Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.     

The sterol composition of Trypanosoma cruzi changes after growth in different culture media and results in different sensitivity to digitonin-permeabilization

Respiration, oxidative phosphorylation. and the corresponding changes in membrane potential (deltapsi) of Trypanosoma cruzi epimastigotes grown either in liver infusion-tryptose (LIT) or brain heart infusion (BHI) culture medium were assayed in situ using digitonin to render their plasma membrane permeable to succinate, ADP, safranine O, and other small molecules. When the cells were permeabilized with 64 microM digitonin, a concentration previously used with epimastigotes, the ability of the cells grown in LIT medium to sustain oxidative phosphorylation was demonstrated by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. In contrast, the cells grown in BHI medium were not able to sustain a stable membrane potential and did not respond to ADP addition. Analyses of oxygen consumption by these permeabilized cells indicated that the rate of basal respiration, which was similar in both cell types, was significantly decreased by 64 microM digitonin. Addition of ADP to the permeabilized cells grown in LIT medium promoted an oligomycin-sensitive transition from resting to phosphorylating respiration in contrast to the cells grown in BHI medium, whose respiration decreased steadily and did not respond either to ADP or CCCP. Titration of the cells grown in BHI medium with different digitonin concentrations indicated that their mitochondria have higher sensitivity to digitonin than those grown in LIT medium. Analysis of the sterol composition of epimastigotes grown in the two different media showed a higher percentage of cholesterol in total and mitochondrial extracts of epimastigotes grown in BHI medium as compared to those grown in LIT medium, suggesting the involvement of this sterol in their increased sensitivity to digitonin-permeabilization.     

Oxidative phosphorylation and rotenone-insensitive malate- and NADH-quinone oxidoreductases in Plasmodium yoelii yoelii mitochondria in situ

Respiration, membrane potential, and oxidative phosphorylation of mitochondria of Plasmodium yoelii yoelii trophozoites were assayed in situ after permeabilization with digitonin. ADP induced an oligomycin-sensitive transition from resting to phosphorylating respiration in the presence of oxidizable substrates. A functional respiratory chain was demonstrated. In addition, the ability of the parasite to oxidize exogenous NADH, as well as the insensitivity of respiration to rotenone and its sensitivity to flavone, suggested the presence of an alternative NADH-quinone (NADH-Q) oxidoreductase. Rotenone-insensitive respiration and membrane potential generation in the presence of malate suggested the presence of a malate-quinone oxidoreductase. These results are in agreement with the presence of genes in P. yoelii encoding for proteins with homology to NADH-Q oxidoreductases of bacteria, plant, fungi, and protozoa and malate-quinone oxidoreductases of bacteria. The complete inhibition of respiration by antimycin A and cyanide excluded the presence of an alternative oxidase as described in other parasites. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin and GTP but was unaffected by carboxyatractyloside. These results provide the first biochemical evidence of the presence of an alternative NADH-Q oxidoreductase and a malate-quinone oxidoreductase and confirm the operation of oxidative phosphorylation in malaria parasites.     

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Mitochondria maintain numerous energy‐consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl‐ and sucrose‐based solutions using Clark oxygen electrode. Respiration of digitonin‐treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate‐stimulated respiration rate did not depend on solution composition. In sucrose‐based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl‐based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose‐based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non‐mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose‐based solution. Copyright © 2012 John Wiley & Sons, Ltd.     

