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At least 1,135 records · Page 63Linked to original sources
PubMed2005

Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins.

YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K+ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K+- and H+-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K+ and H+ transport across the yeast inner mitochondrial membrane. Wild-type SMPs exhibited rapid, reciprocal translocations of K+ and H+ driven by concentration gradients of either of them. K+ and H+ translocations have stoichiometries similar to those mediated by the exogenous K+/H+ exchanger nigericin, and they are shown to be essentially electroneutral and obligatorily coupled. Moreover, [K+] gradients move H+ against its concentration gradient, and vice-versa. These features, as well as the sensitivity of K+ and H+ fluxes to quinine and Mg2+, qualify these activities as K+/H+ exchange reactions. Both activities are abolished when the yeast Yol027p protein is absent (yol027Delta mutant SMPs), indicating that it has an essential role in this reaction. The replacement of the yeast Yol027p by the human Letm1 protein restores K+/H+ exchange activity confirming functional homology of the yeast and human proteins. Considering their newly identified function, we propose to refer to the yeast YOL027c gene and the human LETM1 gene as yMKH1 and hMKH1, respectively.

Calcium-Binding Proteins↗
PubMed2004

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.

2-Methoxyestradiol↗
PubMed2006

Structure-activity relationship studies on UK-2A, a novel antifungal antibiotic from Streptomyces sp. 517-02. Part 5: Roles of the 9-membered dilactone-ring moiety in respiratory inhibition.

Several open-chained analogues of UK-2A, a novel antifungal antibiotic isolated from Streptomyces sp. 517-02, were prepared for structure-activity studies. The in vitro antifungal activities of these compounds against Rhodotorula mucilaginosa IFO 0001 and the inhibition of uncoupler-stimulated respiration in bovine heart submitochondrial particles (SMP) were evaluated. Oxidative potentials were measured by cyclic voltammetry. An analogue prepared from dihexyl L-glutamate showed comparable inhibitory activity as UK-2A.

Animals↗
PubMed2006

Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells.

Oxidative stress is implicated in neurodegenerative diseases including stroke, Alzheimer's disease and Parkinson's disease, and has been extensively studied as a potential target for therapeutic intervention. Pyruvate, a natural metabolic intermediate and energy substrate, exerts antioxidant effects in brain and other tissues susceptible to oxidative stress. We tested the protective effects of pyruvate on hydrogen peroxide (H(2)O(2)) toxicity in human neuroblastoma SK-N-SH cells and the mechanisms underlying its protection. Hydrogen peroxide insult resulted in 85% cell death, but co-treatment with pyruvate dose-dependently attenuated cell death. At concentrations of >or=1 mM, pyruvate totally blocked the cytotoxic effects of H(2)O(2). Pyruvate exerted its protective effects even when its administration was delayed up to 2 h after H(2)O(2) insult. As a scavenger of reactive oxygen species (ROS), pyruvate dose-dependently attenuated H(2)O(2)-induced ROS formation, assessed from 2,7-dichlorofluorescein diacetate fluorescence. Furthermore, pyruvate suppressed superoxide production by submitochondrial particles, and attenuated oxidative stress-induced collapse of the mitochondrial membrane potential. Collectively, these results suggest that pyruvate protects neuronal cells through its antioxidant actions on mitochondria.

Cell Death↗
PubMed2005

The Western immunoblotting pattern of anti-mitochondrial antibodies is independent of the clinical expression of primary biliary cirrhosis.

