[55]
UBIQUINONE 1N SUBMITOCHONDR1AL PARTICLES
[55] E x t r a c t i o n a n d R e i n c o r p o r a t i o n o f U b i q u i n o n e Submitochondrial Particles
By
573
in
LARS ERNSTER, E L Z B I E T A GLASER, and BIRGITTA N O R L I N G
Methods for extraction of ubiquinone from mitochondria and submitochondrial particles are based on the use of organic solvents. An early procedure I employed isooctane to extract ubiquinone from aqueous suspensions of beef-heart mitochondria or submitochondrial particles. Both N A D H and succinate oxidases were inactivated, and the latter could be restored by the addition of either ubiquinone or cytochrome c. Extraction of lyophilized beef- or rat-liver mitochondria with 96% acetone was subsequently shown 2 to result in a preparation that required both ubiquinone and cytochrome c for the restoration of succinate oxidase activity. In 1966 Szarkowska 3 introduced the use of n-pentane to extract ubiquinone from lyophilized beef-heart mitochondria. She was able to restore both N A D H and succinate oxidase activities by the addition of ubiquinone in the form of a phospholipid suspension. Similar findings were made with yeast mitochondria. 4 This method had the advantage that both NADH and succinate oxidase activities could be restored by the addition of ubiquinone and that the latter could not be replaced by cytochrome c. All these procedures had the disadvantage that they involved the addition of relatively large amounts of a water-insoluble quinone to an aqueous particle suspension, often resulting in unstable emulsions with varying degrees of contact between the quinone and the particles, and inevitably causing mixing artifacts and lack of reproducibility in the measurement of enzyme activities. Choice of appropriate phospholipids to stabilize the quinone suspensions (cf. Ernster et al.5) only partially overcomes these problems. The method ~ described below eliminates the above difficulties and allows a quantitative incorporation of controlled amounts of ubiquinone ' F. L. Crane, C. Widmer, R. L. Lester, and Y. Hatefi, Biochi,n. Biophys. Acta 31,476 (1959). 2 R. L. Lester and S. Fleischer, Biochim. Biophys. Acta 47, 358 (1961). :~ L. Szarkowska, Arch. Biochem. Biophys. 113, 519 (1966). 4 T. Ohnishi, G. Sottocasa, and L. Ernster, Bull. Soc. Chim. Biol. 48, 1189 (1%6). L. Ernster, C. P. Lee, and B. Norling, in "The Energy Level and Metabolic Control in Mitochondria" (S. Papa, J. M. Tager, E. Quagliariello, and E. C. Slater, eds.), p. 195. Adriatica Editrice, Bari, 1969; and unpublished results. 6 L. Ernster, I.-Y. Lee, B. Norling, and B. Persson, FEBS Lett. 3, 21 (1969); Eur. J. Biochem. 9, 299 (1969).
574
OUINONES
[55]
and a full restoration of ubiquinone-dependent enzyme activities in ubiquinone-depleted submitochondrial particles. In principle, this metho& is based on a treatment of lyophilized, pentane-extracted submitochondrial particles with a pentane solution containing a suitable concentration of ubiquinone, thus allowing a "repartition" of ubiquinone between the pentane solution and the membrane lipid. Procedure for Extraction and Reincorporation of Ubiquinone Submitochondrial particles are prepared from " h e a v y " beef-heart mitochondria 7 after sonication at pH 8.6-8.8 in the presence of 2 mM EDTA and 0.25 M sucrose, as described by Lee and Ernster. 8 The particles are suspended in 0.15 M KC1 at a concentration of approximately 20 mg of protein per milliliter and lyophilized for at least 9 hr to achieve complete dehydration. To extract ubiquinone, the lyophilized particles are suspended in npentane (analytical grade, dried over anhydrous sodium sulfate) by gentle homogenization, and the suspension is shaken in a glass-stoppered tube for 5 min at 0 °. The pentane extract is removed by centrifugation and saved for determination of ubiquinone; this is done spectrophotometrically at 275 nm with 290 nm as the reference wavelength, using an extinction coefficient of 12.2 M -1 cm-1. 9 The extraction is repeated 4 times to complete the removal of ubiquinone. The ubiquinone-depleted particles are dried in a rotating vacuum evaporator at room temperature to remove residual pentane. The total amount of ubiquinone extracted by this procedure ranges between 3 and 6 nmol per milligram of particle protein (varying from one particle preparation to another) and represents 90-95% of the ubiquinone present in the particles. A more effective removal of ubiquinone can be obtained either by washing the particles extensively with KCI before lyophilization, 1° to remove residual sucrose, or by adding 5-10% acetone to the pentane for the extraction of the lyophilized particles." However, both these modifications result in a partial inactivation of NADH oxidase. For the reincorporation of ubiquinone, the dried, ubiquinone-depleted particles are gently homogenized in a small volume (1-2 ml) of n-pentane containing ubiquinone-50 at a concentration of 40 nmol per milligram of 7 H. L6w and I. Vallin, Biochim. Biophys. Acta 69, 361 (1%3). 8 C. P. Lee and L. Ernster, this series, Vol. I0, p. 543. 9 R. L. Lester, Y. Hatefi, C. Widmer, and F. L. Crane, Biochim. Biophys. Acta 33, 169 (1959). ,0 M. Gutman, C. J. Coles, T. P. Singer, and J. E. Casida, Biochemistry 10, 2036 (1971). " B. Norling, E. Glazek, B. D. Nelson, and L. Ernster, Eur. J. Biochern. 47, 475 (1974).
