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T Higuti

Publications and source records attributed to T Higuti.

53 records · Page 3Linked to original sources
PubMed1983

Triphenyltetrazolium and its derivatives are anisotropic inhibitors of energy transduction in oxidative phosphorylation in rat liver mitochondria.

Triphenyltetrazolium and its derivatives inhibited energy transduction in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. Triphenyltetrazolium incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. Triphenyltetrazolium also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced sites on the membranes for triphenyltetrazolium and its derivatives. The maximum amounts of energy-dependent binding sites for triphenyltetrazolium on membranes energized with succinate and ATP, respectively, were 14 and 4 nmol/mg protein. Triphenyltetrazolium also induced H+ ejection from the energized membranes. The maximum amounts of H+ ejection from membranes energized with succinate and ATP, respectively, were 4 and 2.4 nmol/mg protein. Triphenyltetrazolium also decreased the membrane potential up to about half the control value and caused shrinkage of mitochondria in an energy-dependent fashion. Comparison of the Hammett's sigma constants of triphenyltetrazolium derivatives with various substituents on the 3-benzene ring showed that lower concentrations of triphenyltetrazolium derivatives with a stronger positive charge were required for inhibition of energy transduction. The present findings show that triphenyltetrazolium and its derivatives act as anisotropic inhibitors of energy transduction by binding to negative charges created on the outer side (C-side) of energized mitochondria, and that the positive charge of these inhibitors is one of important factors for their inhibitory activity. These negative charges may be an essential part of the H+ pump.

Adenosine Triphosphate↗
PubMed1981

Photoaffinity labeling of a mitochondrial hydrophobic protein by an anisotropic inhibitor of energy transduction in oxidative phosphorylation.

The monoazide derivative of ethidium, the parent compound of which is an anisotropic inhibitor of energy transduction in oxidative phosphorylation, was synthesized and shown to be useful as a photoaffinity probe. Results showed that monoazide ethidium specifically binds to a hydrophobic protein of mitochondria (with an apparent molecular weight of about 6200 in the presence of 0.1% sodium dodecyl sulfate). The molar binding ratios of monoazide ethidium to protein were about 5 and 17 with protein in the nonenergized and energized states, respectively. This protein differed from the dicyclohexylcarbodiimide-binding protein. We refer to this new hydrophobic protein, anisotropic inhibitor-binding protein, in this paper.

Affinity Labels↗
PubMed1980

Rhodamine 6G, inhibitor of both H+-ejections from mitochondria energized with ATP and with respiratory substrates.

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F1. Rhodamine 6G also inhibited both H+-ejections from mitochondria energized with respiratory substrates and with ATP. The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, Pi, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H+-ejection by redox components and also on F0.

Adenosine Triphosphate↗
PubMed1980

Anisotropic inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by tetraphenylarsonium.

Tetraphenylarsonium (TPA+) inhibited energy transduction in oxidative phosphorylation in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. TPA+ incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. TPA+ also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced binding sites on the membranes for TPA+. The amounts of energy-dependent binding sites for TPA+ on mitochondria energized with succinate and with ATP, respectively, were 90 and 13 nmol/mg of protein. TPA+ also caused shrinkage of mitochondria energized with succinate and with ATP in an energy-dependent fashion. The energy-dependent binding of TPA+, TPA+-induced H+-ejection, TPA+-induced shrinkage of mitochondria, and TPA+-induced inhibition of energy transduction occurred in parallel. The present findings show that TPA+ inhibits energy transduction by binding to negative charges created on lipophilic domains near the surface of the outer side (C-side) of the mitochondrial inner membranes, and that it has no inhibitory activity on the inner side (M-side) of the membranes.

Adenosine Triphosphate↗
PubMed1979

Localized energization of the mitochondrial inner membrane by ATP.

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane. The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F1 . F0) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211-222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F1), reflecting the localized energization of the membrane by ATP.

Adenosine Triphosphatases↗
PubMed1978

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.

Adenosine Triphosphate↗
PubMed1976

Energization of mitochondrial inner membranes caused by L-malate.

It was found that 0.06 mug antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c1 completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.

Adenosine Triphosphate↗
PubMed1976

Polarographic studies in presence of Triton X-100 on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum.

