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PubMed1982

Methyl hydroperoxy-epoxy-octadecenoate as an autoxidation product of methyl linoleate: a new inhibitor-uncoupler of mitochondrial respiration.

A new peroxide compound (ML-X) was isolated from an autoxidation product of methyl linoleate and was determined as methyl 9-hydroperoxy-12, 13-epoxy-10-octadecenoate. This compound inhibited state 3 respiration of rat heart- and liver mitochondria when glutamate and malate were used as substrates, but not when the substrate was succinate. State 4 respiration of mitochondria was not affected when glutamate-malate was used as the substrate, but it was stimulated when the substrate was succinate. ML-X inhibited oxidative phosphorylation of the mitochondria and abolished the membrane potential formed by respiration or by added ATP. NADH oxidase activity of submitochondrial particles was inhibited by ML-X but succinate oxidase activity was not inhibited. NADH-acceptor reductase activities of submitochondrial particles were inhibited by ML-X to the same extents as by rotenone. These findings show that ML-X has dual effects on mitochondrial respiration as (1) an inhibitor of NADH dehydrogenase complex and (2) an uncoupler. Neither methyl linoleate monohydroperoxide nor methyl epoxy stearate has such effects. ML-X is a new type of inhibitor-uncoupler of mitochondrial respiration in which hydroperoxy- and epoxy groups co-operate.

Animals↗
PubMed1979

Effect of ubiquinone-homologs on the sensitivity of mitochondrial ATPase to energy transfer inhibitors.

Short-chain ubiquinone (UQ-3) abolishes oligomycin sensitivity of ATPase in submitochondrial particles and the effect is reversed by long-chain ubiquinone (UQ-7). Ubiquinone-3 also abolishes DCCD sensitivity of ATPase in submitochondrial particles but the effect is not reversed by long-chain ubiquinones. These data suggest that ubiquinone interferes with energy transfer process by interaction with mitochondrial ATPase.

Adenosine Triphosphatases↗
PubMed1976

[Mechanism of action of the specific inhibitor of respiration and phosphorylation in mitochondria--n-(N,N-di-2-chlorethyl)aminophenylacetic acid].

An electrophilous inhibitor, p-(N,N-di-2-chloroethyl)amino-phenylacetic acid (I), specifically disturbs the mechanism of respiration and phosphorylation coupling in mitochondria. I inhibits respiration and ATPase activity in intact mitochondria and does not affect these processes in mitochondria and submitochondrial particles with partially or completely impaired coupling system. The data obtained show that I inhibits protonophoric function of NADH-ferricianide reductase from submitochondrial particles soluble ATPases from bovine heart and Micrococcus lysodeikticus mitochondria adsorded on octane water interface and has no effect on respective enzymes in water solutions. Cation-transferring enzymes are shown to behave with respect to the inhibitor on lipid water interface like respective enzymes in intact mitochondria, while in water solutions they behave like those in systems with the impaired coupling mechanism. Effect of I on protonophoric function of oligomycin-sensitive ATPase and bacteriorhodopsin plaques isolated from Halobacterium halobium is also studied. It is shown that the precence or the absence of I effect is due to a nature of lipid in the enzymatic complex. I is found also to inhibit specifically the transport of Ca2+ from water to octane in the presence of Ca2+-ATP-ase from rabbit sarcoplasmic reticulum.

Adenosine Triphosphatases↗
PubMed1975

Stimulation of rat liver mitochondrial adenosine triphosphatase by anions.

The hydrolysis of MgATP by isolated rat liver mitochondrial ATPase (EC 3.6.1.3) at pH 8.0 was stimulated by various anions. The rate of hydrolysis was increased from 18 to 170 mumol per min per mg, a 9.4-fold stimulation, by HSeO3 at 1 mM MgATP. In the absence of a stimulatory anion, reciprocal plots of initial velocity studies with MgATP as the variable substrate were curved (Hill coefficient approximately 0.5). With the addition of anion, the reciprocal plots became linear. When the substrate was MgITP or MgGTP with the isolated enzyme or MgATP with submitochondrial particles, no curvature of the reciprocal plots was observed. With purified ATPase, anions stimulated the hydrolysis of MgITP, MgGTP, MgUTP or MgCTP only slightly. With submitochondrial particles the stimulation by anions of MgATP hydrolysis was limited to approximately 2-fold. These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.

