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Liver ischemia-reperfusion injury is still an open problem in many clinical circumstances, including surgery and transplantation. This study investigates how mitochondrial structure, mass and oxidative phosphorylation change and may be preserved during a brief period of ischemia followed by a long period of reperfusion, an experimental model that mimics the condition to which a liver is exposed during transplantation. Livers were explanted from rats and exposed for 24 h to three different oxygen availability conditions at 4 °C. Mitochondrial mass, respiration, oxidative phosphorylation (OXPHOS), and levels of OXPHOS complexes were all significantly altered in livers stored under the currently used preservation condition of normoxia. Remarkably, liver perfusion with hyperoxic solutions fully preserved mitochondrial morphology and function, suggesting that perfusion of the graft with hyperoxic solution should be considered in human transplantation.

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q (CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochrome c oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochrome c reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochrome c oxidoreductase is strongly stimulated by added CoQ3, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of the Km for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14 mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ9 and CoQ10.

Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ9/CoQ10 molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt. c reductase, NADH-cyt. c reductase, succinate-DCIP, ubiquinol2-cyt. c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ2H2-cyt. c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23-24 degrees C.

Extraction of endogenous ubiquinone with different methods does not influence ubiquinol oxidase activity in lyophilized mitochondria in terms of KM, although a decrease of Vmax is sometimes observed. Experiments with submitochondrial particles from a UQ-deficient mutant of S. cerevisiae confirm the results with UQ-depleted mitochondria and support the idea that endogenous ubiquinone is not required for the oxidation of exogenous ubiquinols by complex III.

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochrome b extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochrome c was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/mol bc1 complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochrome b extrareduction. Since the initial rates of cytochrome b reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochrome b was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimyristoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18-28 degrees C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots of Km also show breaks in the same temperature interval, with the exception of the dioleoyl-enzyme vesicles, where Km is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24 degrees C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3-18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrial bc1 complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in the bc1 complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochrome c reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of the bc1 complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochrome c reductase activity. However, a clear stimulation of the rate of cytochrome b reduction in parallel to an inhibition of cytochrome b oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on the bc1 complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.

We have studied the effect of general anesthetics on the mobility of two stearic acid spin labels (5-doxyl stearic acid and 16-doxyl stearic acid) in bovine heart mitochondria and in phospholipid vesicles made from either mitochondrial lipids or commercial soybean phospholipids. The general anesthetics used include nonpolar compounds (alcohols, halothane, pentrane, diethyl ether, chloroform) and the amphipathic compound, ketamine. All anesthetics tested increase the mobility of the spin labels in phospholipid vesicles to a limited extent up to a concentration where the ESR spectra become those of free spin labels. On the other hand, anesthetics have a pronounced effect on mitochondrial membranes at concentrations as low as those known to produce general anesthesia; the effect is lower near the bilayer surface (5-doxyl stearic acid) and very strong in the bilayer core (16-doxyl stearic acid). The effects of anesthetics are mimicked by the detergent, Triton X-100. We suggest that the discrepancy between the action of anesthetics in mobilizing the spin labels in lipid vesicles and in membranes results from labilization of lipid protein interactions.

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 109 M−1 sec−1 corresponding to a diffusion coefficient in the range of 10−6 cm2/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Hochli, L., Hochli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA81, 2606–2610, 1984) is 3×10−9 cm2/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theKm of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.

The lipophilic cation tetraphenylphosphonium (TPP+) is accumulated by human skin fibroblasts across both the plasma and mitochondrial membranes. We show here that TPP+ uptake is indeed greatly decreased under conditions leading to de-energization of mitochondria. The TPP+ accumulation in the presence of the proton ionophore FCCP has been used for determination of the plasma membrane potential across the plasma membrane, after correction for potential-independent binding of TPP+ to cellular components. Following this procedure, a value of 75 mV has been obtained. Through the amount of TPP+ released by FCCP treatment, an estimate of the in situ mitochondrial membrane potential has been made. Furthermore, we report that the mitochondrial component of TPP+ accumulation decreases with aging of fibroblast cultures.