk lecithin.
530
ARANDA
AND
GGMEZ-FERNANDEZ
temperatures in a widely used technique in order to study the interaction of proteins and polypeptides with membrane lipids as well as the transport mechanisms of the former. For example, valinomycin, which acts as a mobile carrier, has its activity drastically reduced below T, of the phospholipid (19). On the other hand, gramicidin, which acts by means of a pore mechanism is not inactivated below T, (19). Certainly their mechanisms of action are different but whether they are affected or not in their function by the physical state of the membrane can be taken as an indication of a possible mobile behavior. The same can be said about CoQlo although again its mechanism of action is different from the transport molecules mentioned above. It is thought that CoQlo should have a transbilayer motion to conduct protons and electrons across lipid bilayers. Taking our results of trapped ferricyanide reduction as a measurement of ubiquinone-mediated transport we have shown in this paper how this conduction is greatly dependent on membrane fluidity, being drastically reduced at temperatures below T, of the pure phospholipid. However, we should mention that the change in membrane physical state, from fluid to rigid, should influence the ubiquinone-mediated transport kinetics, not only by hindering the motional properties of CoQs but also by changing their organization in the membrane. More work including kinetic experiments and studies of ubiquinone organization in membranes using physical techniques are needed before we can obtain a clear picture of how CoQs are involved in membrane transport. ACKNOWLEDGEMENTS We wish to thank Dr. Weber and Dr. Jenni, Hoffmen-La Roche, Basel, for kind gifts of ubiquinones. We thank Dr. Goiii, Dr. G. Cermona, and Dr. G. C&rova8 for helpful discussions. This work was supported by CAICYT (Spain).
REFERENCES
1. CRANE, F. L. (1977) Annu. Reu. Biochem. 46, 439-469. 2. MITCHELL, P. (1976) J. Theor. Biol. 62,327-367. 3. FUTAMI, A., HUNT, E., AND HAUSKA, G. (1979) Biochim. Biophys. Acta 547, 583-596. 4. KINGSLEY, P. B., AND FEIGENSON, G. W. (1981) Biochim. Biophys. Acta 636,602-618. 5. QUINN, P. J. (1980) Biochem. Znt. 1, 77-83. 6. QUINN, P. J., AND ESFAHANI, M. A. (1980) Biochem.J. 18!,715-722. 7. ALONSO, A., GOMEZ-FERN&EZ,-J. C., ARANDA, F. J., BELDA, F. J. F., AND GONI, F. M. (1981) FEBSLett. 132,19-22. 8. KATSIKAS, H., AND QUINN, P. J. (1981) FEBS L&t. 133.230-234. 9. G~MEZ-FERN~DEZ, J. C., ARANDA, F., ALONSO, A., AND GoNI, F. M. (1981) in Vectorial Reactions in Electron and Ion Transport in Mitochondria and Bacteria (Palmieri et al., eds.), pp. 179-182, Elsevier/North-Holland, Amsterdam. 10. NOBLE, B. (1968) Numerical Methods, Vol. 2, pp. 157-188, Oliver and Boyd, Edinburgh. 11. BARTLE~, G. R. (1959) J. Biol. Chem. 231,466-
477. 12. CRANE, F. L., AND BARR, R. (1971) in Methods in Enzymology (McCormick, D. B., end Wright, L. D., eds.), Vol. XVIII, part C, pp. 137-164, Academic Press, New York/London. 13. MAYER, H., AND ISLER, 0. (1971) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. XVIII, part C, pp. 182-213, Academic Press, New York/London. 14. NEWMAN, G. C., AND HUANG, C. (1975) Biochemistry 14,3363-33-70. 15. DEAMER, D., AND BANGHAM, A. D. (1976) Biochim. Biophys. Acta 443, 629-634. 16. DIPLOCK,A. T., LUCY, J. A., VERRINDER, M., AND ZIEBNIEWSKI, A. (1977) FEBS Z&t. 82, 341-
344. 17. FUKUZAWA, K., IKENO, H., TOKUMURA, A., AND TSUKATANI, H. (1979) Chem. Phys. Lipids 23, 13-22. 18. LENAZ, G., PASQUALI, P., PARENTI-CASTELLI, G., SECHI, A. M., AND BERTOLI, E. (1977) in Bioenergetic5 of Membranes (Packer, L., et al., eds.), pp. 189-198. Elsevier/North-Holland, Amsterdam. 19. KRASNE, S., EISENMAN, G., AND SZABO, G. (1971) Science 174,412%415.
