Evidence of a phosphate-transporter system in the inner membrane of isolated mitochondria

1. The organic mercurial sodium mersalyl, formaldehyde, dicyclohexylcarbodiimide and tributyltin each blocked respiratory-chain-linked ATP synthesis in rat liver mitochondria. 2. Mersalyl and formaldehyde also blocked a number of other processes dependent on the entry of inorganic phosphate into mitochondria, including mitochondrial respiration and swelling stimulated by cations and phosphate, the substrate-level phosphorylation reaction of the citric acid cycle, and swelling in ammonium phosphate. 3. Dicyclohexylcarbodi-imide and tributyltin did not inhibit the entry of phosphate into mitochondria. 4. Mersalyl and formaldehyde had a relatively slight effect on succinate oxidation and swelling stimulated by cations when phosphate was replaced by acetate, on succinate oxidation stimulated by uncoupling agents, and on swelling in solutions of ammonium salts other than phosphate or arsenate. 5. Formaldehyde blocked the oxidation of NAD-linked substrates in mitochondria treated with 2,4-dinitrophenol and the ATP-dependent reduction of NAD by succinate catalysed by ox heart submitochondrial particles. Both these effects appear to be due to an inhibition by formaldehyde of the NAD-flavin region of the respiratory chain. 6. Concentrations of dicyclohexylcarbodiimide or tributyltin sufficient to abolish ADP-stimulated respiration blocked the dinitrophenol-stimulated adenosine triphosphatase activity, whereas mersalyl and formaldehyde caused only partial inhibition of ATP hydrolysis. 7. When mitochondria were incubated with dinitrophenol and ATP, less than 10% of the total inorganic phosphate liberated was recovered in the mitochondria and no swelling occurred. In the presence of mersalyl or formaldehyde at least 80% of the total inorganic phosphate liberated was retained in the mitochondria and extensive swelling was observed. This swelling was inhibited by oligomycin but not by antimycin or rotenone. 8. The addition of mersalyl to mitochondria swollen by treatment with valinomycin, K(+) and phosphate blocked the contraction induced by dinitrophenol and caused an increase in the phosphate content of the mitochondria, but had no effect on the contraction of mitochondria when phosphate was replaced by acetate. 9. It is concluded that mitochondria contain a phosphate-transporter system, which catalyses the movement of phosphate in either direction across the mitochondrial membrane, and that this system is inactivated by organic mercurials and by formaldehyde. Evidence is presented that the phosphate-transporter system is situated in the inner membrane of rat liver mitochondria and is also present in other types of mammalian mitochondria.     

The preparation and some properties of yeast mitochondria

Summary A comparison of the structural and functional integrity of yeast mitochondria prepared by a mechanical disintegration method with that reported for mitochondria prepared by enzyme methods has been made.The mitochondria prepared by mechanical disruption show at least as great a degree of functional integrity as that reported for enzymically prepared mitochondria. This was judged by the substrates oxidised, the rates of these oxidations, the respiratory control and ADP:0 ratios and difference spectra.The respiration of citrate and of ethanol by the mechanically prepared mitochondria has been shown to be stimulated by exogenous NAD; in the case of the latter substrate a degree of respiratory coupling became apparent as a consequence of this stimulation.The penetration of NADH₂, and of citrate or of ethanol in the presence of exogenous NAD, into the mitochondria has been examined.The results are consistent with the reported existence of a NADH₂ dehydrogenase system located in the outer area of the inner mitochondrial membrane. The NAD stimulation of ethanol respiration also suggests the occurrence of an external alcohol dehydrogenase in the mitochondrial preparation; similar results with citrate suggest external forms of aconitic hydratase and isocitric dehydrogenase to be present.Unlike animal mitochondria the rate of penetration of oxidisable substrates in uninhibited suspensions appeared to exceed respiration and/or outward diffusion rates in this preparation.Evidence of a malate stimulated citrate and isocitrate transport system similar to that reported for animal mitochondria is presented, but the specificity of the system for tricarboxylate anion appears to differ, at least in respect of propane 1.2.3 tricarboxylate (tricarbalyllate).     

