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PubMed1977

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.

Adenine Nucleotides↗

PubMed1992

The mode of action of lipid-soluble antioxidants in biological membranes. Relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles.

The effects of ubiquinol and vitamin E on ascorbate- and ADP-Fe(3+)-induced lipid peroxidation were investigated in beef heart submitochondrial particles before and after extraction of ubiquinone and vitamin E, and after reincorporation of either or both of these components. It is concluded that ubiquinol is capable of inhibiting lipid peroxidation without the mediation of vitamin E, but may also amplify the antioxidant effect of the latter. It is pointed out that ubiquinol is the only known lipid-soluble antioxidant that can be synthesized de novo in animal cells, and for which there exists an enzymic mechanism--the mitochondrial electron-transport system--that can regenerate the antioxidant from its oxidized form resulting from its inhibitory effect on lipid peroxidation. These features, together with its high degree of hydrophobicity and its general occurrence in biological membrane and in low-density lipoprotein, suggest a highly important role of ubiquinol in cellular defense against oxidative damage.

Animals↗

PubMed1978

Proton electrochemical gradient and phosphate potential in submitochondrial particles.

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.

Acridines↗

PubMed1979

The binding of 8-anilinonaphthalene-1-sulphonate to a fixed concentration of unenergised and succinate-energised submitochondrial particles.

The effect of the approximately hyperbolic relationship between fluorochrome concentration and light absorbed on the interpretation of data for the binding of 8-anilinonaphthalene-1-sulphonate to unenergised and succinate-energised submitochondrial particles has been investigated. If this is taken into account plots of fluorescence against fluorescence x [fluorochrome]-1 do not tend towards a maximum fluorescence value. The significance of this findings is discussed.

Anilino Naphthalenesulfonates↗

PubMed1975

Energy dependent hydrogen ion accumulation in submitochondrial particles.

The fluorescence quenching of 9-aminoacridine (9AA) in suspension of beef heart EDTA submitochondrial particles was studied and was used to calculate the pH gradient between these particles and the medium. This pH gradient, which is energy dependent, is also dependent strongly on the presence of anion species in the medium. It is 2.2 pH units in acetate medium and can be as high as 3.6 units in the presence of other highly lyophilic anions. The anions tested were found to be effective in the following order: SCN- greater than I- greater than NO3- greater than Br- greater than Cl-. The validity of the deltapH calculations was confirmed by comparison with deltapH values calculated from NH4+ uptake. In contrast, calculations based on quinacrine (QA) fluorescence quenching under the same assumption used for 9AA did not agree with NH4+ measurements and show quantitative and in some cases even qualitative differences. Both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NH4+ decreased deltapH significantly. When the rate of electron transport is slow, i.e., with succinate as substrate or with NADH and low concentration of rotenone, very low concentration of nigericin (less than 20 ng/ml) decreased deltapH. Under these conditions, valinomycin antagonized the nigericin effect and restored deltapH to its original value. Upon increasing nigericin concentration (greater than 100 ng/ml) the valinomycin effect is gradually replaced by a slower response of further reduction of deltapH.

Acetates↗

PubMed1977

Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors.

1. A procedure for preparation of sonic submitochondrial particles competent for adenine nucleotide transport is described. ADP or ATP transport was assayed, in the presence of oligomycin, in a saline medium made of 0.125 M KCl, 1 mM EDTA, 10 mM 4-morpholinopropane sulfonic acid buffer, pH 6.5. 2. Sonic particles transport ADP and ATP by an exchange diffusion process. Externally added ADP (or ATP) is exchanged with internal ADP and ATP with a stoichiometry of one to one. The V value for ADP transport 5 degrees C was between 2 and 3 nmol/min per mg protein. 3. The transport system in sonic particles is specific for ADP and ATP. It is strongly dependent on temperature. The activation energy between 0 and 9 degrees C is approx. 35 kcal/mol. The optimum pH is 6.5, 4, Like in intact mitochondria, externally added ADP is transported into sonic particles faster at a given concentration than externally added ATP. The V value for ADP transport is 1.5-2 times higher than the V value for ATP transport. 5. The transition from the energized to the deenergized state in sonic particles results in a decrease of the pH gradient across the membrane (internal pH less than external pH) and in a 2-4 fold increase in the Km value for ATP. This latter effect is opposite that found for transport of added ATP in intact mitochondria (Souverijn, J.H.M., Huisman, L.A., Rosing J. and Kemp, Jr., A. (1973) Biochim. Biophys. Acta 305, 185-198). Energization has no effect on the V value of ATP transport in sonic particles. 6. In contrast to intact mitochondria, inhibition of ADP transport in sonic particles by bongkrekic acid does not have any lag-time and does not depend on pH. The inhibition caused by bongkrekic acid is a mixed type inhibition with a Ki value of 1.2 micronM. Atractyloside and carboxyatractyloside do not inhibit ADP transport in sonic particles, unless the particles have been preloaded with these inhibitors during the sonication. 7. Palmityl-CoA added to sonic particles inhibits efficiently ADP transport. The mixed type inhibition found with palmityl-CoA has a Ki value of 1.6 micronM. 8. [3H]Bongkrekic acid binds to sonic particles readily and with high affinity. Bongkrekic acic binding to sonic particles does not depend on pH and it has a saturation plateau, corresponding approximately to 1.3 mol of site per mol of cytochrome a. The number of [3H]atracytloside binding sites is much lower (one-fifth of the bongkrekic acid). External carboxyatractyloside does not compete with [3H]bongkrekic acid for binding to sonic particles. However, when carboxyatractyloside is present inside the particles, it inhibits the binding of [3H]bongkrekic acid.

