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PubMed1996

Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel.

Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 refraction sediments with submitochondrial particles at 150,000 x g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M fully prevents the inhibitory effect of H+ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P. (1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the isomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitution in position 8 while a 4-substituted, nonimmunosuppressive derivative is a as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.

Amino Acid Isomerases↗
PubMed1983

Triphenyltetrazolium and its derivatives are anisotropic inhibitors of energy transduction in oxidative phosphorylation in rat liver mitochondria.

Triphenyltetrazolium and its derivatives inhibited energy transduction in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. Triphenyltetrazolium incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. Triphenyltetrazolium also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced sites on the membranes for triphenyltetrazolium and its derivatives. The maximum amounts of energy-dependent binding sites for triphenyltetrazolium on membranes energized with succinate and ATP, respectively, were 14 and 4 nmol/mg protein. Triphenyltetrazolium also induced H+ ejection from the energized membranes. The maximum amounts of H+ ejection from membranes energized with succinate and ATP, respectively, were 4 and 2.4 nmol/mg protein. Triphenyltetrazolium also decreased the membrane potential up to about half the control value and caused shrinkage of mitochondria in an energy-dependent fashion. Comparison of the Hammett's sigma constants of triphenyltetrazolium derivatives with various substituents on the 3-benzene ring showed that lower concentrations of triphenyltetrazolium derivatives with a stronger positive charge were required for inhibition of energy transduction. The present findings show that triphenyltetrazolium and its derivatives act as anisotropic inhibitors of energy transduction by binding to negative charges created on the outer side (C-side) of energized mitochondria, and that the positive charge of these inhibitors is one of important factors for their inhibitory activity. These negative charges may be an essential part of the H+ pump.

Adenosine Triphosphate↗
PubMed1984

Three proton pumps, morphology and movements.

The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the delta pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 micrograms per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.

Animals↗
PubMed1984

Voltage-driven ATP synthesis by beef heart mitochondrial F0F1-ATPase.

The F0F1-ATPase of the inner mitochondrial membrane catalyzes the conversion of a proton electrochemical energy into the chemical bond energy of ATP (Boyer, P.D., Chance, B., Ernster, L., Mitchell, P., Racker, E., and Slater, E.C. (1977) Annu. Rev. Biochem. 46, 955-1026). To assess the role of the membrane potential (delta psi) in this process and to study the effect of very short pulses on ATP synthesis, we employed a high voltage pulsation method (Kinosita, K., and Tsong, T.Y. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927) to induce a delta psi of controlled magnitude and duration in a suspension of submitochondrial particles and F0F1-ATPase vesicles. Cyanide-treated submitochondrial particles were exposed to electric pulses of 10-30 kV/cm of magnitude (generating a peak delta psi of 150-450 mV) and 1-100 microseconds duration. Net [32P]ATP synthesis from [32P]Pi and ADP was observed with maximal values of 410 pmol/mg X pulse for a 30 kV/cm-100-microseconds pulse. This corresponds to a yield of 10-12 mol of ATP per mol of F0F1 complex per pulse. As many as 4 nmol/mg were produced after pulsing the same sample 8 times. By varying the ionic strength of the suspending medium, and consequently the pulse width, it is clearly shown that the synthesis was electrically driven and did not correlate with Joule heating of the sample. Titrations using specific inhibitors and ionophores were performed. The voltage-induced ATP synthesis was 50% inhibited by 0.11 microgram/mg of oligomycin and 2.4 nmol/mg of N,N'-dicyclohexylcarbodiimide. Ionophores and uncouplers had varying degrees of inhibition. The dependence of ATP synthesis on pulse width was nonlinear, exhibiting a threshold at 10 microseconds and a biphasic behavior above this value. Isolated F0F1-ATPase reconstituted into asolectin vesicles also synthesized ATP when pulsed with electric fields. A 35 kV/cm pulse induced the synthesis of 115 pmol of ATP per mg of protein, which corresponds to approximately 0.34 mol of ATP per mol of F0F1-ATPase. This synthesis was also sensitive to oligomycin and dicyclohexylcarbodiimide. The possibility of turnover of the ATPase in microseconds is considered.