In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate

Mitochondria play a central role in neuronal death during pathological exposure to glutamate (excitotoxicity). To investigate the detailed bioenergetics of the in situ mitochondria, a method is described to monitor continuously the respiration of primary cerebellar granule neuron cultures while simultaneously imaging cytoplasmic Ca(2+) and mitochondrial membrane potential. Coverslip-attached cells were perfused in an imaging chamber with upstream and downstream flow-through oxygen electrodes. The bioenergetic consequences of chronic glutamate exposure were investigated, including ATP supply and demand, proton leak, and mitochondrial respiratory capacity during chronic glutamate exposure. In 25 mM K(+) medium supplemented with 10% dialyzed serum, cells utilized 54% of their respiratory capacity in the absence of receptor activation (37% for ATP generation, 12% to drive the mitochondrial proton leak, and the residual 5% was nonmitochondrial). glutamate initially increased mitochondrial respiration from 51 to 68% of capacity, followed by a slow decline. It was estimated that 85% of this increased respiration was because of increased ATP demand, whereas 15% was attributable to a transient mitochondrial proton leak. N-Methyl-D-aspartate receptor activation was only responsible for 62% of the increased respiration. When adjusted for cell death over 3 h of glutamate exposure, respiration of the viable cells remained near basal and protonophore stimulated respiration to the same extent as control cells. Pyruvate-supplemented media protected cells from glutamate excitotoxicity, although this was associated with mitochondrial dysfunction. We conclude that excitotoxicity under these conditions is not because of an ATP deficit or uncoupling. Furthermore, mitochondria maintain the same respiratory capacity as in control cells.     

Preparation of an inner membrane-matrix fraction from yeast yeast mitochondria.

Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.     

Respiration characteristics of mitochondria in parental and giant transformed cells of the murine Nemeth-Kellner lymphoma

Respiration characteristics of mitochondria of the parental and giant cells of murine NK/Ly (Nemeth-Kellner lymphoma) were studied. The giant cell-enriched ascites were obtained by serial intraperitoneal injections of vinblastine in tumour-bearing mice. Ascites containing >70% giant cells were used. Their diameter of was over 17 μm (~2800 μm(3)), while the diameter of the parental cells was 12.7 μm (1100 μm(3)). The respiration rate of mitochondria in situ was measured by oxygen consumption in intact and digitonin-permeabilized NK/Ly cells. Endogenous respiration of intact giant NK/Ly cells was three times higher compared to the parental ones, roughly in agreement with the volume change. The giant NK/Ly cells were far more resistant to permeabilization with digitonin than the parental cells, as shown by Trypan Blue and LDH (lactate dehydrogenase) release tests. After digitonin permeabilization, oxygen consumption was reduced to a minimal level (0.06 ng atom O/(s × 106 cells) in both types of cells. Addition of α-ketoglutarate or succinate to the incubation medium increased oxygen consumption in the parental cells by 46 and 164% respectively. In the giant NK/Ly cells, the corresponding increases were 164 and 276%. Addition of ADP to α-ketoglutarate- or succinate-supplemented medium further stimulated oxygen consumption of the permeabilized NK/Ly cells; however, the effect of ADP was more pronounced in the giant cells. In addition, indices of respiratory control were significantly higher in the giant cells. Oligomycin suppressed considerably the respiration of the intact giant cells but had a much weaker effect on parental cells. Thus, giant NK/Ly cells possess much higher respiration rates and show tighter coupling between the respiration and oxidative phosphorylation compared with parental cells.     

ATP-sensitive potassium channel in mitochondria of the eukaryotic microorganism Acanthamoeba castellanii

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.     

Chlortetracycline-mediated continuous Ca2+ oscillations in mitochondria of digitonin-treated Tetrahymena pyriformis

Ca2+ transport in mitochondria was studied in situ using digitonin-permeabilized cells of the ciliate protozoan Tetrahymena pyriformis GL. In the presence of oxidizable substrates and inorganic phosphate, mitochondria were able to accumulate a large amount of the added Ca2+ without subsequent uncoupling and mitochondrial damage. However, the maximal Ca2+ uptake dramatically decreased in the presence of micromolar concentrations of the fluorescent calcium indicator, chlortetracycline, which in aerobic conditions caused an uncoupling of the respiration in Ca2+-loaded mitochondria. Moreover, on reaching hypoxia, when the rate of oxygen diffusion from the air to the stirred incubation medium became a limiting factor, continuous Ca2+ oscillations were observed. Ca2+ fluxes were synchronous with the cyclic changes of the membrane potential and were followed with a significant delay by the changes of the membrane-associated fluorescence of Ca-chlortetracycline complexes. Both the chlortetracycline-induced uncoupling of the respiration and the oscillations were prevented by either EGTA or ruthenium red. It is suggested that in conditions of the limited rate of respiration the oscillations are generated as a result of the functioning of the two Ca2+-transport pathways: a Ca2+ uniport and a chlortetracycline-mediated electroneutral Ca2+ efflux.     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