[55]
UBIQUINONE IN SUBMITOCHONDRIAL PARTICLES
575
particle protein, and the suspension is shaken in a glass-stoppered tube in an ice-bath for 30 min. The excess amount of ubiquinone is removed by centrifugation, followed by one gentle rinsing of the pellet with a small volume of pentane. After removal of the fluid, the pellet is dried in a rotating vacuum evaporator. The amount of ubiquinone incorporated is determined spectrophotometrically9 after extraction with methanol-light petroleum according to Kr6ger and Klingenberg. ,2 The particles prepared by this procedure contain virtually the same amount of ubiquinone as those before depletion. For incorporation of varying amounts of ubiquinone, the procedure is modified as follows": Dried, ubiquinone-depleted particles (20-40 mg of protein) are homogenized at 4 ° in a small volume ( - 2 mi) of pentane containing ubiquinone in amounts varying between 0.4 and 25 nmol per milligram of particle protein. The suspension is transferred to a rotating vacuum evaporator, and the pentane is gradually removed under slightly reduced pressure at 4 °. Gradual removal of the pentane (2 ml over a period of about 30 min) is necessary for good incorporation. After removal of the bulk of the pentane, the evaporation is continued at maximally reduced pressure until the particles are completely dry. The amount of ubiquinone incorporated is determined as above. Using this procedure one can incorporate an amount of ubiquinone ranging from 1/20 to 4-fold the original ubiquinone content of the particles.5' The dried, lyophilized particles, either before or after pentane extraction and reincorporation of ubiquinone, can be stored at - 15 to - 2 0 ° in v a c u o for several weeks without apparent loss of enzyme activities. Caution must be taken, however, that the preparations, which are highly hygroscopic, remain absolutely dry. Even the slightest amount of water impairs both the extraction and the reincorporation of ubiquinone.
Effects of Extraction and Reincorporation of Ubiquinone on E n z y m e Activities Extraction of ubiquinone from submitochondrial particles causes an inhibition of NADH and succinate oxidases, of the reduction of cytochromes b, c,, c, a, and a3 by NADH and succinate, and of the rotenonesensitive oxidation of NADH by fumarate; all these activities are restored by the reincorporation of ubiquinone. 6 These findings are consistent with the concept that ubiquinone is an obligatory redox component between NADH and succinate dehydrogenases and the cytochrome system, i.e., ,2 A. Kr6gerand M. Klingenberg,Eur. J. Biochem. 34, 358 (1973).