The responses of isolated plant mitochondria to external nicotinamide adenine dinucleotide

The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2-oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NAD-depletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate K_m of 0.105 ± 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD.     

Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.     

Characterization of mitochondrial membrane fragments resulting from spontaneous swelling: novel stimulation of NADH-dependent respiration by carboxylic acids.

Metabolically induced high amplitude swelling of rat liver mitochondria has been found to result in the formation of a heterogeneous population of mitochondrial membranes consisting of right side-out particles with occluded fumarase activity and inside-out particles/fragments capable of NADH-dependent respiration. This rotenone-sensitive, uncoupler-insensitive, NADH-dependent respiration was specifically and instantaneously stimulated by several ligands such as glutamate and malate (which can be metabolized) and, interestingly, even lactate (which could not be metabolized by the swollen mitochondria). These observations suggest that high amplitude swelling results in a novel type of control of respiration in these fragments.     

Induction of calcium-dependent nonspecific permeability of the inner membrane in liver mitochondria of mammals and birds: A comparative study

The kinetics of the processes accompanying the induction of Ca²⁺-dependent permeability (pore opening) of the inner membrane—swelling of organelles and Ca²⁺ release from the matrix—was studied in isolated liver mitochondria of mammals (mice, rats, and rabbits) and birds (pigeons and guinea fowls). It was found that the mitochondria of rats, pigeons, and guinea fowls of the gray-speckled population (GSP) are similar in terms of respiration and oxidative ATP synthesis, whereas mitochondria of rabbits exhibit a greater degree of coupling of respiration and ATP synthesis, and mitochondria of mice and Zagorskaya White breed (ZWB) guinea fowls, a lower degree of coupling. It was established that mammalian mitochondria energized by succinate oxidation and incubated with 1 mM of inorganic phosphate are able to swell upon the addition of 125 nmol of CaCl₂ per 1 mg protein. Under these conditions, mitochondria of GSP and ZWB guinea fowls and pigeons are capable of swelling upon addition of at least 875, 875 and 1000 nmol of CaCl₂ per 1 mg protein, respectively. Cyclosporin A (CsA, 1 μM) inhibits mitochondrial swelling. It was shown that mitochondria of mammalians and guinea fowls but not of pigeons are able to effectively absorb and retain Ca²⁺ in the matrix. Calcium retention capacity of mitochondria from rats, mice, rabbits, GSP, and ZWB guinea fowls were, respectively, 70, 57, 38, 844 and 793 nmol of CaCl₂ per 1 mg of protein. In the presence of an oxidizing agent tert-butylhydroperoxide (TBH), the induction of the Ca²⁺-dependent pore in the mitochondria was observed upon addition of CaCl₂ in substantially smaller quantities. TBH was most effective in the case of rabbit mitochondria and had the lowest efficiency in the case of guinea fowl and pigeon mitochondria.     

Uncoupling Action of Antibiotic Sporaviridins with Rat-liver Mitochondria

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.     

Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria.

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.     

Tl+ induces both cationic and transition pore permeability in the inner membrane of rat heart mitochondria

Effects of Tl⁺ were studied in experiments with isolated rat heart mitochondria (RHM) injected into 400 mOsm medium containing TlNO₃ and a nitrate salt (KNO₃ or NH₄NO₃) or TlNO₃ and sucrose. Tl⁺ increased permeability of the inner membrane of the RHM to K⁺ and H⁺. This manifested as an increase of the non-energized RHM swelling, in the order of sucrose < K⁺ < NH₄ ⁺, respectively. After succinate administration, the swollen RHM contracted. The Tl⁺-induced opening of the mitochondrial permeability pore (MPTP) in Ca²⁺-loaded rat heart mitochondria increased both the swelling and the inner membrane potential dissipation, as well as decreased basal state and 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ were suppressed by the MPTP inhibitors (cyclosporine A, ADP, bongkrekic acid, and n-ethylmaleimide), activated in the presence of the MPTP inducer (carboxyatractyloside) or mitoK_ATP inhibitor (5-hydroxydecanoate), but were not altered in the presence of mitoK_ATP agonists (diazoxide or pinacidil). We suggest that the greater sensitivity of heart and striated muscles, versus liver, to thallium salts in vivo can result in more vigorous Tl⁺ effects on muscle cell mitochondria.     

