Transfer of phosphatidic acid between microsomal and mitochondrial outer and inner membranes.

A protein fraction from rat liver cytoplasm, precipitable at 50-95% saturation of ammonium sulphate, binds phosphatidic acid from mitochondrial and microsomal membranes. Protein-bound phosphatidic acid was eluted from Sephadex G-75 in fractions corresponding to a molecular weight of about 10 000. No such binding was observed with mitochondrial soluble proteins, either total or precipitated with ammonium sulphate between 50 and 95% saturation. The transfer of phosphatidic acid from microsomes to mitochondria was increased by liver cytoplasmic proteins precipitable at 50-95% saturation of ammonium sulphate but not with mitochondrial soluble proteins. This increase by cytoplasmic proteins was pronounced in 200 mM sucrose but was negligible in 100 mM KCI where the spontaneous transfer was quite high. Cytoplasmic proteins stimulated the synthesis of cardiolipin and phosphatidylglycerol in mitochondria deprived of the outer membrane but not in intact mitochondria when phosphatidic acid was supplied either by microsomes or liposomes. It is suggested that the transfer of phosphatidic acid from the outer to the inner mitochondrial membrane is not mediated by transfer proteins but occurs either by direct contact of the membranes or as free diffusion through the aqueous phase.

Effect of fatty acids on energy coupling processes in mitochondria.

Long-chain fatty acids are natural uncouplers of oxidative phosphorylation in mitochondria. The protonophoric mechanism of this action is due to transbilayer movement of undissociated fatty acid in one direction and the passage of its anion in the opposite direction. The transfer of the dissociated form of fatty acid can be, at least in some kinds of mitochondrion, facilitated by adenine nucleotide translocase. Apart from dissipating the electrochemical proton gradient, long-chain fatty acids decrease the activity of the respiratory chain by mechanism(s) not fully understood. In intact cells and tissues fatty acids operate mostly as excellent respiratory substrates, providing electrons to the respiratory chain. This function masks their potential uncoupling effect which becomes apparent only under special physiological or pathological conditions characterized by unusual fatty acid accumulation. Short- and medium-chain fatty acids do not have protonophoric properties. Nevertheless, they contribute to energy dissipation because of slow intramitochondrial hydrolysis of their activation products, acyl-AMP and acyl-CoA. Long-chain fatty acids increase permeability of mitochondrial membranes to alkali metal cations. This is due to their ionophoric mechanism of action. Regulatory function of fatty acids with respect to specific cation channels has been postulated for the plasma membrane of muscle cells, but not demonstrated in mitochondria. Under cold stress, cold acclimation and arousal from hibernation the uncoupling effect of fatty acids may contribute to increased thermogenesis, especially in the muscle tissue. In brown adipose tissue, the special thermogenic organ of mammals, long-chain fatty acids promote operation of the unique natural uncoupling protein, thermogenin. As anionic amphiphiles, long-chain fatty acids increase the negative surface charge of biomembranes, thus interfering in their enzymic and transporting functions.

Transport of phosphatidic acid within the mitochondrion.

Transfer of phosphatidic acid from the outer to the inner membrane within intact rat liver mitochondria was assessed by measuring the ratio of lipid 32P to the marker enzyme of the outer membrane, rotenone-insensitive NADH-cytochrome c reductase, in the outer and inner membrane fractions obtained after incubation of mitochondria under conditions for net synthesis of [32P]phosphatidic acid. This transfer was found to proceed with time, to occur only under high ionic strength of the external medium and to be insensitive to N-ethylmaleimide and factors reducing the number of contact sites between the two mitochondrial membranes. These results are interpreted as supporting the idea that phosphatidic acid transport within the mitochondrion occurs as free diffusion through the aqueous phase and not being mediated by phospholipid transfer protein(s).

Homeostasis of the protonmotive force in phosphorylating mitochondria.

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.

Energy-storage capacity of the mitochondrial proton-motive force.

Resting state respiration of rat-liver mitochondria in the presence of oligomycin was rapidly blocked with cyanide and the dissipation of the membrane potential was followed with a tetraphenylphosphonium-sensitive electrode. From the rate of this dissipation and the electric capacitance of the mitochondrial membrane the energy stored in form of the membrane potential was calculated as about 7 microJ/mg protein. In the absence of oligomycin, dissipation of the membrane potential was slower, as it was partly compensated by proton ejection by mitochondrial ATPase hydrolyzing endogenous ATP. This allowed to calculate the total energy storage capacity of the proton-motive force. It amounted to the equivalence of 3.3 nmol ATP/mg protein or about 130 microJ/mg protein. The stoichiometry of proton-pumping ATPase utilizing endogenous ATP was estimated as three protons per molecule ATP.