576
QUINONES
[55]
between complexes I, II, and III of the respiratory chain. '3 The antimycin-sensitive oxidation of menadiol by molecular oxygen, involving the aerobic oxidation of reduced cytochrome b, is unaffected by the extraction of ubiquinone. The nicotinamide nucleotide transhydrogenase and ATPase activities are also unaffected.'4 Extraction of ubiquinone alters several properties of both succinate dehydrogenase '~ and the cytochrome bc, complex '4'16-~9 and these effects are also reversed by the reincorporation of ubiquinone. On the basis of these findings it has been suggested '~-'7 that ubiquinone, in addition to its function as a redox catalyst, also exerts a regulatory effect on the succinate-cytochrome Cl segment of the respiratory chain. A further effect of the extraction of ubiquinone, also restored by its reincorporation, is a decrease of the g = 2.00 EPR signal of submitochondrial particles observed in the presence of NADH or succinate. 2°'zl The ubiquinone dependent g = 2.00 signal ~s due to ubisemiquinone, the involvement of which in the respiratory chain has been implicated by several laboratories. 2z-2~ Gradual extraction of ubiquinone from submitochondrial particles has been shown 6 to result in a parallel inactivation of N A D H and succinate oxidases (Fig. 1), indicating that ubiquinone is a common rate-limiting 13 y. Hatefi, in "Comprehensive Biochemistry" (M. Florkin and E. H. Stotz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam, 1%6. ,4 L. Ernster, 1.-Y. Lee, B. Norling, B. Persson, K. Juntti, and U.-B. Torndal, in "Probes of Structure and Function of Macromolecules and Membranes" (B. Chance, C. P. Lee, and J. K. Blasie, eds.), Vol. 1,377. Academic Press, New York, 1971. 15 E. Rossi, B. Norling, B. Persson, and L. Ernster, Eur. J. Biochem. 16, 508 (1970); in "Energy Transduction in Respiration and Photosynthesis" (E. Quagliariello, S. Papa, and C. S. Rossi, eds.), p. 329. Adriatica Editrice, Bari, 1971. 16 B. D. Nelson, B. Norling, B. Persson, and L. Ernster, Biochem. Biophys. Res. Commun. 44, 1312 (1971); Biochem. Biophys. Res. Commun. 44, 1321 (1971). ,7 B. D. Nelson, B. Norling, B. Persson, and L. Ernster, Biochim. Biophys. Acta 267, 205 (1972). ,s I.-Y. Lee and E. C. Slater, in "Dynamics of Energy-Transducing Membranes" (L. Ernster, R. W. Estabrook, and E. C. Slater, eds.), BBA Library, Vol. 13, p. 61. Elsevier, Amsterdam, 1974. ,9 A. Kr6ger and M. Klingenberg, Eur. J. Biochem. 39, 313 (1973). 2o D. B~ickstr6m, B. Norling, A. Ehrenberg, and L. Ernster, Biochim. Biophys. Acta 197, 108 (1970). 2, F. J. Ruzicka, H. Beinert, K. L. Schepler, W. R. Dunham, and R. H. Sands, Proc. Natl. Acad. Sci. U.S.A. 72, 2886 (1975). 22 H. Baum, J. S. Rieske, H. 1. Silman, and S. H. Lipton, Proc. Natl. Acad. Sci. U.S.A. 57, 798 (1%7). 2~ M. K. F. Wikstr6m and J. A. Berden, Biochim. Biophys. Acta 283, 403 (1972). 24 p. Mitchell, FEBS Lett. 56, 1 (1975); FEBS Lett. 59, 137 (1975); J. Theor. Biol. 62, 327 (1976). 2,~W. J. lngledew, J. C. Salerno, and T. Ohnishi, Arch. Biochem. Biophys. 177, 176 (1976).
[55]
UB1QUINONE 1N SUBMITOCHONDRIAL PARTICLES
577
0 ra
>
50
50
*6
c o
100
l i ~ l l l 12 4 5 6 Numberof extractions
100
FIG. 1. Removal of ubiquinone (z%) and decrease in NADH (O) and succinate oxidase (O) activities upon repeated extractions of lyophilized beef-heart submitochondrial particles with n-pentane. From L. Ernster, I.-Y. Lee, B. Norling, and B. Persson, Eur. J. Biochem. 9, 299 (1969).
component of the two oxidases. This conclusion is further supported by the finding" that incorporation of limiting amounts of ubiquinone reactivates N A D H and succinate oxidases in a parallel fashion (Fig. 2). A preferential reactivation of succinate oxidase by the addition of small amounts of ubiquinone to pentane-extracted particles has been reported by Gutman e t a l . lo and by Kr6ger and Klingenberg 12 and has been interpreted as indicating either the involvement of different pools of ubiquinone in the two oxidases 26 or different affinities of the NADH and suc120
"
o"
g 8o , NADH ox i d a s e o Succ oxidase
-
~, 60 4O 2O L
O0 -
-
5 10 1'5 Ubiquinone content (nmol ling protein )
i
20
FIG. 2. Restoration of NADH (O) and succinate oxidase (©) activities of ubiquinonedepleted beef-heart submitochondrial particles as a function of the ubiquinone content. From B. Norling, E. Glazek, B. D. Nelson, and L. Ernster, Eur. J. Biochem. 47, 475 (1974). 2~ G. Lenaz, G. D. Daves, Jr., and K. Folkers, Arch. Biochem. Biophys. 123, 539 (1%8).
