Crucial role of sulfhydryl groups in the mitochondrial inner membrane structure

The mitochondrial inner membrane lost its selectivity for the transport of solutes after reaction of hydrophobic sulfhydryl groups with alkylating agents (maleimide derivatives). The nature of the thiol reagent-induced membrane perturbations was investigated. Modifications of the interactions between membrane components after treatment with thiol reagents were assessed by measuring the binding parameters of 1-anilinonaphtalene-8-sulfonate. An enhancement (about 50%) of the fluorescence intensity, a weak increase of the number of binding sites, and a decrease of the apparent dissociation constant were observed. However, no significant modification of the net surface charge was detected. The osmotic behavior of mitochondria in hypotonic solutions of sucrose was altered after thiol modification. The outer membrane did not seem to influence the matricial volume expansion when thiols were alkylated. After swelling in an isotonic solution of permeant ions, N-butylmaleimide-treated mitochondrial lost one-half of their malate dehydrogenase content, whereas fumarase and glutamate dehydrogenase did not leave the matrix space. Addition of polyethylene glycol of molecular weight below 6000 to swollen mitochondria induced a rapid but transient shrinkage. In swollen mitochondria, the above results indicate a possible holes formation in the membrane structure. The size of these holes was estimated to be about 3 nm. This process which required the presence of the outer membrane, was favored by increasing the temperature and was antagonized by specific effectors of the adenine nucleotide translocator.     

Mitochondrial sulfhydryl group modification by adriamycin aglycones

Induction of Ca2+ release from isolated, preloaded rat heart mitochondria by low concentrations (less than 5 micrM) of adriamycin aglycones, has recently been reported [(1988) Biochem. Pharmacol. 37, 803]. Ca2+ release occurs via a generalized, Ca2+-dependent increase in the permeability of the inner mitochondrial membrane to small molecules. The process is antagonized by dithiothreitol, suggesting thiol involvement. This communication demonstrates modification of mitochondrial sulfhydryl groups, detected as decreased 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) reactivity, by adriamycin aglycones. Ca2+ release and sulfhydryl modification are shown to depend similarly on aglycone concentration and on the C-7 substituent of the anthracycline ring. In addition, DTNB elicits Ca2+ release. It can therefore be proposed that adriamycin aglycones alter mitochondrial membrane permeability by altering mitochondrial thiol status.     

Calcium-dependent release of acetyl-coenzyme A from liver mitochondria

Calcium ions (10 microM) enhance the permeability of the hepatic inner mitochondrial membrane to acetyl-CoA (and CoA). This effect is suppressed by the absence of phosphate ions or by the presence of EGTA, La3+, or ruthenium red. Exposure of mitochondria to Ca2+ for short intervals of time (e.g., 10 min) results in a reversible permeability change with release of acetyl-CoA and retention of sensitivity to EGTA. It is suggested that conditions resulting in an increase of the calcium ion concentration in the cytoplasm may thereby increase the rate of transfer of acetyl-CoA from the mitochondria to the cytoplasm.     

Fluorescamine-induced membrane permeability in mitochondria.

1. Addition of fluorescamine (75 microM) to mitochondria induced an increase in membrane permeability. 2. The leakiness of the inner mitochondrial membrane is characterized by extensive release of accumulated Ca2+, collapse of the transmembrane potential, mitochondrial swelling and efflux of matrix proteins, among them, malate dehydrogenase. 3. These effects were diminished by supplementing the media with 1 mM phosphate, and partially prevented by Mg2+. 4. These results indicate that the primary amino groups of membrane components contribute, partially, to the maintenance of the permeability barrier in mitochondria.     