Adenosine Triphosphatases↗
PubMed1983

Spectral and electron transfer properties of Sepharose 6MB-immobilized cytochrome c.

Cytochrome c has been covalently attached to Sepharose 6MB to study the effects of immobilization on the molecule. A detailed study of the resulting product was conducted based on its three characteristic properties: spectra, oxidation-reduction potential, and biological activity. The spectral properties demonstrated that cytochrome c was essentially the same after attachment. No major conformational changes were indicated. The redox potentials for most samples of immobilized cytochrome c loaded with different amounts of protein were generally 20-25 mV lower than native cytochrome c (270 mV). Heavily loaded samples, however, showed no difference in potential. The Km values for immobilized cytochrome c with cytochrome oxidase and reductase from submitochondrial particles were comparable to the soluble protein. Vmax values are more strongly affected by immobilization, especially for the reductase. It has been demonstrated that the submitochondrial particles cannot penetrate the pores of the support material and therefore only the cytochrome c molecules on the surface are available for reaction. As a support material, Sepharose 6MB, which is CNBr activated, hydrolyzes at a significant rate at the pH of the coupling reaction, and this must be considered in establishing coupling conditions for protein immobilization.

Animals↗
PubMed1991

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Schägger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.

Amino Acid Sequence↗
PubMed1995

Inhibition by Ca2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect.

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszyński, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszyński, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

Adenosine Triphosphate↗
PubMed1998

Calcium ions potentiate lipoxygenase activity of cytochrome c at the physiological pH.

Cytochrome c oxidized linoleic acid, with lipoxygenase-like activity, to 9-hydroperoxy-10,12-octadecadienoic acid (9-HpODE), 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE), 13-keto-9,11-octadecadienoic acid (13-KODE), and hydroxy-epoxy-octadecenoic acids (HEPOs) in the presence of calcium chloride at the physiological pH of 7.5. In the absence of calcium, the reaction was remarkably reduced. Statistically significant increase of 13-KODE was observed 1 min after the reaction was started by adding calcium. Calcium chloride at concentrations as low as 30 microM caused statistically significant increase of 13-KODE production in the presence of 1 microM cytochrome c, and cytochrome c at concentrations as low as 10 nM caused statistically significant increase of 13-KODE production in the presence of 2 mM calcium. The addition of bleomycin enhanced the reaction, and the addition of adriamycin attenuated the reaction. The same oxidized linoleic acid products were found in the submitochondrial particles which were hydrolyzed by phospholipase-A2 in the presence of calcium. Lipoxygenase activity potentiated by calcium may be involved in the lipid peroxidation in the submitochondrial particles hydrolyzed by phospholipase-A2.

Animals↗
PubMed1985

Inhibition of the mitochondrial Mg2+-ATPase by propranolol.

The in vitro effects of propranolol, a commonly used beta-adrenergic blocker, on the membrane structure and function of rat heart mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the drug was added to the assay medium. At the concentration higher than 1.0 X 10(-4) M, propranolol significantly inhibited the State 3 respiration but had little effect on the State 4 respiration of the mitochondria. On the other hand, the drug exhibited noncompetitive inhibitions toward the Mg2+-ATPase activity of submitochondrial particles and purified enzyme preparations at the concentrations ranging from 3.0 X 10(-4) to 1.5 X 10(-3) M. The inhibitory constants of propranolol toward the enzyme activity in submitochondrial particles and in the purified preparation were estimated to be 6.7 X 10(-4) and 1.4 X 10(-3) M, respectively. However, the drug did not show significant effect on the activity of any of the enzyme complexes of the mitochondrial respiratory chain. It is thus concluded that propranolol impairs the mitochondrial respiration and oxidative phosphorylation mainly through its inhibition of the Mg2+-ATPase activity of the mitochondria. This effect of propranolol may explain, at least partly, its depression effects on the cardiac functions of the animal.

Adenosine Diphosphate↗
PubMed1976

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase.

