The interaction of organic cations with the mitochondrial membrane (original) (raw)
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Archives of Biochemistry and Biophysics, 1969
The effect of uncouplers of oxidative phosphorylation on the movements of H+ and K+ between isolated rat liver mitochondria and the suspending medium has been studied as a function of initial pH. Such agents induce uptake of H+ at initial pH below 6.5, but as the pH is increased to 7.0 and above, H+ uptake diminishes to zero. At high initial pH, above 8.0, uncoupling agents do not promote loss of H+ from intact mitochondria to the medium. On the other hand, agents known to increase permeability of the mitochondrial membrane, such as the nonionic detergents Lubrol and Triton X-100, induce H+ movements in either direction depending on the initial pH of the extramitochondrial medium. The movements of H+ induced by uncoupling agents are related to (and are controlled by) the opposite translocation of K+ ions. Below pH 7.0, K+ is freely permeable through the membrane, as was shown by direct measurement of K+ loss from mitochondria. Under these conditions H+ can enter in exchange for intramitochondrial K+. At pH above 7.0 K+ cannot go through the membrane, therefore H+ cannot enter. When K+ permeability was increased by addition of valinomycin, then H+ could be ejected from mitochondria in exchange for K+. Uncouplers have also been found to catalyze the exchange of intramitochondrial Cazf with extramitochondrial H+, while valinomyrin and gramicidin under the same conditions induce the exchange of intramitochondrial Ca2+ with extramitochondrial K+ or Na+, according to their well-known cation specificity.
Permeability of the mitochondrial outer membrane to organic cations
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
Inhibition of mitoc,~mS~al respiration by hydrophobic fluorescent dyes (Rhodamine 6G, Safranine O, Pyronine B) is much less potentiated by digitonin.iysis of the outer membrane than that by polyamines or adriamycin. This situation may be explained by ~mpenneability of the anion-selective channels in the outer mitochocdrial membrane to large cations and by ~qte abi.~ty of hydmp~F~ (but not polar or amphipathic) ions to directly permeate lipid bilayer~.
Role of Substrates in the Regulation of Mitochondrial Function In Situ
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2001
Investigations of mitochondrial oxidative phosphorylation have been mainly carried out in isolated mitochondria, where the experimental conditions can be precisely set. However, in intact living systems oxidative phosphorylation takes place in a complex environment, whose experimental dissection is a major challenge. It has long been recognized that the ef ciency of oxidative phosphorylation depends on the nature of the respiratory substrates, which feed electrons to the respiratory chain at different levels. Yet, the role of substrates in determining mitochondrial function and their response to energetic stress has been largely overlooked. Here we review recent work showing that the nature of the energetic substrates profoundly affects the mitochondrial responses to manipulations of pathophysiological relevance, such as uncoupling and opening of the permeability transition pore (PTP). Uncoupling of intact hepatocytes caused very different metabolic effects depending on whether carbohydrates or lipids were the energy source. With dihydroxyacetone as the substrate dinitrophenol caused a collapse of the mitochondrial membrane potential and of the ATP/ADP ratio, while the respiratory rate was increased only transiently. With octanoate as the substrate, on the other hand, dinitrophenol caused a dramatic stimulation of the respiratory rate, while the mitochondrial membrane potential and ATP/ADP ratio were affected only marginally. We then review results indicating that the activity of complex I directly regulates the PTP, a nding that emphasizes the importance of the respiratory substrates in PTP regulation. IUBMB Life, 52: 221-229, 2001
The Problem of Cation‐Binding Sites in the Energized Membrane of Intact Mitochondria
European Journal of Biochemistry, 1973
Weak bases, such as the acridine and the aminophenazine dyes, which undergo protonation in the chromophoric group, are actively bound in sonicated fragments. On the other hand strong bases such as the divalent cations, the safranine and the cyanine dyes, which carry a permanent charge, are actively bound in intact mitochondria. In the latter case the process results in stimulation of the respiration, H+ ejection, and a large change in absorbance, which is attributed to a stacking of the dyes. The effects are inhibited or reversed by uncouplers, and insensitive to nigericin.Four alternatives are considered: accumulation in the inner aqueous space; binding to matrix proteins; binding to the outer membrane surface; and binding to sites in the membrane fabric. The view that the energized membrane contains high affinity sites for cationic dyes is supported by: comparison of the spectra of the membrane‐bound and polyanion‐bound dye; competition with divalent cations; osmotic activity; and...