Inhibition by Ca2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect.

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszynski, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszynski, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria.

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.

Is monoamine oxidase activity in the outer mitochondrial membrane influenced by the mitochondrial respiratory state?

Monoamine oxidase activity was measured in isolated rat liver mitochondria using the radiochemical assay with [14C]tyramine as substrate. With toluene as the extracting solvent the apparent activity in the resting state (State 4) was much higher than in the active state (State 3) in agreement with Smith and Reid (Smith, G.S. and Reid, R.A. (1978) Biochem. J. 176, 1011-1014). However, with ethyl acetate or diethyl ether as extracting solvents, the activity in both states was almost identical and several times higher than that measured with toluene. p-Hydroxyphenylacetaldehyde, p-hydroxyphenylacetalcohol and p-hydroxyphenylacetic acid were identified as final reaction products, the latter one being hardly extractable with toluene. It is concluded that monoamine oxidase activity is not influenced by the respiratory state of mitochondria and that differences found by Smith and Reid are due to different extractability of secondary reaction products. NADPH-dependent aldehyde reductase was tentatively identified in rat liver mitochondria, its specific activity amounting to about one fourth of that in the cytosol.

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids.

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the 'efficiency' of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a 'safety valve' preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria.

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Relationship between the energy cost of ATP transport and ATP synthesis in mitochondria.

Liver mitochondria form rats kept on a high-protein diet exhibit an increased rate of respiration upon addition of ornithine in the presence of HCO2 and NH+4. This is the manifestation of intramitochondrial utilization of ATP for the synthesis of citrulline. State 3 respiration of these mitochondria could be adjusted to the same rate as that produced by ornithine by either using limiting amounts of hexokinase or titration with atractyloside. Under such conditions, in both systems the proton-motive force, the intramitochondrial ATP/ADP ratio and the redox state of the respiratory chain were the same. In contrast to this, the ATP/O ratio (equal to 2 X citrulline/O ratio) in mitochondria synthesizing citrulline was higher than the glucose 6-phosphate/O ratio in the system where ATP was trapped extramitochondrially. The ratio of these two ratios was close to 1.5 with both glutamate and succinate as respiratory substrates. From these results it can be concluded that the translocation of ATP against ADP and phosphate utilizes an amount of the chemiosmotic proton gradient equal to half of that needed for the synthesis of ATP in the inner compartment.

Influence of different energy drains on the interrelationship between the rate of respiration, proton-motive force and adenine nucleotide patterns in isolated mitochondria.

The respiration of rat liver mitochondria was stimulated by three different ways of energy drain: (a) partial uncoupling (equivalent to direct collapse of the proton-motive force), (b) intramitochondrial utilization of ATP for citrulline synthesis, and (c) extramitochondrial utilization of ATP for glucose phosphorylation. At identical rates of respiration, the intramitochondrial ATP : ADP ratios were the same in all three systems. Furthermore, the proton-motive force was the same in partially uncoupled mitochondria and in the presence of hexokinase plus glucose up to a respiration rate amounting to about 60% of that of the fully active state. However, external ATP : ADP ratios were considerably different in various systems at comparable rates of oxygen uptake, being the lowest under conditions when ATP was being utilized externally. On this basis, it is concluded that the respiratory rate is controlled directly by the proton-motive force and the mitochondrial ATP-synthesizing system operates under near-equilibrium conditions with respect to the membrane energy state parameters. However, a disequilibrium exists at the step of the transport of ATP from mitochondria to the external (cytoplasmic) compartment.

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.

Energetics of Ehrlich ascites mitochondria: membrane potential of isolated mitochondria and mitochondria within digitonin-permeabilized cells.

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.

Regulation of mitochondrial resting state respiration: slip, leak, heterogeneity?