Influence
of Membrane Fluidity on Transport Mediated Ubiquinones through Phospholipid Vesicles
FRANCISCO Departamento
J. ARANDA
Znterfmultatiuo
JUAN
AND
de Bioquimim,
Facultad
Received
January
25, 1982,
C. GGMEZ-FERNANDEZ’
de Medicinn,
Espinardo,
Murcia,
by
Uniuersidad
de Murcia,
Apartado
61,
Spain
and in revised
form
June
6, 1982
Coenzymes Q1,, and Q3 are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ,,, and CoQQ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.
Coenzyme Q10 (ubiquinone, UQ)’ is a component of the electron transport chain which appears to be involved in the transfer of electrons between respiratory complex I (or II) and respiratory complex III in the inner mitochondrial membrane (see (l), for a review). CoQ has been implied in the mechanism of vectorial proton translocation coupled to electron transport in mitochondria (2). However, the exact localization of ubiquinones in the membrane and the molecular mechanisms of electron and proton transport are still a matter of discussion. Hauska and co-workers (3), using ferricyanide trapped inside quinone-containing lipid vesicles, adding dithionite externally have measured the rate of ferricyanide reduction. Their conclusions were that quinones with side chains longer than those of Qz were more efficient in electron and proton translocation. They suggested that the quinones with longer isoprenyl 1 To whom all correspondence should ’ Abbreviations used: Q, ubiquinone; mitoylphosphatidylcholine.
be addressed. DPPC, dipal-
525
side chains formed aggregates within the phospholipid bilayer. NMR studies recently have shown that ubiquinones in lipid vesicles exhibit some degree of local molecular motion, and that CoQs with longer side chains tend to have a deeper localization in the lipid bilayer. At the same time the ubiquinol rings are closer to the membrane surface than the ubiquinone rings. On the other hand, monolayer experiments have shown that ubiquinones (n = 3 or greater) are immiscible with the phospholipid, being squeezed out of the monolayer (5,6). More recently it has been shown by fluorescence and differential scanning calorimetry experiments (7, 8) that CoQlo does not perturb the phospholipid phase transition, therefore suggesting that its localization should not be within the bilayer but rather between both monolayers where the terminal methyl residues of the hydrocarbon chains are located. In this paper we study the effect of lipid fluidity on the ubiquinone ability to transport reducing equivalents across phospholipid bilayers. Some of these results have 0003.9861/82/120525-06$02.00/O Copyright AI1 rights
Q 1982 by Academic Preen. Inc. of reproduction in any form reserved.
526
ARANDA
AND
GGMEZ-FERNANDEZ
I FIG. 1. Traces for the reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine vesicles. The different experiments were done with vesicles containing the Co& indicated and without CoQ in the control, and at the temperatures shown.