Anti-mitochondrial antibodies are the serological markers of primary biliary cirrhosis. We analysed the detailed anti-mitochondrial antibodies patterns to see whether the immunological specificities detected at the time of the diagnosis correlate with the histological, clinical and immunological expression of the disease. One hundred and thirty primary biliary cirrhosis patients were studied at the time of presentation/diagnosis. Anti-mitochondrial antibodies reactivity was dissected and evaluated by Western immunoblotting with bovine heart submitochondrial particles as antigenic source. Six different Western immunoblotting patterns have been identified with the following hierarchy: pattern A (anti-PDC-E2+anti-E3BP, 38.5%), pattern B (anti-PDC-E2+anti-E3BP+anti-OGDC-E2, 20.8%), pattern C (anti-PDC-E2+anti-E3BP+anti-BCOADC-E2+anti-OGDC-E2, 13.1%), pattern D (anti-PDC-E2+anti-E3BP+anti-BCOADC-E2, 6.9%), pattern E (anti-BCOADC-E, 6.1%) and pattern F (anti-mitochondrial antibodies negative primary biliary cirrhosis, 14.6%). The different patterns were neither associated with peculiar clinical, biochemical, histological and immunological features nor with the Mayo Risk Score. The anti-mitochondrial antibodies pattern at presentation is independent of the stage of the liver disease; therefore, the Western immunoblotting characterisation of anti-mitochondrial antibodies does not seem to be helpful in identifying the clinical, biochemical or histological expression of primary biliary cirrhosis at the time of the diagnosis.

Adult↗
PubMed2005

Thermodynamic and EPR studies of slowly relaxing ubisemiquinone species in the isolated bovine heart complex I.

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.

Animals↗
PubMed2005

A novel strong competitive inhibitor of complex I.

Alkaline incubation of NADH results in the formation of a very potent inhibitor of complex I (NADH:ubiquinone oxidoreductase). Mass spectroscopy (molecular mass equal to 696) and absorption spectroscopy suggest that the inhibitor is derived from attachment of two oxygen atoms to the nicotinamide moiety of NADH. The inhibitor is competitive with respect to NADH with a K(i) of about 10(-8)M. The inhibitor efficiently suppresses NADH-oxidase, NADH-artificial acceptor reductase, and NADH-quinone reductase reactions catalyzed by submitochondrial particles, as well as the reactions catalyzed by either isolated complex I or the three subunit flavoprotein fragment of complex I.

Animals↗
PubMed2005

Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain.

Mitochondrial electron transport is a major source of reactive oxygen species (ROS) during cardiac ischemia and reperfusion. In the isolated rabbit heart, 30 and 45 min of ischemia decrease the contents of cardiolipin and cytochrome c in subsarcolemmal mitochondria (SSM) located beneath the plasma membrane. In contrast, interfibrillar mitochondria (IFM) in the interior of the myocyte do not sustain a decrease in cardiolipin. We proposed that the depletion of cardiolipin and the accompanying cytochrome c loss during ischemia were critical events that amplified ROS production by mitochondria. The total production of H2O2 was measured in submitochondrial particles (SMP) prepared from rabbit heart SSM and IFM after 0, 15, 30, and 45 min of ischemia. With NADH as substrate, total H2O2 production was increased only in SMP from SSM after 30 and 45 min ischemia, when ischemia decreased the content of cardiolipin and cytochrome c. In contrast, ischemia did not augment H2O2 generation in SMP from IFM with preserved cardiolipin and cytochrome c content. Thus, during the evolution of ischemic injury, H2O2 production from the electron transport chain increased after depletion of cardiolipin and the loss of cytochrome c.

Animals↗
PubMed2004

Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.

The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.

Adenosine Triphosphatases↗
PubMed1997

Identification of mitochondrial antigens recognized by antibodies in sera of patients with idiopathic dilated cardiomyopathy by two-dimensional gel electrophoresis and protein sequencing.