Adenosine Triphosphate↗
PubMed1978

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.

Adenosine Triphosphate↗
PubMedDate not supplied

Inhibition of succinic dehydrogenase and F0F1-ATP synthase by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 microM DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP<=>[32P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (Ki = 31.7 microM and Ki = 32.7 microM), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F1-ATPase were also inhibited 50% by 31.9 and 20.9 microM DIDS, respectively.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

[Hysteresis behavior of complex I in delta mu H+-dependent reduction of NAD+ succinate].

It was shown that the membrane-bound complex I is fully inactive in the absence of NADH during the reverse electron transfer from succinate to NAD+. The enzyme activation is attained by preincubation of submitochondrial particles with low concentrations of NADH; the activating effect persists after a complete oxidation of the latter during long-term (several hours) aerobic incubation. The experimental results suggest that complex I contains a redox component, whose reduction by NADH and aerobic oxidation are not involved in the overall catalytic reaction. An experimental scheme is proposed, according to which the key role of such a component is ascribed to the tightly bound ubiquinone; the activation and inactivation of the enzyme are due to a slow reversible redox conversion (ubiquinone in equilibrium ubisemiquinone), whereas the catalytic act involves a rapid reversible conversion (ubisemiquinone in equilibrium ubiquinol). It was demonstrated that the "redox" mechanism of the inactivation-activation reaction determines the strong dependence of activity of the reverse electron transfer on the mode of preparation of submitochondrial particles. The coupling properties of the submitochondrial particulate membrane and the activities of enzymes involved in the reverse electron transfer are stable at room temperature for over 14 hours.

Adenosine Triphosphate↗
PubMed2006

The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase.

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.

Animals↗
PubMed1989

Chromium(V) is produced upon reduction of chromate by mitochondrial electron transport chain complexes.

Incubation of chromate with isolated rat liver submitochondrial particles under anaerobic conditions in vitro results in reduction of chromium(VI) and formation of chromium(V). In the presence of NADH, submitochondrial particles (SMPs) were active in reducing chromate as shown by UV-vis spectroscopic studies, and forming a chromium(V) species which was detectable by electron paramagnetic resonance spectroscopy. In the presence of succinate, SMPs were less effective in reducing chromate and forming chromium(V) relative to their NADH-dependent activity. However, SMPs showed a higher rate of oxygen depletion with NADH as compared to succinate as substrate, suggesting that differences in the NADH-dependent versus succinate-dependent chromate-reductase activity of SMPs is probably due to differences in efficiency of electron donation by succinate and NADH. The use of specific electron transport chain inhibitors allowed the sites of chromium(VI) reduction and chromium(V) formation in SMPs to be determined. Rotenone, antimycin and cyanide all produced approximately 40% inhibition of the NADH-dependent chromate-reductase activity. Thus, complex I (NADH:ubiquinone oxidoreductase) appears to be responsible for the inhibitor-insensitive, and complex IV (ferrocytochrome c:oxygen oxidoreductase) for the inhibitor-sensitive NADH-dependent chromium(VI) reduction and chromium(V) formation. Cyanide and antimycin produced approximately 50% inhibition of the succinate-dependent chromate-reductase activity of SMPs, while no detectable inhibition was observed with rotenone. These results confirm the chromate-reductase activity of complex IV, and suggest that complex II (succinate:ubiquinone oxidoreductase) is responsible for the inhibitor-insensitive succinate-dependent chromate-reductase activity of SMPs. Since chromium(VI) is effectively metabolized by electron transport chain complexes of the mitochondrial inner membrane in vitro, and chromium(V) is formed as an intermediate in the process, mitochondria may play a role in chromium(VI) carcinogenesis.

Animals↗
PubMed2000

Proteoliposomes colocalized with endogenous mitochondria in mouse fertilized egg.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Animals↗
PubMed1994

Analysis of factors affecting functional assays for estimating IF1, the mitochondrial ATPase inhibitor.