578
QUINONES
[55]
cinate dehydrogenases to ubiquinone. 12 However, this difference seems to be due primarily to an impaired ability of ubiquinone to reactivate NADH oxidase in ubiquinone-depleted particles under the conditions employed. A selective reactivation of succinate oxidase by certain ubiquinone analogs has been described by Lenaz et al. ~6and interpreted in terms of a difference in quinone specificity between the NADH and succinate dehydrogenases. A recent extension of these studies 27 indicates, however, that the observed phenomenon may be related to a difference in membrane topology of the two dehydrogenases in relation to the cytochrome bcl complex rather than different quinone specificities. A study of the restoration of the NADH, succinate, and a-glycerophosphate oxidase activities of pentane-extracted pig-brain mitochondria by ubiquinone has been reported by Salach and Bednarz. 28 Various aspects of the functions of ubiquinone in mitochondria are discussed in a number of recent reviews. 29-32
Extraction and Reincorporation of Quinones in Other EnergyTransducing Membranes Restoration of NADH oxidase by adding ubiquinone to pentane-extracted membranes of Azotobacter vinelandii has been described by Swank and Burris. 33 Kr6ger et al. 34 have reincorporated ubiquinone and menaquinone into pentane-extracted Proteus rettgeri and demonstrated that ubiquinone reactivates respiration with succinate and formate as substrates, whereas menaquinone restores fumarate reduction. An extraction and incorporation method employing isooctane as solvent has been developed by Horio et al. 35 to study the role of ubiquinone in chromatophores from a blue-green mutant of Rhodospirillum rubrum. 27 G. Lenaz, S. Mascarello, L. Landi, L. Cabrini, P. Pasquali, G. Parenti-Castelli, A. M. Sechi, and E. Bertoli, in "Bioenergetics of Membranes" (L. Packer, G. C. Papageorgiou, and A. Trebst, eds.), p. 189. Elsevier, North-Holland Biomedical Press, Amsterdam, 1977. 28 j. I. Salach and A. J. Bednarz, Arch. Biochem. Biophys. 157, 133 (1973). 29 F. L. Crane, Annu. Rev. Biochem. 46, 439 (1977). a0 j. DePierre and L. Ernster, Annu. Rev. Biochem. 46, 201 (1977). 31 p. D. Boyer, B. Chance, L. Emster, P. Mitchell, E. Racker, and E. C. Slater, Annu. Rev. Biochem. 46, 955 (1977). 32 L. Ernster, in "Biomedical and Clinical Aspects of Coenzyme Q'" (K. Folkers and Y. Yamamura, eds.), p. 15. Elsevier, North-Holland Biomedical Press, Amsterdam, 1977. aa R. T. Swank and R. H. Burris, J. Bacteriol. 98, 311 (1969). 34 A. Kr6ger, V. Dadak, M. Klingenberg, and F. Diemer, Eur. J. Biochem. 21,322 (1971). "~ T. Horio, Y. Horiuti, N. Yamamoto, and K. Nishikawa, this series, Vol. 24, p. 96.
[56]
DETERMINATIONOF Q AND MK
579
Using the same method, Baccarini-Melandri and Melandri 36have recently reported evidence for an involvement of ubiquinone in the cytochrome bc2 segment of the redox chain of Rhodopseudomonas capsulata chromatophores. 34 A. Baccarini-Melandri and B. A. Melandri, FEBS Lett. 80, 459 (1977).
[56] D e t e r m i n a t i o n o f C o n t e n t s a n d R e d o x Ubiquinone and Menaquinone
States of
By A. KROGER Methods for the extraction and determination of ubiquinone (Q) and menaquinone (MK) have been described earlier in this series. 1-3 With the commercial availability of the dual-wavelength photometer, which was first designed by Chance, 4 it is now possible to determine these quinones more sensitively and with greater accuracy and convenience. Furthermore, it is possible to follow the redox states of the quinones as functions of the metabolic states of biological preparations either by extraction or by direct optical recording of the redox reactions of the quinones. Investigation of the function of the quinones in mitochondria and bacteria has revealed that at least 95% of the quinones occur either in the oxidized (quinone) or reduced state (hydroquinone). Other derivatives (chromenols and chromanols) or intermediates (radicals) are not detected by the methods described here. Extraction of Q and M K The extraction method described here is developed for (1) the determination of the total contents of either Q or MK in mitochondria, submitochondrial particles ~'G, cell-free bacterial homogenates and purified E. 2 F. :~ P. 4 B. 5 A. L.
R. Redfearn, this series, Vol. 10, p. 381. L. Crane and R. Barr, this series, Vol. 18C, p. 137. J. Dunphy and A. F. Brodie, this series, Vol. 18C, p. 407. Chance, Rev. Sci. Instrum. 22, 619 (1951). M. Pumphrey and E. R. Redfearn, Biochem. J. 76, 61 (1960). Szarkowska and M. Klingenberg, Biochem. Z. 338, 674 (1963).