Antimitochondrial antibodies in sera of patients with idiopathic dilated cardiomyopathy (IDC) have been described previously, but the corresponding antigens have not been analyzed systematically. We therefore used both 1-dimensional and high-resolution 2-dimensional gel electrophoresis followed by immunoblotting and N-terminal amino acid sequencing to identify the relevant mitochondrial antigens, which are recognized by serum antibodies. Sera were obtained from patients with IDC (n = 75) and healthy controls (n = 182). For detection of antimitochondrial antibodies the mitochondrial antigen fraction, consisting of submitochondrial particles isolated from a bovine heart, was separated on SDS-PAGE and all sera were examined by immunoblot analysis. For further characterization of the mitochondrial epitopes the antigen fraction was separated in the first dimension according to isoelectric points using isoelectric focusing followed by gel electrophoresis. Proteins recognized by serum antibodies in 2-dimensional immunoblots were analyzed by N-terminal amino acid sequencing. In 1-dimensional immunoblot analysis, 51% of patients with IDC and 34% of controls contained serum antibodies reacting with mitochondrial protein bands with molecular weights of about 30, 43, 60, and preferentially 50 to 55 and 70 to 75 kD (p <0.01). We identified a 75-kD subunit of nicotinamide-adenine dinucleotide dehydrogenase (17% in IDC patients vs 5% in controls, p <0.05) and 2 core proteins of ubiquinol-cytochrome-c reductase (core P1: 39% in IDC patients vs 15% in controls, p <0.05; core P2: 20% in IDC patients vs 10% in controls, p <0.1), both enzymes of the respiratory chain, as are most relevant mitochondrial antigens. Furthermore, serum antibodies of patients with IDC were directed against lipoamide-dehydrogenase (15% vs 10% in controls) and a subunit of pyruvate-dehydrogenase (9% vs 3% in controls). Because these antigens play an important role in energy metabolism, the respective antibodies can be more than merely diagnostic markers of cell damage. To attribute them also to pathogenetic relevance appears to be a most attractive but still speculative hypothesis.

Adult↗
PubMed2003

In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.

The channel-forming antibiotic alamethicin was used to permeabilize mitochondrial membranes for the low molecular mass hydrophilic substrates NADH and ATP. Alamethicin-treated mitochondria show high rotenone-sensitive NADH oxidase, NADH-quinone reductase, and oligomycin-sensitive and carboxyatractylate-insensitive ATPase activities. Alamethicin does not affect Complex I and ATPase activities in inside-out submitochondrial particles. Permeabilized mitochondria quantitatively retain their aconitase and iso-citrate dehydrogenase activities. Electron microscopy of alamethicin-treated mitochondria reveals no disruption of their outer and inner membranes. From the results obtained it is recommended, that alamethicin be used for the in situ catalytic assay of intramitochondrially located enzymes.

Alamethicin↗
PubMed2001

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.

Adenosine Diphosphate↗
PubMed1991

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.

Adenosine Triphosphatases↗
PubMed1989

Electron-transfer flavoprotein from anaerobic Ascaris suum mitochondria and its role in NADH-dependent 2-methyl branched-chain enoyl-CoA reduction.

Electron-transfer flavoprotein was purified to apparent homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was 70,000, as estimated by gel filtration, and it migrated as two bands with apparent subunit molecular weights of 37,000 and 31,500 during sodium dodecylsulfate polyacrylamide gel electrophoresis. The enzyme exhibited an absorption coefficient for the bound FAD of 13.5 mM-1.cm-1 at 436 nm and a protein/flavin (270 nm/436 nm) ratio of 5.6. While the ascarid enzyme is similar to its mammalian counterpart, physiologically it functions in the reverse direction, shuttling reducing power from the electron-transport chain to a soluble 2-methyl branched-chain enoyl CoA reductase. Indeed, when A. suum submitochondrial particles were incubated with NADH, 2-methylcrotonyl-CoA and purified A. suum 2-methyl branched-chain enoyl-CoA reductase, 2-methylbutyryl-CoA formation was proportional to the amount of electron-transfer flavoprotein added.

Acyl Coenzyme A↗
PubMed1997

Regulation of the energy coupling in mitochondria by some steroid and thyroid hormones.