Adenine Nucleotides↗

PubMed1977

The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles.

The effects of bathophenanthroline, bathophenanthrolinesulphonate and 2-thenoyltrifluoroacetone on mung-bean mitochondria and submitochondrial particles were investigated. A variety of inhibitory effects on the oxidations of NADH, succinate and malate were observed. The results are discussed in relation to sites of inhibition and their relation to the effects on mammalian mitochondria.

Malates↗

PubMed1975

Dynamic control on the rate of the reduction of the b type cytochromes in submitochondrial particles.

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.

Animals↗

PubMed1975

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.

Animals↗

PubMedDate not supplied

[Interaction of ubisemiquinone with the high-potential iron-sulfur center of submitochondrial particle succinate dehydrogenase. EPR study at 240 and 12 degrees K].

The ESR spectra of beef heart submitochondrial particles were measured in the same samples at 240 degrees and 12 degrees K. There is close similarity between the inhibitory action of alpha-thenoyltrifluoroacetone, ethanol and ferricyanide on the non-saturating free radical signal SQ-2 observed at 240 degree K and peak at g=1.99 (and 2.04) which is visible only at very low temperatures. This result strongly supports our previous proposal that both ESR signals are manifestations of the ubisemiquinone complex with the High-Potential Iron-Sulfur protein of succinate dehydrogenase.

Animals↗

PubMed1979

[Interaction of 3-[3H]-2-n-nonyl-4-hydroxyquinoline-N-oxide with submitochondrial particles of beef heart. II. Determination of the binding sites].

The binding of 3H-NQNO in submitochondrial particles was determined by measuring the radioactivity in the supernatants as well as in the sediments after centrifugation of particles suspensions containing different amounts of 3H-NQNO. From the binding data Scatchard plots were constructed showing a large amount of aspecific binding depending on the particles preparation and concentration. In the presence of saturating concentrations of either antimycin or unlabelled NQNO (2-n-Nonyl-4-hydroxy-quinolinee-N-oxide) that remove or prevent the specific binding of 3H-NQNO, it is possible to evaluate the aspecific component of 3H-NQNO binding and to subtracte it from the experimental binding data by graphyc correction according to (3). The straight line from the corrected points gives the specific binding parameters: number of specific binding sites: about 0,5 moles 3H-NQNO/ moles cytochrome b and KD= 50 nM.

Animals↗

PubMed1976

[Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled submitochondrial particles. General kinetic analysis].

Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled ultrasonic submitochondrial particles at different pH values were studied. The rate constants calculated according to Pring's equation (k1=V/Prpoxt i+1) were found to increase linearly with the increase in the rate of electron transfer. Linearity was observed, however, only at relatively low rates of electron transfer. Several kinetic models were developed and analysed to fit the experimental data on the basis of the suggested activation of respiratory chains induced by their functioning. The best agreement with the experimental data was obtained with the model implying that the rate of activation of the electron carriers is directly proportional to the overall rate of electron transfer and the portion of non-activated respiratory chains in the system. It followed therefrom that electron transfer through already activated chains induced activation of adjacent non-activated chains. This model made it possiple to determine the rate constants for non-activated (ki) and activated (k) carrier states and the life-times of activated carriers (tau).

Animals↗

PubMed1977

Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. I. General kinetic analysis.

Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled ultrasonic submitochondrial particles at different pH values has been studied. Rate constants calculated from the Pring equation (ki' = V/PirPi+1ox) increased with the increase of the rate of the process. As in the previous work (Saks, V. A., Kupriyanov, V. V. and Luzikov, V. N. (1972) Biochim. Biophys. Acta 283, 42-53) this dependence was linear, but only at comparatively low rates of electron transfer. To explain the experimental data several kinetic models, based on the assumption that respiratory chains are activated when functioning, have been proposed and analysed. The best agreement with the experimental data was obtained for the model suggesting that the rate of activation of the carriers is directly proportional to the overall rate of electron transfer and to the proportion of non-activated respiratory chains in the system. Hence it appeared that electron transfer through already activated chains entailed activation of adjacent non-activated chains. This model allowed rate constants for non-activated (ki) and activated (ki) states of the carriers, as well as the life-time of the activated carriers (tau) to be determined.

Animals↗

PubMed1975

A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles.

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

Adenosine Triphosphatases↗

PubMedDate not supplied

[Cytochromes b in submitochondrial particles from beef heart in the presence of redox succinate/fumarate buffer].

Aerobic red-ox titration of cytochromes b from submitochondrial particles (SMP) using red-ox succinate/fumarate couple revealed two components, one of them having E'0=80 mv; n=1 and alpha-band absorption maximum at 562 nm (b562); and the other-E'0=-25 mv; n=1 and the absorption maximum at 565 nm. Energisation of SMP, equilibrated with red-ox succinate/fumarate buffer, brought about a increase in absorption at the cytochromes b region (564-565 nm), which was reversed and prevented by an uncoupler. Energy-dependent reverse electrone transport from ascorbate+TMPD resulted in considerable higher reduction of cytochromes b with summary maximum at 563 nm with the same initial reduction level prior to energisation. The data obtained show that energy dependent reduction of cytochromes b of SMP poised with succinate/fumarate red-ox buffer is presumably to the effect of energisation on the red-ox state of cytochrome b566. It is suggested that the transmembrane electric potential difference, generated upon the energisation of the particles, should result in re-distribution of the semi-quinone Q anion (-Q-) across membrane, thus altering the equilibrium redox-state of respiratory carriers, interacting with redox-couples -Q/Q and QH2/-Q- in the mitochondrial membrane.

Animals↗

PubMed1979

Proteolysis of the products of mitochondrial protein synthesis in yeast mitochondria and submitochondrial particles.

Degradation of mitochondrial translation products in Saccharomyces cerevisiae mitochondria was studied by selectively labelling these entities in vivo in the presence of cycloheximide and following their fate in isolated mitochondria. One-third to one-half of the mitochondrial translation products are shown to be degraded, depending on the culture growth phase, with an approximate half-life of 35 min. This process is shown to be ATP-dependent, enhanced in the presence of puromycin and inhibited by chloramphenicol. Further, the proteolysis is suppressed by detergents and is insensitive to antisera against yeast proteinases A and B when measured in mitochondria or 'inside-out' submitochondrial particles. It is concluded that the breakdown of mitochondrial translation products is most probably due to the action of endogenous proteinase(s) associated with the mitochondrial inner membrane. This proteinase is inhibited by phenylmethanesulphonyl fluoride, leupeptin, antipain and chymostatin.

Adenosine Triphosphate↗

PubMed1975

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles.

1. Succinate dehydrogenase is inhibited by citrate and beta-hydroxy-butyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved. 2. Pyruvate, alpha-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria. 3. Stimulation by alpha-ketoglutarate and glutamate is not influenced by the presence of rotenone. 4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K+ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate. 5. Stimulation by malate is higher in the presence of rotenone.

Animals↗

PubMed1978

Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. II. Influence of pH on kinetics of electron transfer.

pH Dependences of steady-state kinetic parameters of cytochrome chains of submitochondrial particles have been studies. It has been shown that the lifetimes of activated states (tau) of the pairs of cytochromes b leads to c1 and a leads to a3 have different pH dependences; those for the c1 leads to c and c leads to a cytochrome pairs being similar. The rate constants for the non-activated state of the respiratory chains decreased for the b leads to c1 pair and increased for the a leads to a3 pair when the pH value was increased. The values of pK calculated from these dependences for the pairs b leads to c1 and a leads to a3 were 7.2 and 8.9, respectively. It has been supposed that the ratio of activated to non-activated electron carriers may be controlled by the local pH value in the mitochondrial membrane, the latter being dependent upon the rate of electron transfer. The kinetic model based on this assumption allows one to explain the experimental dependences on pH of the rate constants for cytochromes b leads to c, and a leads to a3. The values of the diffusion rate constants for H+ and OH- ions in the mitochondrial membrane estimated from these kinetic data obtained in this study were 10(4)--10(5) s-1 and 10(2)--10(3) s-1, respectively.

Cytochrome c Group↗