Mitochondrial ATP synthesis in permeabilized cells: assessment of the ATP/O values in situ.

We have developed a method to assess the actual mitochondrial ATP synthesis in cells (fibroblasts and Ehrlich ascites tumor cells). This method relies on gentle permeabilization of the cells, inhibition of nonmitochondrial ATP synthesis systems (glycolysis and adenylate kinase), and the inhibition of all ATPase activities (ATP hydrolysis) by decreasing the cytosolic magnesium concentration with EDTA. We have simultaneously measured the rate of respiration, and the ATP/O values obtained with this method are similar to those obtained with isolated mitochondria using the same respiratory substrates.

Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation: dependence upon both fluxes and forces.

The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation.

Effects of the local anesthetic bupivacaine on mitochondrial energy metabolism: change from uncoupling to decoupling depending on the respiration state.

The local anesthetic bupivacaine has been found to uncouple oxidative phosphorylation. However, the precise mechanisms of bupivacaine uncoupling have not been elucidated. In the present paper, we demonstrate that the uncoupling effect of the local anesthetic depends on the respiration state.

Effects of the anaesthetic propofol on the calcium-induced permeability transition of rat heart mitochondria: direct pore inhibition and shift of the gating potential.

Mitochondrial calcium exchanges are involved in intracellular calcium homeostasis and in the contraction-relaxation process in myocytes. The calcium-induced permeability transition of the heart mitochondria inner membrane appears to be an important calcium efflux mechanism involved in some physiological and pathological situations. The negative inotropic effect of the anaesthetic propofol results in part from a decrease in intracellular calcium availability.

Mechanistic stoichiometry of yeast mitochondrial oxidative phosphorylation.

This study investigates the relationships between the efficiency of oxidative phosphorylation (ATP/O) and respiratory flux in yeast mitochondria. To manipulate the electron flux through the respiratory chain, different substrates leading to NAD(P)H were used. By testing the effect of ADP either on respiratory rate in the presence or absence of oligomycin or on the level of NAD(P)H, on one hand, and the effects of uncouplers on respiration, on the other, we distinguished several categories of substrates: those for which the low respiration rate was mainly controlled by dehydrogenase activities and others for which the respiration was high and controlled downstream from the dehydrogenases.

Antibodies against subunits of F0 sector of ATP synthase from Saccharomyces cerevisiae. Stimulation of ATP synthase by subunit-8-reactive antibodies and inhibition by subunit-9-reactive antibodies.

Polyclonal antibodies against the three purified proteolipids of the F0 sector [subunit 6 (Su6), subunit 8 (Su8), subunit 9 (Su9)] and against the beta subunit (F1) of ATP synthase were raised in rabbits. All antisera showed ELISA reactivities with F0F1-ATPase. Antisera used to immunoblot partially purified ATP synthase labeled a single band migrating with the same molecular mass as that of the purified protein.

Respiration in non-phosphorylating yeast mitochondria. Roles of non-ohmic proton conductance and intrinsic uncoupling.

Respiratory rate, protonmotive force and charge/O ratio were measured under two different kinds of steady state in non-phosphorylating yeast mitochondria: (i) when the electron flux was modulated by a variable limitation in electron supply or (ii) when oxygen consumption was decreased by respiratory chain inhibitor titration. We showed that the relationships between either delta p or charge/O ratio and respiratory rate are different under the two kinds of steady state, indicating different degrees of intrinsic uncoupling in respiratory chain. Moreover, we observed a non-ohmic dependence between H(+)-conductance and delta p.

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine.

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria.

Almitrine, a new kind of energy-transduction inhibitor acting on mitochondrial ATP synthase.

At low concentrations, almitrine inhibits yeast cell multiplication by acting on oxidative metabolism. Studies on isolated mitochondria display the following features: (i) almitrine inhibits ATPase activity and decreases ATP/O ratio during oxidative phosphorylation; (ii) no direct effect on respiration can be evidenced; (iii) ATP/O value decreases without any change in the magnitude of delta p; (iv) the higher the ATP synthesis and respiratory fluxes, the larger is the decrease in ATP/O ratio induced by almitrine. These results indicate that almitrine does not act as a classical protonophoric uncoupler nor as previously studied non protonophoric uncouplers (e.