UBIQUINONE 1N SUBMITOCHONDR1AL PARTICLES
[55] E x t r a c t i o n a n d R e i n c o r p o r a t i o n o f U b i q u i n o n e Submitochondrial Particles
By
573
in
LARS ERNSTER, E L Z B I E T A GLASER, and BIRGITTA N O R L I N G
Methods for extraction of ubiquinone from mitochondria and submitochondrial particles are based on the use of organic solvents. An early procedure I employed isooctane to extract ubiquinone from aqueous suspensions of beef-heart mitochondria or submitochondrial particles. Both N A D H and succinate oxidases were inactivated, and the latter could be restored by the addition of either ubiquinone or cytochrome c. Extraction of lyophilized beef- or rat-liver mitochondria with 96% acetone was subsequently shown 2 to result in a preparation that required both ubiquinone and cytochrome c for the restoration of succinate oxidase activity. In 1966 Szarkowska 3 introduced the use of n-pentane to extract ubiquinone from lyophilized beef-heart mitochondria. She was able to restore both N A D H and succinate oxidase activities by the addition of ubiquinone in the form of a phospholipid suspension. Similar findings were made with yeast mitochondria. 4 This method had the advantage that both NADH and succinate oxidase activities could be restored by the addition of ubiquinone and that the latter could not be replaced by cytochrome c. All these procedures had the disadvantage that they involved the addition of relatively large amounts of a water-insoluble quinone to an aqueous particle suspension, often resulting in unstable emulsions with varying degrees of contact between the quinone and the particles, and inevitably causing mixing artifacts and lack of reproducibility in the measurement of enzyme activities. Choice of appropriate phospholipids to stabilize the quinone suspensions (cf. Ernster et al.5) only partially overcomes these problems. The method ~ described below eliminates the above difficulties and allows a quantitative incorporation of controlled amounts of ubiquinone ' F. L. Crane, C. Widmer, R. L. Lester, and Y. Hatefi, Biochi,n. Biophys. Acta 31,476 (1959). 2 R. L. Lester and S. Fleischer, Biochim. Biophys. Acta 47, 358 (1961). :~ L. Szarkowska, Arch. Biochem. Biophys. 113, 519 (1966). 4 T. Ohnishi, G. Sottocasa, and L. Ernster, Bull. Soc. Chim. Biol. 48, 1189 (1%6). L. Ernster, C. P. Lee, and B. Norling, in "The Energy Level and Metabolic Control in Mitochondria" (S. Papa, J. M. Tager, E. Quagliariello, and E. C. Slater, eds.), p. 195. Adriatica Editrice, Bari, 1969; and unpublished results. 6 L. Ernster, I.-Y. Lee, B. Norling, and B. Persson, FEBS Lett. 3, 21 (1969); Eur. J. Biochem. 9, 299 (1969).
574
OUINONES
[55]
and a full restoration of ubiquinone-dependent enzyme activities in ubiquinone-depleted submitochondrial particles. In principle, this metho& is based on a treatment of lyophilized, pentane-extracted submitochondrial particles with a pentane solution containing a suitable concentration of ubiquinone, thus allowing a "repartition" of ubiquinone between the pentane solution and the membrane lipid. Procedure for Extraction and Reincorporation of Ubiquinone Submitochondrial particles are prepared from " h e a v y " beef-heart mitochondria 7 after sonication at pH 8.6-8.8 in the presence of 2 mM EDTA and 0.25 M sucrose, as described by Lee and Ernster. 8 The particles are suspended in 0.15 M KC1 at a concentration of approximately 20 mg of protein per milliliter and lyophilized for at least 9 hr to achieve complete dehydration. To extract ubiquinone, the lyophilized particles are suspended in npentane (analytical grade, dried over anhydrous sodium sulfate) by gentle homogenization, and the suspension is shaken in a glass-stoppered tube for 5 min at 0 °. The pentane extract is removed by centrifugation and saved for determination of ubiquinone; this is done spectrophotometrically at 275 nm with 290 nm as the reference wavelength, using an extinction coefficient of 12.2 M -1 cm-1. 9 The extraction is repeated 4 times to complete the removal of ubiquinone. The ubiquinone-depleted particles are dried in a rotating vacuum evaporator at room temperature to remove residual pentane. The total amount of ubiquinone extracted by this procedure ranges between 3 and 6 nmol per milligram of particle protein (varying from one particle preparation to another) and represents 90-95% of the ubiquinone present in the particles. A more effective removal of ubiquinone can be obtained either by washing the particles extensively with KCI before lyophilization, 1° to remove residual sucrose, or by adding 5-10% acetone to the pentane for the extraction of the lyophilized particles." However, both these modifications result in a partial inactivation of NADH oxidase. For the reincorporation of ubiquinone, the dried, ubiquinone-depleted particles are gently homogenized in a small volume (1-2 ml) of n-pentane containing ubiquinone-50 at a concentration of 40 nmol per milligram of 7 H. L6w and I. Vallin, Biochim. Biophys. Acta 69, 361 (1%3). 8 C. P. Lee and L. Ernster, this series, Vol. I0, p. 543. 9 R. L. Lester, Y. Hatefi, C. Widmer, and F. L. Crane, Biochim. Biophys. Acta 33, 169 (1959). ,0 M. Gutman, C. J. Coles, T. P. Singer, and J. E. Casida, Biochemistry 10, 2036 (1971). " B. Norling, E. Glazek, B. D. Nelson, and L. Ernster, Eur. J. Biochern. 47, 475 (1974).