been published form (9). MATERIALS
already
AND
in preliminary
METHODS
Coenzyme Q10 and coenzyme Qs were the kind gifts of Dr. Weber and Dr. Jenni, Hoffmann-La Roche, Basel. Dipalmitoylphosphatidylcholine (DPPC) was from Fluka, Buchs. Egg yolk lecithin from Sigma Chemical Company (Poole, Dorset, U. K.). Phospholipid vesicles containing ferricyanide were prepared essentially as described by Futami and co-workers (3). Briefly, phospholipids and CoQs were mixed in chloroform. A phospholipid:CoQ ratio of 5O:l (w/w) was used in these experiments. After evaporation to dryness under Nz in the dark, the last traces of solvent were removed in a vacuum dessicator. Small unilamellar vesicles were formed by sonicating the dry lipids in a 0.2 M ferricyanide solution, buffered at pH 8.0 with 50 mM tricine/NaOH, 0.3 M KCl. Sonication was performed under Nz. Temperature was kept between 50 and 60°C during the sonication. The external ferricyanide was removed on a 50 X 1.5~cm Sephadex G-50 column, equilibrated and eluted with the same buffer. Absorbance of the eluted fractions was measured at 550 nm, and fractions diluted less than twofold with respect to the unfractionated sample were collected. Reduction of trapped ferricyanide by external 20 mM dithionite, in the same buffer used for sonication, was studied by a stopped flow technique; a dual wavelength Aminco DW2a spectrophotometer equipped with an Aminco Morrow stopped flow accessory was used. Syringe A contained the lipid vesicle suspension and syringe B the dithionite solution. Data were ac-
quired and digitally stored by means of an Aminco DASAR unit which allows the transfer of the curves to the spectrophotometer recorder. Reduction was monitored at 420 with the reference at 600 nm. The high excess of dithionite should make the reaction of pseudo-first order as studied in (3). Initial rates were obtained by a numerical procedure described in (10). Phospholipid concentration was measured as inorganic phosphate by the Bartlett procedure (11). Ubiquinone-10 concentration was assayed spectrophotometrically at 275 nm, comparing the oxidized and the reduced states, using an extinction coefficient of 14.6 M-‘-cm-’ (12). Ubiquinone-3 was assayed in petroleum ether at 270 nm, using an extinction coefficient of 12.6 M-i *cm-i (13). Millipore immersible CX-10 ultrafilters were used to investigate the possible leakiness of the vesicles for the trapped ferricyanide. These filters have a nominal exclusion molecular weight of 10,000 for proteins. After filtration through the Sephadex column to eliminate the nontrapped ferricyanide, aliquots of the lipid vesicle suspension were ultrafiltered at 20°C and at 50-55”C, i.e., below and above of the phase transition of the pure DPPC. The sample and the ultrafiltration system were introduced in a temperature-controlled cupboard in order to carry out the experiments at 5055V. The first 4 ml of ultratiltrate were ignored in all the measurements, in order to avoid dilution with the water retained in the system. The fifth milliliter was used for ferricyanide assay using a 1.04 X 10’ M-l. cm-i extinction coefficient at 420 nm. Comparisons were made with a control sample treated with 2% Triton X-100 to dissolve the lipid vesicles. To exclude any possibility of specific retention of free ferricyanide by the filter another control, consisting of a lipidfree ferricyanide solution, was run through the ultrafiltration system. It was found that free ferricyanide is not retained at all by the filter. The total amount of trapped ferricyanide for both dipalmitoylphosphatidylcholine and egg yolk lecithin vesicles containing Co&,0 was calculated in a way similar to that described in (14). DPPC/CoQis vesicles contained 0.35 liter/m01 DPPC and those of egg yolk lecithin/CoQ,, 0.50 liter/m01 phospholipid. These values are in the range of others described in the literature for small unilamellar vesicles, contrasting with the notably higher values of up to approximately 1.8 liters/m01 of phospholipid, found for multilamellar vesicles (15).