Succinate and phenylsuccinate as modifiers of sulfhydryl groups of inner mitochondrial membrane protein. Study by EPR

Paramagnetic labels specific for sulfhydryl (SH) groups have been used to study the conformational changes of the inner mitochondrial membrane. The EPR spectra of the SH-groups spin-labeled with maleimide or iodoacetamide show the existence of two populations of sulfhydryl groups, differing in their mobility (one weakly, the other strongly immobilized). The incubation with succinate or phenylsuccinate decreased the binding of these labels of the weakly immobilized sites while the number of total SH groups was the same before and after the incubation. These results suggest that succinate or phenylsuccinate induce a reversible change in protein conformation or in protein arrangement within the inner mitochondrial membrane. This change is concomitant to the protein movement between inner membrane and perimembranal space induced by either of these two molecules.     

Detection and functional role of local gradients of H+-ions on the intracellular mitochondrial membrane with covalently linked pH-probe

The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.     

The effect of Ca2+ and acyl coenzyme A:lysophospholipid acyltransferase inhibitors on permeability properties of the liver mitochondrial inner membrane

We have reported previously that a number of metabolites and toxins which cause Ca2+ release from mitochondria do so by increasing the permeability of the inner membrane. The metabolic basis of this permeability change is proposed to be perturbation of a phospholipid deacylation-reacylation cycle which results in an accumulation of free fatty acids and lysophospholipids (see Broekemeier, K. M., Schmid, P. C., Schmid, H. H. O., and Pfeiffer, D. R. (1985) J. Biol. Chem. 260, 105-113 and references therein). This hypothesis predicts that inhibitors of acyl-CoA:lysophospholipid acyltransferase would be among those agents which increase membrane permeability and that their effects on permeability could occur in the absence of pyridine nucleotide oxidation or of an accumulation of glutathione disulfide. The hypolipidemic drugs WY-14643 and clofibric acid inhibit the mitochondrial acyl-CoA:lysophospholipid acyltransferase and have the predicted effects on mitochondrial permeability properties. The development of increased permeability due to WY-14643 and clofibric acid requires accumulated Ca2+ specifically, is sensitive to inhibitors of phospholipase A2, and results in a pattern of solute release and swelling which is typical of other Ca2+-releasing agents. Neither agent promotes pyridine nucleotide nor sulfhydryl glutathione oxidation in the absence of Ca2+. In addition, the swelling response to hypolipidemic drugs is not significantly inhibited by dithiothreitol. In the presence of Ca2+, both agents promote an accumulation of free fatty acids. The composition of these lipid degradation products suggests that mitochondria treated with hypolipidemic drugs retain an active lysophospholipase whereas this enzyme is inactivated by Ca2+-releasing agents which alter mitochondrial sulfhydryl groups.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.     

Effect of palmitoyl-CoA binding with adenine nucleotide translocase on energization of mitochondria]

Under conditions of inhibiting oxidative phosphorylation of oligomycin palmitoyl-CoA (p-CoA) decreases the rate of energy dependent reduction of acetoacetate and Ca2+-capacity of mitochondria in a phosphate medium. Energy independent osmotic swelling of mitochondria in NH4NO3, which depends on H+ permeability of the inner mitochondrial membrane is inhibited by ADP and acclereated by p-CoA. Carnitin and competitive ADP abolish all the effects of p-CoA. It is concluded that decreased energization induced by p-CoA is related to an increase in the inner mitochondrial membrane permeability b- H+ as a result of the inhibitor bindings with adenine nucleotide translocase.     

Resting state respiration of mitochondria: reappraisal of the role of passive ion fluxes

Rat liver mitochondria respiring under resting state conditions in the presence of oligomycin were rapidly blocked with cyanide and the dissipation of the membrane potential, measured with a tetraphenylphosphonium-sensitive electrode, was followed over time. The plot of the rate of membrane potential dissipation versus the actual value of the membrane potential was nonlinear and identical to the plot of resting state respiration (titrated with small amounts of a respiratory inhibitor) versus the membrane potential. The relationship between the respiratory chain activity and the proton-motive force in mitochondria oxidizing succinate with either oxygen or ferricyanide as electron acceptors was also found to be identical. These results are interpreted as an indication that the passive permeability of the inner mitochondrial membrane toward ions is far more significant in maintaining resting state respiration than is the molecular slippage of the pumps in the respiratory chain. These results also confirm the non-ohmic characteristics of the inner mitochondrial membrane.     

