On the Antimycin-sensitive Cleavage of Complex III of the Mitochondrial Respiratory Chain

Rieske, John S.; Baum, Harold; Stoner, Clinton D.; Lipton, S. H.

doi:10.1016/s0021-9258(18)99450-0

Cited by 116 publications

(12 citation statements)

References 32 publications

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“…With each of these detergents, we were never successful in generating homogeneous monomeric cytochrome bc\ unless the complex was first stabilized by inhibition with antimycin A, an inhibitor known to stabilize the cytochrome bc\ complex (Rieske et al, 1967). After inhibition by antimycin A, high concentrations of these detergents no longer cause disruption of the complex and instead produce a significant amount of monomeric complex having a protein molecular weight of approximately 230 000.…”

Section: Discussionmentioning

confidence: 99%

“…However, the quality of the extracted enzyme is significantly improved when Triton X-100 is used in the initial extraction of the enzyme from Keilin-Hartree particles, i.e., a procedure similar to that used for the extraction of the enzyme from freshly prepared mitochondria. Subsequent purification of complex III from deoxycholate and cholate by the method of Rieske (1967) then becomes routine and reproducible.…”

Section: Discussionmentioning

confidence: 99%

“…The resulting turbid suspension was centrifuged for 75 min at 41 500 rpm and the dark red pellet resuspended by homogenization in 20 mM Tris-Cl buffer, pH 8.0, containing 0.66 M sucrose and 0.001 M histidine (TSH buffer) with a glass/Teflon homogenizer. This resuspended product is equivalent to the precipitated S-1 fraction of Rieske (1967) and was further purified by ammonium acetate and ammonium sulfate precipitation from deoxycholate and cholate as described by Rieske (1967). After the final ammonium acetate precipitation, the floating layer of red protein was carefully removed, dissolved in the TSH buffer, dialyzed 2-3 h against TSH buffer, and frozen at -70 °C until it was used.…”

Section: Methodsmentioning

confidence: 99%

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Detergent-Solubilized Monomeric and Dimeric Cytochrome bc1 Isolated from Bovine Heart

Musatov

Robinson²

1994

Biochemistry

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Mitochondrial cytochrome bc1 complex, isolated from frozen bovine heart, was solubilized with five different "non-denaturing" detergents: dodecyl maltoside, octaethylene glycol monododecyl ether (C12E8), Triton X-100, Tween 20, and sodium cholate. The hydrodynamic properties of the solubilized complex III's were then investigated by sedimentation analysis. Complex III exists as a stable and monodisperse dimer when it is solubilized in low ionic strength buffer with a low concentration of any of the five detergents. At pH 7.8, 20 degrees C, the protein sediments as a homogeneous species with an s(obs) of about 14 S. The protein molecular weight of the 14S particle, after correction for bound detergent, is 465,000 +/- 30,000 as measured by sedimentation equilibrium analysis. The aggregation state and/or homogeneity of cytochrome bc1 is strongly dependent upon the concentration of the solubilizing detergent and ionic strength. The enzyme becomes a homogeneous, monomeric complex with a protein molecular weight of 235,000 +/- 20,000 and an s(obs) of 10-10.5 S after it is solubilized in high concentrations of Tween 20 (more than 5 mg/mg of protein) and sodium chloride (more than 0.5 M). However, a heterogeneous mixture of subcomplexes is produced upon solubilization of the complex with high concentrations of the other detergents and 0.5 M NaCl. Monomerization of cytochrome bc1 by Tween 20 and 0.5 M NaCl has no effect on either the spectral properties, the subunit composition, or the enzymatic activity and is reversible since the dimeric 14S particle is regenerated upon removal of the high concentration of salt.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Detergent-Solubilized Monomeric and Dimeric Cytochrome bc1 Isolated from Bovine Heart

