In Phosphorylating Acanthamoeba castellanii Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

2005 ◽  
Vol 37 (2) ◽  
pp. 97-107 ◽  
Author(s):  
Wieslawa Jarmuszkiewicz ◽  
Aleksandra Swida ◽  
Malgorzata Czarna ◽  
Nina Antos ◽  
Claudine M. Sluse-Goffart ◽  
...  
Keyword(s):  
Redox State ◽  
Uncoupling Protein ◽  
Acanthamoeba Castellanii ◽  
Protein Activity

Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides

2008 ◽  
Vol 413 (2) ◽  
pp. 359-367 ◽  
Author(s):  
Aleksandra Swida ◽  
Andrzej Woyda-Ploszczyca ◽  
Wieslawa Jarmuszkiewicz
Keyword(s):  
Respiratory Rate ◽  
Redox State ◽  
Uncoupling Protein ◽  
Acanthamoeba Castellanii ◽  
Inhibitory Effect ◽  
Proton Conductance ◽  
Purine Nucleotides ◽  
Protein Activity ◽  
Mitochondrial Uncoupling ◽  
Quinone Reduction

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.

scholarly journals Uncoupling proteins in mitochondria of plants and some microorganisms.

2001 ◽  
Vol 48 (1) ◽  
pp. 145-155 ◽  
Author(s):  
W Jarmuszkiewicz
Keyword(s):  
Higher Plants ◽  
Uncoupling Protein ◽  
Physiological Role ◽  
Acanthamoeba Castellanii ◽  
Uncoupling Proteins ◽  
Protein Activity ◽  
Functional Connection ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Carrier Family

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.

Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions

2013 ◽  
Vol 394 (5) ◽  
pp. 649-658 ◽  
Author(s):  
Andrzej Woyda-Ploszczyca ◽  
Wieslawa Jarmuszkiewicz
Keyword(s):  
Respiratory Rate ◽  
Atp Synthase ◽  
Redox State ◽  
Uncoupling Protein ◽  
Acanthamoeba Castellanii ◽  
Complex Iii ◽  
Dependent Manner ◽  
Mitochondrial Uncoupling ◽  
Concentration Dependent Manner ◽  
Succinate Oxidation

Abstract The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μm), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

scholarly journals Physicochemical considerations for bottom-up synthetic biology

2019 ◽  
Vol 3 (5) ◽  
pp. 445-458 ◽  
Author(s):  
Wojciech Mikołaj Śmigiel ◽  
Pauline Lefrançois ◽  
Bert Poolman
Keyword(s):  
Redox State ◽  
Life Sciences ◽  
Metabolic Energy ◽  
Molecular Crowding ◽  
Protein Activity ◽  
Bottom Up ◽  
Internal Ph ◽  
Synthetic Cells ◽  
Molecular Components ◽  
The Impact

The bottom-up construction of synthetic cells from molecular components is arguably one of the most challenging areas of research in the life sciences. We review the impact of confining biological systems in synthetic vesicles. Complex cell-like systems require control of the internal pH, ionic strength, (macro)molecular crowding, redox state and metabolic energy conservation. These physicochemical parameters influence protein activity and need to be maintained within limits to ensure the system remains in steady-state. We present the physicochemical considerations for building synthetic cells with dimensions ranging from the smallest prokaryotes to eukaryotic cells.

Coenzyme Q induces GDP-sensitive proton conductance in kidney mitochondria

2001 ◽  
Vol 29 (6) ◽  
pp. 763-768 ◽  
Author(s):  
K. S. Echtay ◽  
M. D. Brand
Keyword(s):  
Superoxide Dismutase ◽  
Fatty Acid ◽  
Coenzyme Q10 ◽  
Redox State ◽  
Uncoupling Protein ◽  
Coenzyme Q ◽  
Liver Mitochondria ◽  
Uncoupling Protein 2 ◽  
Proton Conductance ◽  
Kidney Mitochondria

Addition of coenzyme Q10 (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

Biologia ◽  
2013 ◽  
Vol 68 (3) ◽  
Author(s):  
Mario Soccio ◽  
Maura Laus ◽  
Daniela Trono ◽  
Donato Pastore
Keyword(s):  
Durum Wheat ◽  
Uncoupling Protein ◽  
Hyperosmotic Stress ◽  
K Channel ◽  
Acid Extraction ◽  
Atp Content ◽  
Protein Activity ◽  
Simple Method ◽  
Wheat Seedlings ◽  
Stress Dependent

AbstractThe assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

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