RESULTS
AND
DISCUSSION
Figure 1 shows the spectrophotometric traces for the reduction of trapped ferricyanide, by externally added dithionite. They were obtained with CoQlo and CoQs reconstituted in dipalmitoylphosphatidyl-
MEMBRANE
FLUIDITY
AND
UBIQUINONE
choline vesicles, at temperatures both above and below the main T, transition temperature of the pure phospholipid (41’C). Controls consisting of vesicles including ferricyanide but not Co& were also studied. One of the controls, at a temperature above T,, is shown in Fig. 1. It can be seen that no ferricyanide reduction takes place in the control case, in the time scale of the experiment. Different reduction patterns are found at temperatures above and below T,. Similar experiments, but using CoQlo reconstituted with egg yolk lecithin, are shown in Fig. 2. In this case the reduction of trapped ferricyanide by externally added dithionite is almost unaffected by the changes in temperature; this phospholipid does not show any thermotropic phase transition in the temperature range under study. We conclude that the physical state of the membrane is important in determining the rate of UQmediated ferricyanide reduction. In order to confirm that the observed initial ferricyanide reduction does not correspond to ferricyanide which has leaked out of the vesicles, ultrafiltration experiments were carried out using Millipore ultrafilters. Vesicle suspensions were filtered at different times after their passage through the Sephadex column, as described under Methods. Less than 3% of the total ferricyanide leaks out of the DPPC/CoQi,-, vesicles after 6 h at either 20 or 50°C; 6 h was the maximum period of time during which these vesicles were used after Sephadex filtration. Identical results are obtained for egg yolk lecithin/CoQ,, vesicles at 20°C; however, some more ferricyanide leaks after 6 hours at 5O”C, but always less than 7%. We deduce that this small leak is not important enough to influence our conclusions. Other arguments can be presented against the possibility of significant leakage of ferricyanide out of the vesicles. One of them is based on the comparison of rate constants. The lz value for the reduction of ferricyanide by dithionite without the membrane barrier was estimated from measurements with vesicle suspensions in the presence of 2% Triton X-100 to be higher than 300 s-’ (3). However, the UQ-
MEMBRANE
TRANSPORT
527
DlthlcdF I !------““‘ro’~Ls’c
FIG. 2. Traces for the reduction of ferricyanide trapped in egg yolk vesicles, including Co&,, or without CoQ (control) and at the temperatures shown.
mediated ferricyanide reduction in intact lipid vesicles has k values of l-2 s-l as calculated from initial rates (see below). Furthermore, the possibility of ubiquinone having a detergent-like effect increasing membrane permeability to dithionite or ferricyanide was also discussed and excluded by those authors, on the basis of experiments involving analog compounds, that do not catalyze the reaction. This is the case, among others, of l-nonprenyl2,3,4,5-tetramethoxy-6-methyl benzene which is the dimethyl ether of QHz-9. Furthermore, it has been shown for other structural analogs of ubiquinones, such as tocopherols, that they do not increase, but rather decrease membrane permeability (16, 17). The stopped-flow traces of both CoQlo and CoQg (Figs. 1 and 2) show complex patterns with a fast absorbance decay followed by a phase of slower ferricyanide reduction. It is interesting to note that for CoQi,, and CoQB reconstituted in DPPC vesicles above T,, the initial rapid phase accounts for the reduction of most of the ferricyanide. However, the second, slow, phase makes an important contribution to that reduction below T,. The dependence
528
ARANDA
TIME
FIG. 3. Pseudo-first-order reduction of ferricyanide phatidylcholine vesicles, temperatures shown.
AND
GGMEZ-FERNANDEZ
I s~cardsl
plots of the traces for the trapped in dipalmitoylphosmediated by CoQlo, at the
of this behavior on temperature will be discussed later. But first, attention will be paid to the order of the reaction which is important in order to understand its mechanism. An excess of dithionite, 10 mM, is used in this reaction (3). Therefore, if the reaction is of second order it should be made of pseudo-first order for Co&, due to the excess dithionite and a straight line should be found in a semilogarithmic plot of the stopped-flow traces. Figure 3 shows such a plot and a straight line is not found and therefore the reaction is of an order higher than two. This might suggest that not all the CoQ molecules in the membrane react in the same way. In turn, this diversity could be due to a heterogeneous distribution of CoQ in the membrane (3). Given this complex kinetics, rate constant calculations are difficult. Consequently initial rates are used in order to
-A
l/T x 16~
FIG. 4. Arrhenius plots of initial rates of reduction of ferricyanide trapped in lipid vesicles containing CoQrs. Filled circles correspond to egg yolk phosphatidylcholine vesicles and open circles to dipalmitoylphosphatidylcholine vesicles.