t-Butylhydroperoxide-induced Ca2+ efflux from liver mitochondria in the presence of physiological concentrations of Mg2+ and ATP

Isolated rat liver mitochondria, energized either by succinate oxidation or by ATP hydrolysis, present a transient increase in the rate of Ca2+ efflux concomitant to NAD(P)H oxidation by hydroperoxides when suspended in a medium containing 3 mM ATP, 4 mM Mg2+ and acetate as permeant anion. This is paralleled by an increase in the steady-state concentration of extramitochondrial Ca2+, a small decrease in delta psi and an increase in the rate of respiration and mitochondrial swelling. With the exception of mitochondrial swelling all other events were found to be reversible. If Ca2+ cycling was prevented by ruthenium red, the changes in delta psi, the rate of respiration and the extent of mitochondrial swelling were significantly diminished. In addition, there was no significant decrease in the content of mitochondrial pyridine nucleotides. Mitochondrial coupling was preserved after a cycle of Ca2+ release and re-uptake under these experimental conditions. It is concluded that hydroperoxide-induced Ca2+ efflux from intact mitochondria is related to the redox state of pyridine nucleotides.     

Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.     

Toxicity of emestrin,a new macrocyclic dithiodioxopiperazine mycotoxin,to mitochondrial function

The effect of emestrin, a new macrocyclic epidithiodioxopiperazine mycotoxin from severalEmericella species, on mitochondrial reactions was studied using isolated rat liver mitochondria to gain insight into the molecular mechanism for itsin vivo toxicity to rat and mouse. Emestrin was found to inhibit ATP synthesis in mitochondria causing an uncoupling of oxidative phosphorylation and a depression of respiration in isolated mitochondria. In addition to these effects on mitochondrial respiration, emestrin elicited a dratsic structural alteration (swelling) of mitochondria as observed in thein vivo system. The mitochondrial swelling was significantly enhanced by the subsequent addition of calcium ion. Emestrin B, in which dithio group is replaced by trithio group, exerted an uncoupling effect on oxidative phosphorylation without accompanying such depressive effect on state 3 respiration as observed for emestrin.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

Brain mitochondria from rats treated with sulforaphane are resistant to redox-regulated permeability transition

Oxidative stress promotes Ca²⁺-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent cell death in many paradigms, including those related to acute brain injury. One approach to pre-conditioning against oxidative stress is pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by agents such as sulforaphane (SFP). This study tested the hypothesis that administration of SFP to normal rats increases resistance of isolated brain mitochondria to redox-sensitive PTP opening. SFP or DMSO vehicle was administered intraperitoneally to adult male rats at 10 mg/kg 40 h prior to isolation of non-synaptic brain mitochondria. Mitochondria were suspended in medium containing a respiratory substrate and were exposed to an addition of Ca²⁺ below the threshold for PTP opening. Subsequent addition of tert-butyl hydroperoxide (tBOOH) resulted in a cyclosporin A-inhibitable release of accumulated Ca²⁺ into the medium, as monitored by an increase in fluorescence of Calcium Green 5N within the medium, and was preceded by a decrease in the autofluorescence of mitochondrial NAD(P)H. SFP treatment significantly reduced the rate of tBOOH-induced Ca²⁺ release but did not affect NAD(P)H oxidation or inhibit PTP opening induced by the addition of phenylarsine oxide, a direct sulfhydryl oxidizing agent. SFP treatment had no effect on respiration by brain mitochondria and had no effect on PTP opening or respiration when added directly to isolated mitochondria. We conclude that SFP confers resistance of brain mitochondria to redox-regulated PTP opening, which could contribute to neuroprotection observed with SFP.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Effect of KCl Medium on Malate Oxidation in Mitochondria of Sugar Beet Taproot