Protection by thiols of the mitochondrial complexes from 4-hydroxy-2-nonenal
Free Radical Biology and Medicine, 2001
In the present study, the effects of 4-hydroxy-2-nonenal (HNE) on highly purified pyruvate dehydrogenase complex (PDC) and its catalytic components in vitro and on PDC, ␣-ketoglutarate dehydrogenase complex (KGDC), and the branched-chain ␣-keto acid dehydrogenase complex (BCKDC) activities in cultured human HepG2 cells were investigated. Among the PDC components, the activity of the dihydrolipoamide acetyltransferase-E3-binding protein subcomplex (E2-E3BP) only was decreased by HNE. Dihydrolipoamide dehydrogenase (E3) protected the E2-E3BP subcomplex from HNE inactivation in the absence of the substrates. In the presence of E3 and NADH, when lipoyl groups were reduced, higher inactivation of the E2-E3BP subcomplex by HNE was observed. Purified PDC was protected from HNE-induced inactivation by several thiol compounds including lipoic acid plus [LA-plus; 2-(N,Ndimethylamine)ethylamidolipoate . HCl]. Treatment of cultured HepG2 cells with HNE resulted in a significant reduction of PDC and KGDC activities, whereas BCKDC activity decreased to a lesser extent. Lipoyl compounds afforded protection from HNE-induced inhibition of PDC. This protection was higher in the presence of cysteine and reduced glutathione. Cysteine was able to restore PDC activity to some extent after HNE treatment. These findings show that thiols, including lipoic acid, provide protection against HNE-induced inactivation of lipoyl-containing complexes in the mitochondria.
Actions of bis(guanylhydrazones) on isolated rat liver mitochondria
Biochemical Pharmacology, 1981
The effects of the anticancer bis(gu~ylhydrazones), methylglyoxal-bis(guanylhydrazone) (MGBG) and 4,4'-diacetyldiphenylurea-b~s(guanylhydrazone) @DUG), on parameters related to the bioenergetic function of isolated rat liver mitochondria were investigated, At concentrations comparable to those attained intracellularly, both bis(guanylhydrazones) si~ificantly inhibited state 4 respiration but had less of an inhibitor effect on state 3 or uncoupled respiration. DDUG was more effective than MGBG, requiring 0.34 mM to achieve a 25 per cent inhibition of respiration as compared to 6.0 mM for MGBG. The inhibition was prevented by potassium cations and was enhanced in mitochondria "de-energized" with valinomycin, a potassium cationophore. This suggested drug competition for potassium-binding sites, possibly membrane phospholipids. Addition of 1.25 mM MGBM or 0.025 mM DDUG to suspended mitochondria caused a rapid aggregation of organelles and an increase in the optical density of the suspension. Pretreatment with either bis(guanylhydrazone) protected mitochondria against non-specific swelling action of 0.0015% Triton X-100, suggesting membrane binding. By electron microscopy, MGBG-or DDUG-treated mitochondria appeared swollen and the spaces between cristae membranes or inner and outer membranes were collapsed, obliterating the outer mitochon~ial compartment. The activity of monoamine oxidase A, an outer membrane marker enzyme, was reduced considerably by 4 mM MGBG or 0.075 mM DDUG. Mobility of mitochondria toward the anode in an electrophoretic field was slowed 50 per cent by 2.5 mM MGBG or 0.1 mM DDUG, These data suggest that positively charged bis(~uanylhydr~ones) neutralize the net negative surface potential of rat liver mitochondria by binding to sites (possibly phospholipids) at the inner mitochondrial membrane. Subsequent interference with cation binding and/or transport results in inhibition of bioenergetic
Journal of Biological Chemistry
Electrophoretic uniport of anions through the inner mitochondrial membrane can be activated by alkaline pH or by depleting the matrix of divalent cations. It has also been suggested that, in the presence of valinomycin and potassium, respiration can also activate anion uniport. We have proposed that a single pathway is responsible for all three of these transport processes (Garlid, K. D., and Beavis, A. D. (1986) Biochim. Biophys. Acta 863, 187-204). We now present evidence that like the "pH-dependent" pore the divalent cation-regulated pore and the "respiration-induced" pore are blocked by N,N'-dicyclohexylcarbodiimide (DCCD). Moreover, the kinetics of inhibition of the latter two pathways are identical and exhibit a second order rate constant of 2.6 % lo-' (nmol DCCD/mg)-'. min-'. DCCD inhibits the uniport of C1-, phosphate, malate, and other lipophobic anions completely, but it has no effect on the classical electroneutral phosphate and dicarboxylate carriers. In Mg+-depleted mitochondria DCCD partially inhibits the transport of SCN-; however, in Mg2+-containing mitochondria and at low pH, no inhibition is observed. Furthermore, in DCCD-treated mitochondria, even following depletion of M 8 + , the transport of SCN-is independent of pH. These results lead us to conclude that two pathways for anion uniport exist: a specific, regulated pathway which can conduct a wide variety of anions and a nonregulated pathway through the lipid bilayer which only conducts lipid-soluble ions.
FEBS Letters, 2001
The effect of non-esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large-amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non-ionic detergent Triton X-100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K + and Cl 3 in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate-induced Cl 3permeation (measured with KCl medium plus valinomycin) was inhibited by N,NP P-dicyclohexylcarbodiimide, quinine or ATP. Myristate-or dodecyl sulfate-initiated swelling was paralleled by the lowering of endogenous Mg 2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg 2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187-induced swelling has identical inhibiting characteristics on Cl 3-permeation with respect to N,NP P-dicyclohexylcarbodiimide, quinine and ATP as the myristate-stimulated swelling. Therefore, we conclude that nonesterified fatty acids increase the permeability of mitochondria to K + and Cl 3 at alkaline pH by activating Mg 2+-dependent ionconducting pathways in IMM.