Bernard Guérin

UNILATERAL BRAIN INJURY IN THE RABBIT; REVERSIBLE AND IRREVERSIBLE DAMAGE OF THE MEMBRANAL ATPases

Modifications of some membranal enzymatic activities in rabbit brain edema induced by cold injury were studied. The edema was characterized by the tissue H2O content and the K+/Na+ ratio. Comparison of the respiratory rate of isolated mitochondria in the state 3 and 4 and the ADP/O ratio suggested an alteration in the ATP synthesis mechanism. The oligomycin sensitive ATPase activity was severely reduced in mitochondria isolated from edematous cells.

Phosphate transport and ATP synthesis in yeast mitochondria: Effect of a new inhibitor: the tribenzylphosphate

We have shown that phosphate transport tn yeast tnttochondrta follows two mechantsms d well-defined cartter-tnedtated process (with a relatively low KT) and a ltke-dtffuston process [ 1.21. both wete tnhtbtted by mersalyl (see [3.4] ) The carrtet-mediated transport was dependent on the tnttortbosomal protein synthesis [Z] and the tmpltcdtton of a P,-btndtng proteoltptd. having also a mttortbosomal ortgtn. was therefore expected [5] The existence of these two systems for transport allowed us to define thetr role tn the metabolic pathway of P, tn mttochondrta Here we describe the action of a new tnhtbttor of both Pitransport and ATP synthesis Our results are consistent wtth the tdea that the carrier-mediated P,-transport IS spectftcally tmpltcdted tn the oxtdattve phosphorylatton process as suggested for tnammahan mttochondrta [61

Polyphosphates as a source of high energy phosphates in yeast mitochondria: A 31P NMR study

Suspensions of purified yeast mitochondria were analyzed under bubbling oxygen by 31P NMR at 161.9 MHz. The recorded spectra indicate that polyphosphates (poly(P)) are present in mitochondrial preparations. These poly(P) further characterized by the NMR study of mitochondrial perchloric acid extracts have an average chain length of 14 ± 1 residues per chain and correspond to 10% of the total content of cellular poly(P) detected by NMR. The stability of mitochondrial poly(P) was increased by the presence of oligomycin, suggesting that this compound may play a role in the energetic metabolism of yeast mitochondria.

Energetic and morphological plasticity of C6 glioma cells grown on 3-D support; effect of transient glutamine deprivation.

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.

Perméabilité de la membrane interne des mitochondries de levure: étude des relations entre structure et activité

Summary In order to investigate the possible relations between the anionic permeability and the functions (or the structure) of the inner mitochondrial membrane, three types of organelles isolated from S. cerevisiae were tested: mitochondria (aerobic culture), promitochondria (anaerobic culture) and CAP-mitochondria (aerobic culture with chloramphenicol added). By using the technique of swelling in isoosmotic potassium salts, after a determination of the isotonic conditions, it was possible to discriminate between an electrogenic (valinomycin induced) or an electroneutral (both valinomycin and uncoupler induced) translocation. o 1) Mitochondria : The permeability properties of mitochondria are energy dependent: a) Respiring mitochondria are permeable to Cl − ; Mg 2+ , however, inhibits this translocation. Phosphate transport seems to be exclusively electrogenic and mersalyl insensitive, but swelling inhibition by that thiol reagent is restored by Mg 2+ . b) Non respiring mitochondria are impermeable to Cl − , but ATP addition restores the permeability. Thiocyanate permeates as the anionic form and acetate as the undissociated form. The phosphate transport, sensitive to mersalyl, seems to be partially electrogenic. 2) Promitochondria : Deficient of respiratory enzymes but containing an oligomycin sensitive ATPase, they are impermeable to Cl − only when Mg 2+ is added. In these conditions, an electrogenic phosphate transport, sensitive to mersalyl, is observed. 3) CAP-mitochondria : Although CAP-mitochondria are cytochrome deficient and contain an oligomycin insensitive ATPase, they are also impermeable to Cl − in presence of Mg 2+ . As in fully differenciated mitochondria, an electroneutral phosphate entry is observed; Mg 2+ is required for mersalyl sensitivity.

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). (Mol Cell Biochem.

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. We concluded that the high rate of static-head respiration in yeast mitochondria was determined both by the non-ohmic proton conductance of the inner membrane and the saturation of the redox proton pump slipping.

Dependence of flux size and efficiency of oxidative phosphorylation on external osmolarity in isolated rat liver mitochondria: role of adenine nucleotide carrier

The aim of this work was a thermodynamic and kinetic study of the influence of varying external osmolarity on overall oxidative phosphorylations in isolated rat liver mitochondria. When external osmolarity is increased from 100 to 400 mosM by using a non-penetrant sugar: (i) matrix volume diminishes, (ii) state 3 respiratory rate decreases when state 4 slightly varies, (iii) states 3 and 4 protonmotive force and NAD(P)H level increase, whereas oxidative phosphorylation efficiency (ATP/O) decreases. Indeed, respiratory flux versus protonmotive force relationships depend on the osmolarity considered: the lower the external osmolarity, the higher the span of overall driving force necessary for the same respiratory rate. To further investigate the mechanism of the decrease in respiratory and ATP synthesis flux leading to a lowering in oxidative phosphorylation efficiency, we determined the adenine nucleotide carrier control coefficient on respiratory and ATP synthesis rates respectively. The main result is that the adenine nucleotide carrier control coefficient on respiratory rate decreases, and conversely that adenine nucleotide carrier control on ATP synthesis rate increases, from iso- to hyperosmolarity. Furthermore, whatever the osmolarity, when state 3 respiratory rate is titrated with carboxyatractyloside, the same relationship is observed between ATP/O ratio and respiratory flux. From many previous studies, it has been shown that an increase in external osmolarity and a consequent decrease in matrix volume inhibits almost all mitochondrial proton pumps (coupling site 1 and 2 of respiratory chain, ATPase) in different ways. In this work, we show that in phosphorylating mitochondria, the adenine nucleotide carrier plays a key role: its inhibition as the external osmolarity increases lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency.

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. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.

Studies on anionic transport in yeast mitochondria and promitochondria. Swelling in ammonium phosphate, glutamate, succinate and fumarate solutions

The transport of some anionic components into mitochondria appeared to be mediated by a series of exchange-diffusion carriers, localized in the internal membrane. Moreover, rat liver mitochondria have been shown to contain at least eight specific carriers [ 141 . One of the most useful tools, in identifying some of these carriers was the osmotic swelling of mitochondria suspended in the isotonic ammonium salt of the anion to be tested [S] . For instance, rat liver mitochondria spontaneously swelled in ammonium phosphate or glutamate, suggesting an anion-hydroxyl exchange. But, with succinate or malate, phosphate was necessary before swelling occurred. However, only the existence of specific inhibitors is a probe for a mediated-carrier transport. With yeast mitochondria and promitochondria , Kolarov et al. [6] noted likewise a swelling in ammonium phosphate, but they reported that succinate uptake did not require phosphate as it did for malate: these data suggested the presence of a succinate carrier distinct of the malate carrier. In order to determine if, in yeast mitochondria (aerobically grown cells) and promitochondria (anaerofically grown cells), the phosphate uptake was carrier mediated, we studied the mersalyl effects on the swelling. We also examined the glutamate, succinate and fumarate transport in these organelles.