study the effect of temperature on the reaction. Arrhenius plots of initial rates of ferricyanide reduction in CoQlO-dipalmitoylphosphatidylcholine, and CoQ,,,-egg yolk lecithin are shown in Fig. 4. Sharp changes in CoQlo functionality can be seen in the first case, near the main T, phase transition of the pure dipalmitoylphosphatidylcholine. No sharp change, i.e., discontinuity, can be detected in the second case, with CoQJegg yolk lecithin vesicles, where no phospholipid phase transition is observed in the range of temperatures under study. Similar results are observed in the case of CoQ, reconstituted with dipalmitoyllecithin (Fig. 5). Below T,, similar rates are observed for Co&, and CoQlo in DPPC membranes (approx. 45 Adz0 U/min) but, above T,, they are higher for CoQ,,, (approx. 145 A42,, U/min) than for CoQB (approx. 100 A420 U/min) . In general, our results show the same behavior for CoQQ and for CoQi,, with respect to the influence of membrane fluidity on their transport ability. However, in agreement with previous authors (3), we find that CoQlo is more efficient than CoQg in this transport. This agrees as well with what was found in mitochondria reconstituted with ubiquinones where long side chains were found to be necessary for adequate functionality (17). It was already mentioned that the patterns of the stopped-flow traces were quite different above and below the phase transition both for CoQlo and CoQg reconstituted in DPPC (Fig. 1). The decrease in absorbance corresponding to the initial (rapid) phase is plotted against temperature in Fig. 6. The form of measuring this decrease is defined in the insert of Fig. 6. Results obtained with CoQlo reconstituted in both DPPC and egg yolk lecithin vesicles, are used for these plots. Figure 6 shows that, below T,, the amount of trapped ferricyanide which is reduced during the first (rapid) phase, is only slightly increased when the temperature is raised. However, near T,, a sharp increase is observed. From this temperature up, the increase is again small. In the
MEMBRANE
FLUIDITY
AND
UBIQUINONE
FIG. 5. Arrhenius plot of initial rates of reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine vesicles containing CoQs.
case of CoQlo reconstituted in egg yolk lecithin vesicles the temperature dependence is small in all cases, as expected. There is a striking similarity between the behavior of CoQlo in fluid and rigid DPPC membranes (Fig. 1) and that of ubiquinones with long and short isoprenyl chains (see Fig. 1, Ref. (3)). Looking at the ferricyanide reduction patterns, CoQS (long isoprenyl chain) in a fluid membrane can be compared to CoQlo in DPPC above T,, and a first (rapid) phase with a large ferricyanide reduction followed by a second (slower) phase, are observed. On the other hand, CoQp (short isoprenyl chain) also in a fluid membrane, can be compared to CoQlo in DPPC membranes below T,, and a first (rapid) phase of ferricyanide reduction is also observed; however, the amount of ferricyanide reduced in this phase is lower than in the previous cases and in consequence most of the ferricyanide is reduced here in the second (slower) phase. By making this comparison we do not necessarily mean that the same explanation can be applied to both experiments. In any case it seems that at least two different mechanisms are implied in the reduction of ferricyanide included in ubiquinonecontaining vesicles by externally added dithionite. The first of them would imply a rapid reaction with a high order for ubiquinone and the second a reaction of pseudofirst order (see Fig. 3 in this paper and Fig. 6 of Ref. (3)). Although the available data do not allow a molecular explanation for all the events described above, it is clear that the localization of CoQs in the membrane must be an important clue to it. Recent studies (7,
MEMBRANE
529
TRANSPORT