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids. 2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition. 3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols. 4. It is concluded that mitochondrial transhydrogenase, catalyzing the nonenergy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.

Animals↗
PubMed1976

Proteins required for the binding of mitrochondrial ATPase to the mitochondrial inner membrane.

1. Isolated F1 (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 muM. 2. About one-third of the F1 and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles). 3. After further treatment with alkali of urea-treated particles, binding of F1 requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc2 are added. The concentration of F1-binding sites in the presence of both OSCP and Fc2 is about the same as that in TU particles. 4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F1, unless Fc2 is also present. Fc2 induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive. 5. It is tentatively concluded that OSCP is the binding site for F1 and Fc2 is the binding site for OSCP.

Adenosine Triphosphatases↗
PubMed1990

Mitochondrial adenosine triphosphatase in the oxyphil cells of a renal oncocytoma.

Oxyphil cells are characterized by cytoplasm packed with large numbers of mitochondria. Study of these unusual cells may provide information about the regulation of mitochondrial biogenesis. Although it has been suggested that this is a compensatory proliferation due to a mitochondrial dysfunction, no such dysfunction has been well documented. In this study we considered the possibility of dysfunction in the mitochondrial enzyme F1/Fo-adenosine triphosphatase(ATPase) as a stimulating factor involved in the mitochondrial proliferation of oxyphil cells. Mitochondria isolated from frozen tissue of a renal oncocytoma showing structural integrity and purity by electron microscopy were studied. Submitochondrial particles formed by sonic disruption showed the presence of the F1 component of mitochondrial ATPase with electron microscopy which was functionally active. The oligomycinsensitive ATPase activity from the renal oncocytoma was 0.133 mumol/min.mg submitochondrial particle protein which was higher than the readings obtained from normal kidney tissue (0.091 mumol/min.mg SMP protein) obtained from hamsters. Normal human renal tissue obtained at autopsy contained only nonfunctional mitochondria and therefore could not be used as control tissue. Mitochondrial ATPase dysfunction does not appear to be the inciting factor in the proliferation of mitochondria seen in oxyphil cell metaplasia and future studies should consider other possibilities. Preliminary functional studies of this nature can be performed with properly prepared frozen surgical tissue.

Adenoma↗
PubMed1983

Reserpine as an uncoupler of oxidative phosphorylation and the relevance to its psychoactive properties.

Many drugs differing widely in chemical structure uncouple mitochondrial oxidative phosphorylation in vitro. This observation has led to the hypothesis that in vivo uncoupling is the basis of their pharmacological activity. Serpasil, a parenteral preparation of reserpine, recently has been shown to uncouple oxidative phosphorylation in vervet monkey kidney mitochondria. Although the drug exhibits some properties of a "classical" uncoupler, our studies show that it has a dual effect on energy conservation. Reserpine released respiratory control in rat liver mitochondria only when dissolved in organic solvents (as in Serpasil) or when deprotonated. Reserpine also released the oligomycin-induced respiratory control in beef heart submitochondrial particles, and inhibited energized uptake of Ca2- by rat liver mitochondria. Reserpine had a dual effect on mitochondrial ATPase: It (a) enhanced ATP hydrolysis by intact liver mitochondria, and (b) inhibited ATP hydrolysis by submitochondrial particles of beef heart. On a molar basis, reserpine was less effective than carbonyl cyanide 3-chlorophenylhydrazone in all bioenergetic reactions examined. Homogenates and mitochondria isolated from brain and liver of rats stuporous from intraperitoneally injections of Serpasil exhibited no detectable abnormalities in respiratory states and responded to known uncouplers in the expected manner. There was no evidence of in vivo uncoupling of oxidative phosphorylation as a basis of the pharmacological activity of reserpine, although interference with energy transfer may be involved in toxic manifestations of the drug. The results indicate the need for caution in interpreting the action of drugs formulated in complex pharmaceutical preparations and based solely on in vitro experiments.

2,4-Dinitrophenol↗
PubMed1999

Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase.

The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.

Catalysis↗
PubMed1988

Effect of oleic acid on mitochondrial cytochrome c oxidase activity.