Male sex hormones [dihydrotestosterone (DTS), and testosterone] and progesterone, when added to the isolated rat liver mitochondria before or after some protonophores, lower the respiration rate and increase the delta psi level, i.e., reverse the protonophore-induced uncoupling. Such a recoupling ability shows specific structural requirements correlating with hormonal activity of steroids studied. For instance, epiandrosterone, a DTS isomer of very low hormonal activity, and deoxycorticosterone, differing from progesterone by additional OH-group and possessing quite different hormonal activity, as well as female sex hormones (estron and estradiol) show no recoupling effect. Like 6-ketocholestanol (kCh), male sex hormones and progesterone recouple mitochondria uncoupled by low concentrations of SF6847, FCCP and CCCP, but not by high concentration of these uncouplers or by any concentration of DNP, palmitate and gramicidin. In contrast to recoupling by kCh, hormonal recoupling requires addition of serum albumin and is inhibited by low concentrations of palmitate. Recoupling can also be shown on the heart and skeletal muscle mitochondria, being absent from the heart muscle submitochondrial particles, the bacterial chromatophores and the cytochrome oxidase proteoliposomes. In mitochondria it does not depend upon the oxidation substrate used (succinate or PMS + ascorbate were tested). Pronounced seasonal effect upon the DTS recoupling degree was revealed. The recoupling is maximal in January, February and from June to November, being minimal in the spring months and in December. In spring, the in vivo administration of thyroxine, di- or triiodothyronine improves the recoupling ability of DTS. 2 x 10 - 6 M. Thyroxine, when added in vitro, does not affect energy coupling if SF6847 was absent. In the presence of small amounts of SF6847, thyroxine stimulates the uncoupling in a DTS-sensitive fashion, di- and triiodothyronines being less effective. Addition of thyroxine to azide-inhibited mitochondria (oligomycin is present) stimulates respiration and normalizes the delta psi level. In this system, triiodothyronine is much less effective, whereas diiodothyronine is not effective at all. In the intact cells (thymocytes and the Krebs-II cells were tested), DTS lowers the respiration rate stimulated by low concentrations of SF6846 or FCCP. In this case, serum albumin is not required. It is suggested that recoupling effects of male sex hormones and progesterone are involved in their anabolic action just as uncoupling takes part in the catabolic activity of thyroid hormones.

Animals↗
PubMed1998

Iron-sulfur clusters/semiquinones in complex I.

NADH-quinone 1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain. Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million. Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8. These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I. From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned. Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I. Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron-sulfur clusters. Therefore, it has become possible to examine which subunits contain individual iron-sulfur clusters, their location within the enzyme and what their ligand residues are. The discovery of g=2.00 EPR signals arising from two distinct species of semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress. The intensity of semiquinone signals is sensitive to DeltamicroH+ and is diminished by specific inhibitors of Complex I. To date, semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems. This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron-sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts. Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit. The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin-spin interactions between cluster N2 and two distinct Complex I-associated species of semiquinone. Based on the splitting of the g1 signal of the cluster N2 and concomitant strong enhancement of the semiquinone spin relaxation, one semiquinone species was localized 8-11 A from the cluster N2 within the inner membrane on the matrix side (N-side). Spin relaxation of the other semiquinone species is much less enhanced, and thus it was proposed to have a longer distance from the cluster N2, perhaps located closer to the other side (P-side) surface of the membrane. A brief introduction of EPR technique was also described in Appendix A of this mini-review.

Animals↗
PubMed1998

Contraction transitions of F1-F0 ATPase during catalytic turnover.

Strong acoustic pressure was applied to submitochondrial particles (SMP) from bovine heart in order to drive ATP synthesis by F1-F0 complex for the account of sound waves. We observed a net ATP production at two narrow frequency ranges, about 170 Hz and about 340 Hz, that corresponds to the resonance oscillations of experimental cuvette when the acoustic pressure had a magnitude of 100 kPa. The results can be explained quantitatively by contractive conformational changes of F1-F0 complex during catalytic turnover. Negative staining electron microscopy of SMP preparations was used to visualize the ADP(Mg2+)-induced conformational changes of F1-F0 complex. In the particles with high ATPase activity in the presence of phosphate the factors F1 and F0 formed a congregated domain plunged into the membrane without any observable stalk in between. The presence of ADP(Mg2+) caused a structural rearrangement of F1-F0 to the essentially different conformation: the domains F1 and F0 were dislodged distinctly from each other and connected by a long thin stalk. The latter conformation resembled well the usual bipartite profile of ATPase. The data indicate that besides rotation, the catalytic turnover of ATP synthase is also accompanied by stretch transitions of F1-F0 complex.

Acoustics↗
PubMed1999

Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).

Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1 reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Ki of 1x10-5 M (pH 8.0, 25 degrees C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5x10-5 M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+ reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1 reductase activity of SMP is inhibited non-competitively by added Q1 whereas a simple competition between Q1 and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.

Animals↗