Functional assays for IF1 have been in use for more than 30 years, i.e., since the initial report of Pullman and Monroy in 1963 on the inhibition of soluble F1-ATPase by the purified bovine heart inhibitor. However, beginning with the report of Horstman and Racker in 1970 and for approximately 17 years thereafter, workers in many laboratories routinely used IF1-depleted bovine heart submitochondrial particles (SMP) prepared from slaughterhouse material for the assay of IF1-containing extracts and preparations. Then, in 1987 we introduced the use of submitochondrial particles prepared from a species naturally poor in IF1 for this purpose. Thus, rat heart SMP which are largely depleted of IF1 in their native state were found to allow the performance of particularly linear and reproducible IF1 titration assays regardless of the species source of the IF1 titrated on them. The present study presents the first systematic comparison of the effects of a variety of factors upon functional assays for IF1. These include variations in IF1 functional assays due to seasonal effects on bovine A particles as well as to the species source of the IF1-depleted particles used in the assays. Interestingly, bovine heart A particles prepared during cold weather were considerably more active than those prepared during warmer weather. Moreover, the more active cold-weather particles allowed the performance of better IF1 assays. Also, the larger the species of origin of the IF1-depleted heart muscle SMP, the less IF1 was required to produce a given amount of ATPase inhibition regardless of both seasonal effects and the species source of the IF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hydroxyl radical generation during mitochondrial electron transfer and the formation of 8-hydroxydesoxyguanosine in mitochondrial DNA.

Production of hydroxyl radicals (HO.) by substrate-supplemented beef heart submitochondrial particles was studied by electron paramagnetic resonance in conjunction with the spin trap 5,5'-dimethyl-1-pirroline-N-oxide (DMPO). Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation hydroxyl-, alpha-hydroxyethyl-, and methyl radical adducts. The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfoxide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by superoxide dismutase and abolished by catalase. Further support for the production of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger. This approach yielded a kappa SCAVENGER/kappa DMPO value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. The scission of H2O2 to HO. requires consideration of a Fenton chemistry, i.e., the participation of metals or redox active metal pools in mitochondria to drive this reaction. The effect of several metal chelators on the formation of both HO. and H2O2 was examined. Bathophenantroline, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal and increased accumulation of H2O2. Conversely, EDTA or diethylenetriaminepentaacetic acid substantially increased the DMPO-HO. signal intensity and decreased H2O2 accumulation. These different results were rationalized in terms of the reduction potential of the redox couples involved, i.e., that of the ligated metal and those encompassed in the one-electron reduction of superoxide radical and of hydrogen peroxide. The formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was examined under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological significance of H2O2 and HO. formation during mitochondrial electron transfer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in adrenal cortex mitochondria and microsomes.

3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of delta 5-3 beta-hydroxysteroids to delta 4-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD+ and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD+, was inhibited by reduction of intramitochondrial NAD(P)+, and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). However, mersalyl blocked the effects of exogenous NADH and NAD+ and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD+ activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD+ and contained none or 2.9-7.3 nmol NAD+/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD+ for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD+ was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD+. The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD+ or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.

Adrenal Cortex↗
PubMed1981

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of th respiratory chain.

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c1 segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.

Animals↗
PubMed1975

Electrode measurement of oxygen tension with 1-ms time resolution.

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neurtralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation o of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.

Computers↗
PubMed1981

2-n-nonyl-4-hydroxy[3-3H]quinoline N-oxide: interaction with the mitochondrial membrane.

1. Synthesized 2-n-nonyl-4-hydroxy[3-3H]quinoline N-oxide ([3H]NoHOQnO) is a powerful inhibitor of both NADH oxidase and succinate oxidase activities in submitochondrial particles from beef heart. 2. The binding of [3H]NoHOQnO to submitochondrial particles reveals one specific binding site, which is sensitive to antimycin, and a large amount of aspecific binding. 3. The content of [3H]NoHOQnO-specific binding sites is related to the cytochrome b content in the particles. 4. The binding of [3H]NoHOQnO to the specific site causes the inhibition of electron transfer. 5. The relation between the inhibition of NADH oxidation and [3H]NoHOQnO-specific binding is hyperbolic.

Animals↗
PubMed1986

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter.

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.

Animals↗
PubMed1988

Inhibition by suramin of mitochondrial ATP synthesis.

Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.

Adenosine Triphosphatases↗