Both Km and Vmax values of cytochrome c oxidase for cytochrome c were elevated in oleic acid-incorporated mitochondria, whereas the amount of oleic acid incorporated into submitochondrial particles was smaller than that into mitochondria and the fatty acid had little effect on the enzyme activity. The degree of change in the bulk membrane fluidity was, however, almost the same in mitochondria and submitochondrial particles. Solubilized cytochrome c oxidase was insensitive to the effect of oleic acid. Oleic acid may act as a modifier of the interaction between cytochrome c oxidase and membrane lipids.

Animals↗
PubMed1983

Inhibition of oxidative phosphorylation by an oligomer of prostaglandin B1, PGBx.

PGBx is a synthetic, oligomeric derivative of prostaglandin B1 that has been shown to protect rat liver mitochondria from the deleterious effects of aging. In fresh mitochondria, PGBx inhibits reactions involving the F1F0-ATPase. It prevents the stimulation of respiration by ADP and inhibits ATP-driven Ca2+ transport. It has no effect, however, on Ca2+-stimulated respiration and associated proton movements, or on respiration-driven Ca2+ transport, indicating that PGBx is not an inhibitor of the electron transport chain. The ATPase activity of submitochondrial particles is inhibited by PGBx with mixed type kinetics in which both Km and V are affected. PGBx has no effect on the ATPase activity of soluble F1, but induces respiratory control in F1-deficient submitochondrial particles, indicating a mode of action similar to oligomycin and DCCD. The binding of DCCD to its proteolipid receptor is inhibited by PGBx, suggesting that this is the binding site for PGBx. It is concluded that PGBx inhibits reactions involving the F1F0-ATPase by binding at or near the DCCD-binding protein and blocking proton conduction through the F0 moiety of the complex.

Adenosine Triphosphatases↗
PubMed1993

Inhibition by 2,3-butanedione-monoxime of mitochondrial ADP-dependent respiration and muscle contraction.

2,3-Butanedione-monoxime (BDM) inhibits tension development in skeletal, cardiac and smooth muscle. Experiments on isolated single fibres of frog-muscle have shown a use dependent intermittent failure of twitching during continued stimulation. Our analysis of the effect of BDM on rabbit liver mitochondria and submitochondrial particles revealed a reduction of oxygen consumption in coupled mitochondria stimulated by adding of ADP, while in uncoupled mitochondria the oxygen consumption was not inhibited by BDM. Furthermore the substance did not inhibit ATP hydrolysis by submitochondrial particles. Based on these findings we suggest that BDM inhibits the activity of the mitochondrial ATP/ADP translocase.

Adenosine Diphosphate↗
PubMed1981

Interaction of certain cationic dyes with the respiratory chain of rat liver mitochondria.

Cationic dyes of the cyanine type have been observed to specifically inhibit NAD-linked respiration in rat liver mitochondria, with 50% inhibition occurring at about 0.2 mumol/g of mitochondrial protein. The dyes show no effect on succinate oxidation or coupled phosphorylation in the range which completely inhibits NADH oxidation. This specific inhibition was found with all cyanine dyes tested, but, with the possible exception of pyronin B, was not observed with other cationic dyes such as safranine O, rhodamine dyes, or with simple derivatives of the quinaldinium ring structure. The inhibition was observed to be both time- and concentration-dependent, with the half-time for full inhibition determined to be on the order of 15 to 30 s at 25 degrees C. Furthermore, the inhibition was totally dependent on the energization of the mitochondrial membrane by either substrate oxidation or the presence of ATP. The explanation of the energy dependence of the inhibition by cyanine dyes as the simple requirement for energy-linked dye concentration within the mitochondria is not supported by the relatively slow onset of inhibition as compared with the very rapid rate of dye uptake observed. Furthermore, inhibition of energy-requiring, succinate-linked NAD reduction in submitochondrial particles was observed to be inhibited by dyes, while fumarate reduction by NADH was found to be inhibited significantly only in the presence of ATP in addition to the dye. The energy requirement for electron transport inhibition in submitochondrial particles indicates that energy-dependent accumulation of the dyes by mitochondria cannot alone explain the energy requirement for the cyanine dye inhibition of NADH oxidation.

Animals↗