[55]
UBIQUINONE IN SUBMITOCHONDRIAL PARTICLES
575
particle protein, and the suspension is shaken in a glass-stoppered tube in an ice-bath for 30 min. The excess amount of ubiquinone is removed by centrifugation, followed by one gentle rinsing of the pellet with a small volume of pentane. After removal of the fluid, the pellet is dried in a rotating vacuum evaporator. The amount of ubiquinone incorporated is determined spectrophotometrically9 after extraction with methanol-light petroleum according to Kr6ger and Klingenberg. ,2 The particles prepared by this procedure contain virtually the same amount of ubiquinone as those before depletion. For incorporation of varying amounts of ubiquinone, the procedure is modified as follows": Dried, ubiquinone-depleted particles (20-40 mg of protein) are homogenized at 4 ° in a small volume ( - 2 mi) of pentane containing ubiquinone in amounts varying between 0.4 and 25 nmol per milligram of particle protein. The suspension is transferred to a rotating vacuum evaporator, and the pentane is gradually removed under slightly reduced pressure at 4 °. Gradual removal of the pentane (2 ml over a period of about 30 min) is necessary for good incorporation. After removal of the bulk of the pentane, the evaporation is continued at maximally reduced pressure until the particles are completely dry. The amount of ubiquinone incorporated is determined as above. Using this procedure one can incorporate an amount of ubiquinone ranging from 1/20 to 4-fold the original ubiquinone content of the particles.5' The dried, lyophilized particles, either before or after pentane extraction and reincorporation of ubiquinone, can be stored at - 15 to - 2 0 ° in v a c u o for several weeks without apparent loss of enzyme activities. Caution must be taken, however, that the preparations, which are highly hygroscopic, remain absolutely dry. Even the slightest amount of water impairs both the extraction and the reincorporation of ubiquinone.
Effects of Extraction and Reincorporation of Ubiquinone on E n z y m e Activities Extraction of ubiquinone from submitochondrial particles causes an inhibition of NADH and succinate oxidases, of the reduction of cytochromes b, c,, c, a, and a3 by NADH and succinate, and of the rotenonesensitive oxidation of NADH by fumarate; all these activities are restored by the reincorporation of ubiquinone. 6 These findings are consistent with the concept that ubiquinone is an obligatory redox component between NADH and succinate dehydrogenases and the cytochrome system, i.e., ,2 A. Kr6gerand M. Klingenberg,Eur. J. Biochem. 34, 358 (1973).