Characteristics of Ca2+ transport by Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane has allowed the study of Ca2+ transport and oxidative phosphorylation in mitochondria in situ (R. Docampo and A. E. Vercesi (1989) J. Biol. Chem. 264, 108-111). The present results show that these mitochondria are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Addition of Ca2+ or ethylene glycol bis (beta-aminoethyl ether) N-N'-tetraacetic acid to these preparations under steady-state conditions was followed by Ca2+ uptake or release, respectively, tending to restore the original Ca2+ "set point" at about 0.9 microM. In addition, large amounts of Ca2+ were retained by T. cruzi mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butyl hydroperoxide, diamide, and the 1,2-naphthoquinone beta-lapachone. However, when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine in the presence of antimycin A was used as subtrate, beta-lapachone caused pyridine nucleotide oxidation, and Ca2+ accumulation by these mitochondria was considerably lower than in control preparations, this effect being dose-dependent.     

Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles.

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17,400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.     

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.     

Accumulation of D-arginine by rat liver mitochondria

The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate-malate as substrates.     

Estrogens prevent calcium-induced release of cytochrome c from heart mitochondria

We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium-induced release of cytochrome c from mitochondria. 10 nM-10 microM 17beta-estradiol or 4-hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17beta-estradiol and 4-hydroxytamoxifen blocked high calcium-induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium-induced release of cytochrome c.     

Effects of hydrogen peroxide and hypochlorite on membrane potential of mitochondria in situ in rat heart cells.

Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 +/- 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium.     

Regulation of the reaction rate of ATP synthesis in intact mitochondria

High concentrations of respiration inhibitors are known to sharply decrease the membrane potential in mitochondria. The effect of relatively low concentrations of oxidative phosphorylation inhibitors on the value of membrane potential of intact mitochondria and on the rate of respiration and phosphorylation as well was studied. It has been found that within a certain concentration range the inhibitors of oxidative phsophorylation--malonic acid, sodium cyanide m-chlorophenyl hydrozonecarbonylcyanide, sharply decrease the phosphorylation rate (by 70 divided by 90%) but do not practically a affect the membrane potential value of intact mitochondria in the state 3 according to Chance.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.     

The response of plant mitochondria to media of high solute content

The state-3 rate of respiration of potato tuber mitochondria is inhibited by concentrations of KCl or NaCl above 125 mM, and by concentrations of sucrose, lactose, or maltose above 500 mM, but not at all by mannitol, glucose, glycine, or proline up to a concentration of 1500 mM in the medium. Mitochondria from cauliflower, beetroot, cucumber, rock melon, and watermelon behave very similarly to those from potato tuber. The variable response to different solutes proves that the reduction in respiration is not a simple function of the chemical potential of water in the medium. Disruption of potato mitochondria by ultrasonic vibration does not relieve the inhibition of succinate oxidation caused by KCl or sucrose. However, treatment with detergent abolishes completely the inhibition of respiration by sucrose. Inhibition of succinate dehydrogenase [Succinate:PMS, oxidoreductase (EC.1.3.99.1)] and malate dehydrogenase [L-Malate:NAD oxidoreductase (EC.1.1.1.37)] activities by sucrose is less than the inhibition of succinate- and malate-dependent oxygen uptake by the potato mitochondria. Limited substrate uptake and, alternatively, reduced electron flow as a consequence of a direct effect of solute on the mitochondrial membrane are considered as possible mechanisms of inhibition.     

Studies on the relationship between the inner and outer membranes of rat liver mitochondria as determined by subfractionation with digitonin

The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.