Polarographic studies on oxidation-reduction components bound with chromatophores from Rhodospirillum rubrum were carried out at 24 degrees. 1. Using a carbon-paste electrode as the working electrode, polarographic waves characteristic of oxidation-reduction components were observed in the presence, but not in the absence of Triton X-100; these waves were therefore measured in the presence of the detergent. 2. At least two kinds of oxidation-reduction components were detectable, having different half-wave potentials (E1/2); at pH 7, one had an E1/2 value of +275 mV (POC+275) and the other had a value of +60 mV (POC+60). 3. POC+275 was reduced by succinate and by NADH. Both reductions were almost completely inhibited by antimycin A, which hardly affected the reductions of ubiquinone-10 by succinate and by NADH. Most POC+275 molecules were not reduced by the substrates when quinones were extracted from the chromatophores, and the reductions were mostly restored when ubiquinone-10 was re-added. This indicates that POC+275 is functional between ubiquinone-10 and cytochrome c2 in the electron transport system. 4. POC+60 was reduced by succinate, but hardly at all by NADH. The reduction of POC+60 was not influenced either by the addition of antimycin A or by the extraction of quinones. This suggests that POC+60 is functional in the process from succinate dehydrogenase [EC 1.3.99.1] to ubiquinone-10 in the electron transport system. 5. Of the POC+275 reducible by dithionite, approximately 70% could be reduced in the absence of Triton X-100, provided that the potential of the working electrode immersed in chromatophore suspensions was set at potentials of 0 mV or lower and that the electrochemical reaction was carried out at pH 7.5. When the potential of the electrode was set at +50 mV (the same as the E1/2 value of ubiquinone-10 bound with chromatophores), and the suspension was allowed to stand for various lengths in the presence of the detergent, it was found that approximately half of the electrochemically reducible POC+275 was rapidly reduced, followed by a slow reduction. The discrepancy in the oxidation-reduction equilibrium on the basis of the E1/2 values of ubiquinone-10 and POC+275 is discussed.

Antimycin A↗
PubMed1975

Stepwise reduction of cytochromes b-562, b-566 and b-558 in rat liver mitochondria.

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria with out addition of substrate caused reduction of cytochromes b-562 (having an alpha-band at 562 nm at room temperature), c + c1, and a + a3. The effect of KCN on cytochrome b-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromes c+c1 and a+a3 was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 muM KCN under conditions were cytochromes b-562, c+c1 and a+a3 were reduced, caused reduction of cytochrome b-566. The absorbance spectrum of cytochrome b-566 had an alpha-band at 565.5 nm, a beta-band at 538 nm and a gamma-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 cytochromes b-566, b-562, c+c1 and a+a3 were already fully reduced, caused reduction of cytochrome b-558 (having an alpha-band at 558 nm, a beta-band at 527 nm and a gamma-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochrome b-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochrome b-566, but is due to a different species from cytochrome b-566.

Adenosine Triphosphate↗
PubMed1975

Polarographic studies on ubiquinone-10 and rhodoquinone bound with chromatophores from Rhodospirillum rubrum.

Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode.

Bacterial Chromatophores↗
PubMed1975

Role of ubiquinone-10 in electron transport system of chromatophores from Rhodospirillum rubrum.

The role of ubiquinone-10 in the activities for the reduction of free cytochrome c2 and bound cytochrome cc' by succinate was studied with chromatophores from a blue-green mutant (G-9) of Rhodospirillum rubrum. 1. By a single extraction with isooctane, approximately 90% of ubiquinone-10 was easily removed from the chromatophores. In the extracted chromatophores, the activity for succinate-cytochrome c2 reduction decreased to 5-10% of the original activity. This depressed activity was mostly restored by adding ubiquinone-10. The remaining quinone was hardly extractable, even by repeated extractions. With repeatedly extracted chromatophores, the activity for succinate-cytochrome c2 reduction was mostly restored to the same extent as with once-extracted chromatophores, whereas the extent of inhibtion of the activity by antimycin A gradually fell. 2. In isooctane-extracted chromatophores, the activity for the reduction of bound cytochrome cc' by succinate under anaerobic conditions decreased to 35 to 95% of the original level. With chromatophores in which the remaining activity was as low as 40% of the original level, the activity was partially restored by adding ubiquinone-10, but this was not the case with chromatophores in which the remaining activity was higher than approximately 50% of the original level.

Alkanes↗