8) using CoQ reconstituted in liposomes, have suggested that most of the CoQlo would be situated between both monolayers in the region where the terminal methyl groups of the fatty acids are also found. This would be true, at least for the type of ubiquinone:phospholipid ratio used in our experiments. According to the suggestion made in Fig. 3 of Ref. (8), CoQlo molecules will be more heterogeneously distributed and more freely mobile above than below T,; with the membrane in the rigid condition the CoQlo molecules would be predominately sandwiched between the gel phase monolayers. It is interesting to relate these suggestions with the NMR observations (4), that quinones with long isoprenyl chains could form ubiquinone-rich phases, rather than be uniformly distributed in fluid phospholipid bilayers. The inability of short chain quinones to build such ubiquinone-rich domains could be an explanation for their lower transport efficiencies (3, 4, 18) and perhaps for the transport mechanism somewhat different to that of long-chain quinones (Q3 or longer). Another interesting question is why CoQ behaves differently in fluid and rigid membranes. Reconstitution in membranes with fully saturated phospholipids undergoing phase transitions at physiological
a2-
I
20
.
30 lemperatws
40
.
.
50
(‘C )
FIG. 6. Plot of the decrease in A4% during the first (rapid) phase of reduction of ferricyanide trapped in dipalmitoylphosphatidylcholine and egg yolk lecithin vesicles containing Co&i,,, at different temperatures. The magnitude of the decrease in this first phase is calculated as indicated in the insert. l , CoQ&DPPC; 0, CoQ&egg yolk lecithin.
530
ARANDA
AND
GGMEZ-FERNANDEZ
temperatures in a widely used technique in order to study the interaction of proteins and polypeptides with membrane lipids as well as the transport mechanisms of the former. For example, valinomycin, which acts as a mobile carrier, has its activity drastically reduced below T, of the phospholipid (19). On the other hand, gramicidin, which acts by means of a pore mechanism is not inactivated below T, (19). Certainly their mechanisms of action are different but whether they are affected or not in their function by the physical state of the membrane can be taken as an indication of a possible mobile behavior. The same can be said about CoQlo although again its mechanism of action is different from the transport molecules mentioned above. It is thought that CoQlo should have a transbilayer motion to conduct protons and electrons across lipid bilayers. Taking our results of trapped ferricyanide reduction as a measurement of ubiquinone-mediated transport we have shown in this paper how this conduction is greatly dependent on membrane fluidity, being drastically reduced at temperatures below T, of the pure phospholipid. However, we should mention that the change in membrane physical state, from fluid to rigid, should influence the ubiquinone-mediated transport kinetics, not only by hindering the motional properties of CoQs but also by changing their organization in the membrane. More work including kinetic experiments and studies of ubiquinone organization in membranes using physical techniques are needed before we can obtain a clear picture of how CoQs are involved in membrane transport. ACKNOWLEDGEMENTS We wish to thank Dr. Weber and Dr. Jenni, Hoffmen-La Roche, Basel, for kind gifts of ubiquinones. We thank Dr. Goiii, Dr. G. Cermona, and Dr. G. C&rova8 for helpful discussions. This work was supported by CAICYT (Spain).
REFERENCES
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344. 17. FUKUZAWA, K., IKENO, H., TOKUMURA, A., AND TSUKATANI, H. (1979) Chem. Phys. Lipids 23, 13-22. 18. LENAZ, G., PASQUALI, P., PARENTI-CASTELLI, G., SECHI, A. M., AND BERTOLI, E. (1977) in Bioenergetic5 of Membranes (Packer, L., et al., eds.), pp. 189-198. Elsevier/North-Holland, Amsterdam. 19. KRASNE, S., EISENMAN, G., AND SZABO, G. (1971) Science 174,412%415.