576
QUINONES
[55]
between complexes I, II, and III of the respiratory chain. '3 The antimycin-sensitive oxidation of menadiol by molecular oxygen, involving the aerobic oxidation of reduced cytochrome b, is unaffected by the extraction of ubiquinone. The nicotinamide nucleotide transhydrogenase and ATPase activities are also unaffected.'4 Extraction of ubiquinone alters several properties of both succinate dehydrogenase '~ and the cytochrome bc, complex '4'16-~9 and these effects are also reversed by the reincorporation of ubiquinone. On the basis of these findings it has been suggested '~-'7 that ubiquinone, in addition to its function as a redox catalyst, also exerts a regulatory effect on the succinate-cytochrome Cl segment of the respiratory chain. A further effect of the extraction of ubiquinone, also restored by its reincorporation, is a decrease of the g = 2.00 EPR signal of submitochondrial particles observed in the presence of NADH or succinate. 2°'zl The ubiquinone dependent g = 2.00 signal ~s due to ubisemiquinone, the involvement of which in the respiratory chain has been implicated by several laboratories. 2z-2~ Gradual extraction of ubiquinone from submitochondrial particles has been shown 6 to result in a parallel inactivation of N A D H and succinate oxidases (Fig. 1), indicating that ubiquinone is a common rate-limiting 13 y. Hatefi, in "Comprehensive Biochemistry" (M. Florkin and E. H. Stotz, eds.), Vol. 14, p. 199. Elsevier, Amsterdam, 1%6. ,4 L. Ernster, 1.-Y. Lee, B. Norling, B. Persson, K. Juntti, and U.-B. Torndal, in "Probes of Structure and Function of Macromolecules and Membranes" (B. Chance, C. P. Lee, and J. K. Blasie, eds.), Vol. 1,377. Academic Press, New York, 1971. 15 E. Rossi, B. Norling, B. Persson, and L. Ernster, Eur. J. Biochem. 16, 508 (1970); in "Energy Transduction in Respiration and Photosynthesis" (E. Quagliariello, S. Papa, and C. S. Rossi, eds.), p. 329. Adriatica Editrice, Bari, 1971. 16 B. D. Nelson, B. Norling, B. Persson, and L. Ernster, Biochem. Biophys. Res. Commun. 44, 1312 (1971); Biochem. Biophys. Res. Commun. 44, 1321 (1971). ,7 B. D. Nelson, B. Norling, B. Persson, and L. Ernster, Biochim. Biophys. Acta 267, 205 (1972). ,s I.-Y. Lee and E. C. Slater, in "Dynamics of Energy-Transducing Membranes" (L. Ernster, R. W. Estabrook, and E. C. Slater, eds.), BBA Library, Vol. 13, p. 61. Elsevier, Amsterdam, 1974. ,9 A. Kr6ger and M. Klingenberg, Eur. J. Biochem. 39, 313 (1973). 2o D. B~ickstr6m, B. Norling, A. Ehrenberg, and L. Ernster, Biochim. Biophys. Acta 197, 108 (1970). 2, F. J. Ruzicka, H. Beinert, K. L. Schepler, W. R. Dunham, and R. H. Sands, Proc. Natl. Acad. Sci. U.S.A. 72, 2886 (1975). 22 H. Baum, J. S. Rieske, H. 1. Silman, and S. H. Lipton, Proc. Natl. Acad. Sci. U.S.A. 57, 798 (1%7). 2~ M. K. F. Wikstr6m and J. A. Berden, Biochim. Biophys. Acta 283, 403 (1972). 24 p. Mitchell, FEBS Lett. 56, 1 (1975); FEBS Lett. 59, 137 (1975); J. Theor. Biol. 62, 327 (1976). 2,~W. J. lngledew, J. C. Salerno, and T. Ohnishi, Arch. Biochem. Biophys. 177, 176 (1976).
[55]
UB1QUINONE 1N SUBMITOCHONDRIAL PARTICLES
577
0 ra
>
50
50
*6
c o
100
l i ~ l l l 12 4 5 6 Numberof extractions
100
FIG. 1. Removal of ubiquinone (z%) and decrease in NADH (O) and succinate oxidase (O) activities upon repeated extractions of lyophilized beef-heart submitochondrial particles with n-pentane. From L. Ernster, I.-Y. Lee, B. Norling, and B. Persson, Eur. J. Biochem. 9, 299 (1969).
component of the two oxidases. This conclusion is further supported by the finding" that incorporation of limiting amounts of ubiquinone reactivates N A D H and succinate oxidases in a parallel fashion (Fig. 2). A preferential reactivation of succinate oxidase by the addition of small amounts of ubiquinone to pentane-extracted particles has been reported by Gutman e t a l . lo and by Kr6ger and Klingenberg 12 and has been interpreted as indicating either the involvement of different pools of ubiquinone in the two oxidases 26 or different affinities of the NADH and suc120
"
o"
g 8o , NADH ox i d a s e o Succ oxidase
-
~, 60 4O 2O L
O0 -
-
5 10 1'5 Ubiquinone content (nmol ling protein )
i
20
FIG. 2. Restoration of NADH (O) and succinate oxidase (©) activities of ubiquinonedepleted beef-heart submitochondrial particles as a function of the ubiquinone content. From B. Norling, E. Glazek, B. D. Nelson, and L. Ernster, Eur. J. Biochem. 47, 475 (1974). 2~ G. Lenaz, G. D. Daves, Jr., and K. Folkers, Arch. Biochem. Biophys. 123, 539 (1%8).