Musatov

Robinson²

1994

Biochemistry

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“…On the other hand, antimycin and HQNO make b566 reducible, while other complex III inhibitors, including BAL, UHDBT, ethoxyformic anhydride (Yagi et al, 1982), and the inhibitors described by Becker et al (1981), do not. Another important property of antimycin is that it makes complex III structurally more stable and resistant to cleavage by guanidine (Rieske et al, 1967;Hatefi and Hanstein, 1974). Thus, it is possible that the ability of antimycin to make b566 reducible is associated with the fact that it causes certain conformation changes in complex III which lead, among other things, to a more stable structure.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of cytochrome b oxidation in antimycin-treated submitochondrial particles

Hatefi¹,

Yagi²

1982

Biochemistry

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It has been shown that in bovine heart submitochondrial particles, antimycin and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) inhibit the oxidation of NADH, succinate, and reduced ubiquinone incompletely, the uninhibited rate being about 20-40 nmol of substrate oxidized min-1 (mg of protein)-1. By contrast, rotenone, cyanide, BAL (2,3-dimercaptopropanol), and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole [Trumpower, B. L., & Haggerty, J. G. (1980) J. Bioenerg. Biomembr. 12, 151-164] caused essentially complete inhibition when added alone or after maximal inhibition by antimycin or HQNO. Having thus ascertained that the electron leak through the antimycin block appeared to follow the normal path through complex III (ubiquinol: cytochrome c oxidoreductase) and cytochrome oxidase, the reduction of the b cytochromes by substrates and their oxidation through the leak in the antimycin block by molecular oxygen were studied. It was shown that at normal electron flux from NADH and succinate, both cytochromes b562 and b566 were reduced in antimycin-treated submitochondrial particles. Their oxidation after substrate exhaustion was biphasic, however. At 565 minus 575 nm, 56% of the total reduced cytochrome b was oxidized through the leak in the antimycin block at a more rapid rate, while the remaining 44% was oxidized about 10 times slower. When electron flux from substrates to complex III was slowed down by the use of inhibitors or substrates at less than or equal to 0.1 Km concentration, then only reduced b562 accumulated in antimycin-treated particles. The oxidation of b562 after substrate exhaustion or inhibition of substrate oxidation by an appropriate inhibitor occurred at a rate comparable to that of the slower reoxidation phase described above. These results indicated, therefore, that cytochromes b566 and b562 are oxidized through the leak in the antimycin block at two different rates, the reoxidation rate of b566 being about 10 times faster than that of b562. The implications of these findings on the kinetic relationship of these two cytochromes in the respiratory chain have been discussed.

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“…Antimycin A has been shown to be a potent inhibitor of the mitochondrial electron transfer chain (Ahmad et al, 1950;Potter and Reif, 1952) acting in the span between cytochromes b and ci (Chance, 1952; Chance and Williams, 1956). The amount of antimycin A required to inhibit completely succinate (or NADH) oxidase activity is stoichiometric with the cytochrome content being 1:1 for substrate-reducible cytochrome (Chance, 1958;Estabrook, 1962) and 1:2 for dithionite-reducible cytochrome (Pumphrey, 1962;Rieske et al, 1967;Takemori and King, 1964;Berden and Slater, 1970). Antimycin A also causes marked changes in the cytochrome spectra, particularly an increase in absorption at 565 nm (Chance, 1952; Chance and Williams, 1956; Chance, 1958;Slater and Colpa-Boonstra, 1961;Pumphrey, 1962).…”

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confidence: 99%

Effect of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide on the respiratory chain of submitochondrial particles of beef heart

Brandon¹,

Brocklehurst²,

Lee³

1972

Biochemistry

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On the Antimycin-sensitive Cleavage of Complex III of the Mitochondrial Respiratory Chain

Cited by 116 publications

References 32 publications

Detergent-Solubilized Monomeric and Dimeric Cytochrome bc1 Isolated from Bovine Heart

Detergent-Solubilized Monomeric and Dimeric Cytochrome bc1 Isolated from Bovine Heart

Kinetics of cytochrome b oxidation in antimycin-treated submitochondrial particles

Effect of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide on the respiratory chain of submitochondrial particles of beef heart

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