scholarly journals Monitoring of the Membrane Potential in Proteoliposomes with Incorporated Cytochrome-c Oxidase Using the Fluorescent Dye Indocyanine

1996 ◽  
Vol 151 (3) ◽  
pp. 247-259 ◽  
Author(s):  
Yu. A. Ivashchuk-Kienbaum
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Fluorescent Dye

scholarly journals The mechanism for the ATP-induced uncoupling of respiration in mitochondria of the yeast Saccharomyces cerevisiae

1995 ◽  
Vol 307 (3) ◽  
pp. 657-661 ◽  
Author(s):  
S Prieto ◽  
F Bouillaud ◽  
E Rial
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Kinetic Properties ◽  
Yeast Saccharomyces Cerevisiae ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Low Efficiency ◽  
Respiratory Chain Activity

We have recently reported that ATP induces an uncoupling pathway in Saccharomyces cerevisiae mitochondria [Prieto, Bouillaud, Ricquier and Rial (1992) Eur. J. Biochem. 208, 487-491]. The presence of this pathway would explain the reported low efficiency of oxidative phosphorylation in S. cerevisiae, and may represent one of the postulated energy-dissipating mechanisms present in these yeasts. In this paper we demonstrate that ATP exerts its action in two steps: first, at low ATP/Pi ratios, it increases the respiratory-chain activity, probably by altering the kinetic properties of cytochrome c oxidase. Second, at higher ATP/Pi ratios, an increase in membrane permeability leads to a collapse in membrane potential. The ATP effect on cytochrome c oxidase corroborates a recent report showing that ATP interacts specifically with yeast cytochrome oxidase, stimulating its activity [Taanman and Capaldi (1993) J. Biol. Chem. 268, 18754-18761].

scholarly journals Defective kinetics of cytochrome c oxidase and alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres (‘MERRF’) at position 8344 nt

1999 ◽  
Vol 342 (3) ◽  
pp. 537-544 ◽  
Author(s):  
Hana ANTONICKÁ ◽  
Daniel FLORYK ◽  
Petr KLEMENT ◽  
Leona STRATILOVÁ ◽  
Jana HEŘMANSKÁ ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Control Analysis ◽  
Hybrid Cells ◽  
Myoclonus Epilepsy ◽  
Increased Sensitivity ◽  
Hybrid Clones

We have investigated pathogenic effects of the tRNALys A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased Vmax for both the low-affinity and high-affinity phases; however, the Km values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, δΨ, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in δΨ at state 4 and an increased sensitivity of δΨ to an uncoupler in fibroblasts from the patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship between heteroplasmy of the MERRF mutation, COX activity and δΨ. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and δΨ was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and δΨ in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of δΨ inhibition.

Control of proteoliposomal cytochrome c oxidase: the partial reactions

1990 ◽  
Vol 68 (9) ◽  
pp. 1135-1141 ◽  
Author(s):  
Peter Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Redox State ◽  
Ph Gradient ◽  
Transfer Model ◽  
State Reduction

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith–Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by ΔpH and by ΔΨ can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.Key words: proteoliposomes, cytochrome c, cytochrome oxidase, membrane potential, pH gradient, cytochrome a, electron transfer.

scholarly journals Fatty acids as modulators of cytochrome c oxidase in proteoliposomes

1996 ◽  
Vol 320 (2) ◽  
pp. 557-561 ◽  
Author(s):  
Martyn SHARPE ◽  
Ivano PERIN ◽  
John WRIGGLESWORTH ◽  
Peter NICHOLLS
Keyword(s):  
Fatty Acids ◽  
Enzyme Activity ◽  
Oleic Acid ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Palmitic Acid ◽  
Cytochrome C Oxidase ◽  
Respiratory Control ◽  
Similar Concentration

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (ΔΨ) and by pH gradient (ΔpH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that ΔpH exerts a greater influence than does an equivalent ΔΨ. Oleic acid at 100 µM removes all ΔΨ and ΔpH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H+/K+ exchangers, but neither fatty acid alone markedly affects ΔpH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes ΔΨ, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in ΔΨ- and ΔpH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254, 130–145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo.

Time-resolved generation of a membrane potential by ba 3 cytochrome c oxidase from Thermus thermophilus

FEBS Letters ◽  
1999 ◽  
Vol 457 (1) ◽  
pp. 98-102 ◽  
Author(s):  
S. Siletskiy ◽  
T. Soulimane ◽  
N. Azarkina ◽  
T.V. Vygodina ◽  
G. Buse ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Thermus Thermophilus ◽  
Time Resolved
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