Isolated mitochondria were obtained from growing and stored sugar beet (Beta vulgaris L.) taproots. These preparations were used to monitor the mitochondrial matrix volume and malate oxidation after the replacement of sucrose with KCl in the reaction medium. The transfer of mitochondria from sucrose-containing isolation medium to the isoosmotic KCl solution initiated spontaneous or energy-dependent (in the presence of respiratory substrate) swelling whose kinetic parameters (the initial rate and amplitude) were virtually independent of the plant age. At the same time, effects of KCl-induced swelling on oxidative and phosphorylating activities of mitochondria were age-dependent. In mitochondria from growing taproots, K⁺ ions stimulated nonphosphorylating malate oxidation, thereby decreasing the respiratory control ratio and the ADP/O coefficient. The incubation of mitochondria from stored taproots in KCl solution induced a short-term activation and subsequent progressive inhibition of malate oxidation but did not inhibit the oxidation of exogenous NADH. The inhibition of malate oxidation was not released by adding ADP or uncouplers and was enhanced in the presence of valinomycin. The swelling of mitochondria in KCl solutions did not impair the integrity of mitochondrial membranes and did not preclude stimulation of malate oxidation by exogenous NAD. It is supposed that the KCl-induced inhibition of respiration is related to a large increase in the matrix volume and a drastic decrease in the concentration of a coenzyme NAD. Previous studies with isolated mitochondria from stored taproots showed that the mitochondrial NAD level was a rate-limiting factor of malate oxidation assayed in the sucrose-containing media. A possible role of K⁺-transporting mechanisms in regulation of mitochondrial matrix volume and metabolic activity of plant mitochondria is discussed.     

Ultrastructural transformations in bean inner mitochondrial membranes

The ultrastructure of inner membrane-matrix mitochondria isolated from bean (Phaseolus vulgaris) shoots was examined in different metabolic states. Gross ultrastructural transformations analogous to the condensed-to-orthodox configurational changes reported in mammalian mitochondria are observed on transistion from nonrespiring to respiring metabolism. With the induction of oxidative phosphorylation, the particles remain in the orthodox configurational state. The reverse orthodox-to-condensed configurational changes observed in mammalian preparations does not occur. Optically monitored absorbancy studies with bean particles show a substrate-supported P_i-induced swelling under the same conditions that induce the condensed-to-orthodox ultrastructural transformation. The swelling is associated with the net uptake of K⁺ and P_i as well as a small P_i-induced respiratory stimulation. When phosphorylation is initiated with these swollen particles, the optically monitored volume remains unchanged. Thus a positive correlation exists between the ultrastructural configuration and the osmotic volume changes, which supports the conclusion that configurational changes reflect internal osmotic adjustments.     

Respiration induces variable porosity to polyols in the mitochondrial inner membrane

The osmotic basis of low and high amplitude swelling in mitochondria was investigated in detail using sucrose and mannitol as external osmolytes. Osmotic behaviour of mitochondria in various respiratory states was consistent with significant changes in the porosity of the inner membrane corresponding to the rate of respiration. The stoichiometry of oxidative phosphorylation was confirmed to be dependent on the physical state (i.e., osmotic stretch) of the inner membrane regardless of the external polyol used. High amplitude swelling in polyol media was shown to arise from a sequential disruption of the outer and inner mitochondrial membranes, due to a dynamic instability induced by a combination of respiration, unscreened (fixed) surface charge density and the consequent variable porosity of the inner membrane. These novel experimental findings based on the physical theory of osmosis emphasize the need to define the fine structural changes of the inner membrane associated with oxidative phosphorylation to arrive at a comprehensive mechanism.     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Membrane instability in respiring mitochondria: role of phosphate.

Metabolically-induced (spontaneous) high amplitude swelling of mitochondria has been shown to be due to a serial disruption of the mitochondrial membranes [D. Sambasivarao & V. Sitaramam (1985), Biochim Biophys Acta, 806, 195-209]. Phosphate- and arsenate-induced swelling was investigated in mitochondria to evaluate the role of phosphate transport in the instability created in the mitochondrial membranes. Phosphate-induced swelling in respiring mitochondria was similar to spontaneous swelling. Both represent essentially colloidal swelling due to the variable porosity induced in the inner membrane to polyols by respiration. Swelling of non-respiring mitochondria at high ammonium phosphate concentrations was, on the other hand, primarily due to high permeability to phosphate. This membrane instability created by phosphate transport in the surrounding lipid involves neither the endogenous nor the exogenous Ca2+.