Saburo Muraoka

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile; a new powerful uncoupler of respiratory-chain phosphorylation

Abstract 3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) was found to be one of the most powerful uncouplers of respiratory-chain phosphorylation ever reported. Structure-activity studies of the derivatives indicated the role of three functional groups in SF 6847: (1) the malononitrile group as an electron withdrawing group, which may cause uncoupling by interaction with a primary energy conservation site, (ii) a free hydroxyl group as an electron donator, and (iii) bulky tertiary butyl groups of hydrophobic character located at a certain spacial distance from the electron withdrawing center.

Stepwise reduction of cytochromesb-562,b-566 andb-558 in rat liver mitochondria

Abstract 1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria without addition of substrate caused reduction of cytochromesb-562(having an α-band at 562 nm at room temperature),c + c 1 anda + a 3 . The effect of KCN on cytochromeb-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromesc+c 1 anda+a 3 was not reversed by this uncoupler. 2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 μM KCN under conditions where cytochromesb-562,c+c 1 anda+a 3 were reduced, caused reduction of cytochromeb-566. The absorbance spectrum of cytochromeb-566 had an α-band at 565.5 nm, a β-band at 538 nm and a γ-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromesb-566,b-562,c+c 1 anda+a 3 were already fully reduced, caused reduction of cytochromeb-558 (having an α-band at 558 nm, a β-band at 527 nm and a γ-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochromeb-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochromeb-566, but is due to a different species from cytochromeb-566.

The minimum effective amount of uncouplers for rat liver mitochondria

In a previous report [I], SF 6847 (3,5-di-tert-butyl4-hydroxy-benzylidenemalononitrile) was shown to be one of the most effective uncouplers of respiratorychain phosphorylation ever reported, less than 1.2 nmol/g mitochondrial protein being sufficient for halfmaximal uncoupling activity. However, the concentration of SF 6847 causing 5% or 10% uncoupling was found to vary according to the experimental methods and conditions used. Further studies showed that this variation in, the effective concentration of uncoupler was mainly due to variation in the concentration of mitochondria in the reaction mixture and that the effective amount of SF 6847 decreased on increasing the concentration of mitochondrial protein. As pointed out by Kraayenhof [2], the uncoupling effects of some weak uncouplers, such as dinitrophenol, are mainly determined by the uncoupler concentration and are slightly dependent on the protein concentration, while the uncoupling effects of potent uncouplers, such as salicylanilide, benzimidazole and carbonylcyanide derivatives are dependent on the protein concentration. Since relatively little is known about the quantitative relationship between uncoupling effect and protein concentration (but see [3] and [4]), we determined the effects of some uncouplers, such as SF 6847, 2,4_dinitrophenol, pentachlorophenol and flufenamic acid [5-71 at various concentrations of mitochondrial protein. The effective amounts of uncouplers were determined as the amounts per g of protein causing 5% inhibition of the ATP-j2 Pi exchange reaction and some hypotheses on the uncoupling mechanism were evaluated on the basis of the results.

Redox changes of cytochrome a-607 and NAD(P)H in rat liver mitochondria induced by L-malate under anaerobic conditions☆

Dawson et al. [l] demonstrated that C and D-malate induced the oxidation of b-type cytochrome absorbing at 559 nm in Arum spadii mitochondria and rat liver mitochondria. The present work showed that Lmalate induced oxidation of cytochrome a-607 in anaerobic rat liver mitochondria. The effect of Lmalate on cytochrome a-607 was abolished in four ways: by respiratory inhibitors, such as rotenone and antlrnycin A, by redox dyes, such as menadione and phenazine methosulfate, by alkaline pH and by uncouplers. Malate caused similar changes of nicotinamide nucleotides to those of cytochrome a-607 and the reason for the anomalous changes of cytochrome a-607 in the presense of Lmalate is discussed.

Reduction and oxidation of longer-wavelength cytochrome b (b566) in rat liver mitochondria under the influence of external oxidants

Abstract The reduced form of longer-wavelength cytochrome b ( b 566 ) was identified in rat liver mitochondria in State 5 on addition of ferricyanide. Addition of menadione caused oxidation of b 566 and reduction of cytochrome c 1 (+ c ). In the presence of menadione, ferricyanide induced reduction of b 566 and oxidation of cytochrome c 1 + c , and exhaustion of the added ferricyanide resulted in oxidation of b 566 and reduction of cytochrome c 1 + c . Antimycin A enhanced the extent of the ferricyanide-dependent redox change of b 566 from 20 to 68% of the total absorbance at 566 nm. Uncouplers hardly affected the change of b 566 in the presence of antimycin A, but lowered the reduced level in the absence of antimycin A.

New sensitive method for the spectral analysis of respiratory-chain components in mitochondria

Abstract A new analytical method (two-wavelength/double beam method) for identification of respiratory-chain components in mitochondria was developed using a commercially available two-wavelength spectrophotometer. Cancellation of the scattered light due to the turbidity of the sample and correction of baseline due to the errors in the appartus were achieved using an optical attenuator for the reference cuvette over a wide range of wavelength ( λ x ) taking λ 1 as the reference wavelength. Then the same beams were passed through the saple cuvette and the difference between the absorbance of the sample and reference cuvettes at λ x was obtained. Difference spectra between reduced and oxidized rat-liver mitochondria were obtained on a full scale of A 0–0.01 or 0.03 in varying wavelengths of 300–650 nm. Using this method the α-bands of cytochrome a (+ a 3 ), b and c , the Soret bands of cytochrome a 3 (+ a ) and b , and the bands of flavoprotein and pyridine nucleotides were detected in a quite sensitive manner with reasonable accuracy. A discussion is given on the theory and application of this method.

Effect of pH on the redox state of cytochrome a in anaerobic, ATP-treated intact mitochondria

Abstract Addition of ATP to anaerobic, glutamate-reduced coupled mitochondria from rat liver or heart caused oxidation of cytochrome a having a peak at 608 nm. Subsequent increase in pH from 7.0 to 8.4 reversed the effect of ATP and subsequent decrease in pH from 8.4 to 7.0 induced reoxidation. This reversible effect of pH on the redox state of cytochrome a may reflect an electrochemical event in the energy-conserving mechanism of the terminal coupling site.