578
QUINONES
[55]
cinate dehydrogenases to ubiquinone. 12 However, this difference seems to be due primarily to an impaired ability of ubiquinone to reactivate NADH oxidase in ubiquinone-depleted particles under the conditions employed. A selective reactivation of succinate oxidase by certain ubiquinone analogs has been described by Lenaz et al. ~6and interpreted in terms of a difference in quinone specificity between the NADH and succinate dehydrogenases. A recent extension of these studies 27 indicates, however, that the observed phenomenon may be related to a difference in membrane topology of the two dehydrogenases in relation to the cytochrome bcl complex rather than different quinone specificities. A study of the restoration of the NADH, succinate, and a-glycerophosphate oxidase activities of pentane-extracted pig-brain mitochondria by ubiquinone has been reported by Salach and Bednarz. 28 Various aspects of the functions of ubiquinone in mitochondria are discussed in a number of recent reviews. 29-32
Extraction and Reincorporation of Quinones in Other EnergyTransducing Membranes Restoration of NADH oxidase by adding ubiquinone to pentane-extracted membranes of Azotobacter vinelandii has been described by Swank and Burris. 33 Kr6ger et al. 34 have reincorporated ubiquinone and menaquinone into pentane-extracted Proteus rettgeri and demonstrated that ubiquinone reactivates respiration with succinate and formate as substrates, whereas menaquinone restores fumarate reduction. An extraction and incorporation method employing isooctane as solvent has been developed by Horio et al. 35 to study the role of ubiquinone in chromatophores from a blue-green mutant of Rhodospirillum rubrum. 27 G. Lenaz, S. Mascarello, L. Landi, L. Cabrini, P. Pasquali, G. Parenti-Castelli, A. M. Sechi, and E. Bertoli, in "Bioenergetics of Membranes" (L. Packer, G. C. Papageorgiou, and A. Trebst, eds.), p. 189. Elsevier, North-Holland Biomedical Press, Amsterdam, 1977. 28 j. I. Salach and A. J. Bednarz, Arch. Biochem. Biophys. 157, 133 (1973). 29 F. L. Crane, Annu. Rev. Biochem. 46, 439 (1977). a0 j. DePierre and L. Ernster, Annu. Rev. Biochem. 46, 201 (1977). 31 p. D. Boyer, B. Chance, L. Emster, P. Mitchell, E. Racker, and E. C. Slater, Annu. Rev. Biochem. 46, 955 (1977). 32 L. Ernster, in "Biomedical and Clinical Aspects of Coenzyme Q'" (K. Folkers and Y. Yamamura, eds.), p. 15. Elsevier, North-Holland Biomedical Press, Amsterdam, 1977. aa R. T. Swank and R. H. Burris, J. Bacteriol. 98, 311 (1969). 34 A. Kr6ger, V. Dadak, M. Klingenberg, and F. Diemer, Eur. J. Biochem. 21,322 (1971). "~ T. Horio, Y. Horiuti, N. Yamamoto, and K. Nishikawa, this series, Vol. 24, p. 96.
[56]
DETERMINATIONOF Q AND MK
579
Using the same method, Baccarini-Melandri and Melandri 36have recently reported evidence for an involvement of ubiquinone in the cytochrome bc2 segment of the redox chain of Rhodopseudomonas capsulata chromatophores. 34 A. Baccarini-Melandri and B. A. Melandri, FEBS Lett. 80, 459 (1977).
[56] D e t e r m i n a t i o n o f C o n t e n t s a n d R e d o x Ubiquinone and Menaquinone
States of
By A. KROGER Methods for the extraction and determination of ubiquinone (Q) and menaquinone (MK) have been described earlier in this series. 1-3 With the commercial availability of the dual-wavelength photometer, which was first designed by Chance, 4 it is now possible to determine these quinones more sensitively and with greater accuracy and convenience. Furthermore, it is possible to follow the redox states of the quinones as functions of the metabolic states of biological preparations either by extraction or by direct optical recording of the redox reactions of the quinones. Investigation of the function of the quinones in mitochondria and bacteria has revealed that at least 95% of the quinones occur either in the oxidized (quinone) or reduced state (hydroquinone). Other derivatives (chromenols and chromanols) or intermediates (radicals) are not detected by the methods described here. Extraction of Q and M K The extraction method described here is developed for (1) the determination of the total contents of either Q or MK in mitochondria, submitochondrial particles ~'G, cell-free bacterial homogenates and purified E. 2 F. :~ P. 4 B. 5 A. L.
R. Redfearn, this series, Vol. 10, p. 381. L. Crane and R. Barr, this series, Vol. 18C, p. 137. J. Dunphy and A. F. Brodie, this series, Vol. 18C, p. 407. Chance, Rev. Sci. Instrum. 22, 619 (1951). M. Pumphrey and E. R. Redfearn, Biochem. J. 76, 61 (1960). Szarkowska and M. Klingenberg, Biochem. Z. 338, 674 (1963).