The contribution of molecular slippage of proton pumps, of proton leak and of coupling heterogeneity of mitochondrial population to the well-known non-linear interrelationship between resting state respiration and the protonmotive force is discussed in view of the following experimental findings. (1) After blocking mitochondrial respiration with cyanide, the rate of dissipation of the membrane potential is non-linearly dependent on the actual membrane potential, similarly to the resting state respiration in mitochondria titrated with small amounts of an inhibitor. In contrast, delta pH dissipates proportionally to its actual value. (2) The rate of electron flow from succinate to ferricyanide depends upon the protonmotive force, similarly to the flow from succinate to oxygen. This strongly suggests that the H+/e- stoichiometry in complexes III and IV of the respiratory chain is constant. (3) Mitochondria 'in situ', in permeabilized Ehrlich ascites cells, exhibit the same non-linear flux/force relationship as isolated mitochondria. These results strongly suggest that the non-ohmic characteristics of the inner mitochondrial membrane, with respect to protons driven by the membrane potential but not by the concentration gradient, is the main factor responsible for the nonlinear flux/force relationship in resting state mitochondria.

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system.

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.

Synthesis of phospholipids in mitochondria and other membrane fractions of rabbit reticulocytes.

1. Reticulocytosis of 40-50% was obtained in rabbits by daily bleeding. Reticulocytes (plus erythrocytes) were subfractionated into plasma membrane fraction, mitochondria and the post-mitochondrial fraction. 2. In all fractions, fatty acids were incorporated into phospholipids. This process was ATP dependent and represented acylation of lysophospholipids. 3. Incorporation of fatty acids into lysophosphatidic and phosphatidic acids occurred only in the presence of sn-glycerol 3-phosphate and was observed in mitochondria and the post-mitochondrial fraction. It represents a two-step acylation of sn-glycerol 3-phosphate. 4. Incorporation of phosphorylcholine from CDPcholine into phosphatidylcholine was observed in the mitochondrial and the post-mitochondrial fractions. This activity was correlated with NADPH-cytochrome c reductase and was probably connected with the remnants of the endoplasmic reticulum.

The role of cytoplasmic [Ca2+] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect.

The effect of Ca2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (delta psi) and alkalinization of the medium. This typical behaviour is drastically changed if Ca2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of delta psi, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 microM Ca2+ and a preincubation for 2 min with 0.4-1.0 microM Ca2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca2+ concentrations as low as 0.3 microM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyz A. Evtodienko YuV. Duszynski J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148-1154; Czyz A. Teplova VV. Sabala P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539-544], the present results suggest that cytoplasmic Ca2+ plays a crucial role in the Crabtree effect.

Changes in the mitochondrial surface potential during cuprizone-induced formation of megamitochondria.

The formation of megamitochondria upon treatment of mice with cuprizone was studied in relation to the surface potential of mitochondria. The latter was monitored by binding of 8-anilino-1-naphthalene sulphonate to membranes, by kinetics of monoamine oxidase and by free-flow electrophoresis of the particles. It was found that the surface potential of megamitochondria was by about 20 mV less negative than that of normal mitochondria whereas no change of the surface potential upon cuprizone treatment was observed in microsomes. It is suggested that a partial neutralization of the negative surface charge of mitochondrial membranes may promote fusion or inhibit division of mitochondria, thus resulting in formation of giant structures.

Mitochondrial aldehyde reductase: identification and characterization in rat liver and kidney cortex.

Aldehyde reductase (EC 1.1.1.2) has been regarded so far as an exclusively cytosolic enzyme. The present investigation shows that mitochondria of rat liver, kidney cortex and, tentatively, heart also contain an enzyme catalyzing oxidation of NADPH by aldehydes, p-nitrobenzaldehyde, methylglyoxal and glyceraldehyde. Activity of the mitochondrial enzyme can only be measured after the organelles are disrupted by sonication or solubilized with nonionic detergents. Mitochondrial aldehyde reductase activity contributed to about 4.6% and 2.5% of the total cellular activity in liver and kidney cortex, respectively. However, the specific activity in liver mitochondria was about one third and in kidney cortex mitochondria one tenth of that in the cytosol of the corresponding organ. The mitochondrial enzyme resembled the cytosolic one by its absolute specificity towards NADPH as the electron donor, a similar profile of aldehydic electron acceptors and identical Km values. Mitochondrial aldehyde reductase differed from the cytosolic enzyme by low sensitivity to known inhibitors of cytosolic aldehyde reductase, AL-1576, AL-4114 and ONO-2235. In liver, about 60% of the mitochondrial activity was tightly bound to the membranes whereas about 40% was present in the mitochondrial matrix. The membrane-bound activity was inactivated by digestion of mitoplasts with trypsin, alpha-chymotrypsin or papain, thus pointing to exposition of the substrate-binding site at the external surface of the inner membrane. On the other hand, latency of the enzyme in intact mitochondria indicates that the NADPH-binding site is located at the inner surface. These data provide the first direct evidence for the existence of aldehyde reductase in mitochondria of some rat tissues.