valinomycin and myxothiazol

In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.

Topics: Adenosine Triphosphate; Animals; Brain; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Dicyclohexylcarbodiimide; Electron Transport; Hydrolysis; Kinetics; Male; Methacrylates; Mitochondria; Oligomycins; Phosphates; Proton-Motive Force; Rats; Rats, Wistar; Staining and Labeling; Subcellular Fractions; Substrate Specificity; Temperature; Thiamine Triphosphate; Thiazoles; Valinomycin

Opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoK(ATP)-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the K(ATP) channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K(+) influx as K(ATP) channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K(+) influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoK(ATP)-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.

Topics: Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Antifungal Agents; Cromakalim; Cyclic GMP-Dependent Protein Kinases; Decanoic Acids; Diazoxide; Electron Transport Complex I; Fluorescent Dyes; Hydrogen-Ion Concentration; Hydroxy Acids; Ionophores; Male; Methacrylates; Mitochondria, Heart; Mitochondria, Liver; Models, Biological; Potassium Channels; Protein Kinase C; Rats; Rats, Sprague-Dawley; Superoxides; Thiazoles; Valinomycin; Vasodilator Agents

Inside-out subcellular vesicles of Azotobacter vinelandii are found to produce delta pH and delta psi (interior acidic and positive) when oxidising malate or menadiol. These effects are inherent in both Cyd+ Cyo- (lacking the o-type oxidase) and Cyd- Cyo+ (lacking the bd-type oxidase) strains. They appear to be myxothiazol-sensitive in the Cyd- Cyo+ strain but not in the Cyd+ Cyo- strain. The H+/e- ratio for the terminal part of respiratory chain of a bd-type oxidase overproducing strain is established as being close to 1. It is also shown that NADH oxidation by the vesicles from the Cyd- Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+ Cyo- strain is resistant to these Q-cycle inhibitors. It is concluded that (i) the bd-type oxidase of A. vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase and (ii) Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A. vinelandii respiratory chain and does not in the bd-type quinol oxidase terminated branch. These relationships are discussed in the context of the respiratory protection function of the bd-type oxidase in A. vinelandii.

Topics: Anaerobiosis; Azotobacter vinelandii; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Cytochrome b Group; Cytochromes; Electron Transport Chain Complex Proteins; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Malates; Methacrylates; Oxidoreductases; Subcellular Fractions; Thiazoles; Valinomycin; Vitamin K

In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.

Topics: Animals; Cells, Cultured; Electron Transport; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Liver; Membrane Potentials; Methacrylates; Mitochondria, Liver; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Rats; Thiazoles; Valinomycin