The effect of cyclosporin A and oligomycin on the nonspecific permeability of the mitochondria inner membrane

The effect of oligomycin and cyclosporine A on Ca(2+)-induced nonspecific permeability of the inner mitochondrial membrane was under study. Both oligomycin and cyclosporine A were able to prevent the activation of nonspecific permeability; however, but cyclosporine A was the only agent which could restore the initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was not shown to be mediated through redistribution of Ca2+ ions between different subpopulations of mitochondria.     

Permeability of inner mitochondrial membrane and oxidative stress

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.     

The inhibition of succinate, beta-oxybutyrate and glutamate transport in the liver mitochondria of hibernating susliks

Studies have been made on the permeability of the inner membrane of the liver mitochondria from hibernating and active ground squirrels for succinate, glutamate, hydroxybutyrate and inorganic phosphate. The permeability was calculated from the rate of mitochondrial swelling in 100 mM ammonium salts of the substrates and phosphate. It was shown that the rate of mitochondrial swelling in hibernating animals is 2--3 times lower than in active ones, being essentially identical in a solution of ammonium phosphate. It was concluded that the permeability of the inner mitochondrial membrane for the substrates decreases in hibernating animals, remaining unaffected for phosphate. Calcium-induced activation of membrane phospholipase A2 facilitates the transport of oxidative substrates into the mitochondria of hibernating ground squirrels, significant increase in the mitochondrial respiration being simultaneously observed. The data obtained suggest that inhibition of transport of oxidative substrates is one of the main factors which account for a low respiration rate in the mitochondria of hibernating animals.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Virus-induced permeability transition in mitochondria

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.     

Interactions of Adriamycin aglycones with mitochondria may mediate Adriamycin cardiotoxicity

Adriamycin and related anthracyclines are potent oncolytic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone metabolites of Adriamycin (5–20 μM) induce a Ca²⁺-dependent increase in the permeability of the inner mitochondrial membrane of both heart and liver mitochondria to small (< 1500 Da) solutes; this phenomenon is accompanied by release of mitochondrial Ca²⁺, mitochondrial swelling, collapse of the membrane potential, oxidation of mitochondrial pyridine nucleotides [NAD(P)H], uncoupling, and a transition from the condensed to the orthodox conformation and is inhibited by ATP, dithiothreitol, the immunosuppressant cyclosporin A, and the ubiquitous polyamine spermine. Aglycones also modify mitochondrial sulfhydryl groups and induce a Ca²⁺ independent oxidation of mitochondrial NAD(P)H which appears to reflect electron transport from NADH to oxygen, mediated by the aglycones and resulting in the production of Superoxide (O₂⁻). Selenium deficiency and butylated hydroxytoluene inhibit aglycone-induced Ca²⁺ release from liver, but not heart, mitochondria, suggesting that the interactions of the aglycones with mitochondria diner in these two tissues. It can be proposed that the effects of Adriamycin aglycones on heart mitochondria are responsible for the cardiotoxicity of the parent drug.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Shedding light on the mitochondrial permeability transition

The mitochondrial permeability transition is an increase of permeability of the inner mitochondrial membrane to ions and solutes with an exclusion size of about 1500Da. It is generally accepted that the permeability transition is due to opening of a high-conductance channel, the permeability transition pore. Although the molecular nature of the permeability transition pore remains undefined, a great deal is known about its regulation and role in pathophysiology. This review specifically covers the characterization of the permeability transition pore by chemical modification of specific residues through photoirradiation of mitochondria after treatment with porphyrins. The review also illustrates the basic principles of the photodynamic effect and the mechanisms of phototoxicity and discusses the unique properties of singlet oxygen generated by specific porphyrins in discrete mitochondrial domains. These experiments provided remarkable information on the role, interactions and topology of His and Cys residues in permeability transition pore modulation and defined an important role for the outer membrane 18kDa translocator protein (formerly known as the peripheral benzodiazepine receptor) in regulation of the permeability transition.