2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.
3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg2+, K+, Ca2+ or 2,4-dinitrophenol, but is reversed by cytochrome c.
4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes. 相似文献
2. The respiratory control thus revealed is most prominent at the NADH-flavin coupling site but is also present at the cytochrome b region coupling site.
3. Uncoupler concentrations inducing maximal respiratory rates exceed those abolishing the phosphorylative capacity by one order of magnitude or more.
4. The addition of glucose, hexokinase and ADP prior to that of uncoupler reduces the uncoupler-induced respiratory stimulation.
5. A respiratory stimulation initiated by Ca2+ is additive to the uncoupler-nduced effect both in the NADH and succinate oxidase systems. 相似文献
2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low Km. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.
3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c1 (552 nm) and c (545 and 516 nm).
4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.
5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.
6. The responses of yeast mitochondria to Ca2+ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate. 相似文献
2. The amounts of cytochromes c1 and aa3 are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.
3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH2-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.
4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.
5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.
6. Cytochromes aa3, c and c1 are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.
7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin. 相似文献
2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.
3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.
4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.
5. It is proposed that the steady-state concentration of endogenous Pi may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous Pi. 相似文献
2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.
3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.
4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.
5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.
6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation. 相似文献
The transhydrogenase reaction dependent on succinate oxidation was not appreciably inhibited by energy-transfer inhibitors, although oleate and pentachlorophenol were almost equally effective in both reactions. Pi did not stimulate the succinate-supported activity.
From the effects of thyroxine and its derivatives on the energy-dependent and independent reductions of NAD+ by NADPH, the involvement of the same transhydrogenase enzyme in both reactions was suggested.
These and other results indicated that the energy-transfer system of M. denitrificans was very similar to, though not identical with, that of mammalian mitochondria. 相似文献
2. In heart mitochondria Nagarse (EC 3.4.4.16) destroys the ability to activate palmitate. When, however, heart mitochondria are oxidizing palmitate, they are protected from the inactivating action of Nagarse.
3. Although treatment of liver mitochondria with Nagarse causes the loss of about 95 % of the palmitoyl-CoA synthetase activity, no influence is observed on palmitate oxidation.
4. Adenosine inhibits palmitoyl-CoA synthetase in liver and heart mitochondria. Adenosine is a competitive inhibitor with respect to ATP with an apparent Ki of 0.1 mM. The residual palmitoyl-CoA synthetase in Nagarse-treated liver mitochondria is much less sensitive to adenosine.
5. 2 mM adenosine or 2 mM adenosinesulfate inhibit palmitate oxidation (in the presence of 2.5 mM ATP) in heart mitochondria 60–90 %.
6. The data obtained are consistent with the concept of a palmitoyl-CoA synthetase localized on the outside of the outer membrane of rat heart and liver mitochondria, with an additional locus of (ATP-dependent) palmitoyl-CoA synthesis in the inner membrane matrix compartment of liver mitochondria. 相似文献
2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50c value with increasing respiratory rate.
3. The fraction of the normoxic respiration that is observed at p50c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.
4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50PN) varies less than p50c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.
5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca2+ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50PN). 相似文献
2. Assuming Michaelis-Menten kinetics in the respiration, theoretical curves for oxygen tension in the liquid and oxidation level of the terminal oxidase during a linear increase of the oxygen tension in the gas were calculated.
3. Measurements were performed on rat liver mitochondria. Steady-state curves for oxygen tension in the liquid and oxidation level of the terminal oxidase, cytochrome a3, obtained with coupled mitochondria resembled the theoretical curves. For uncoupled mitochondria the cytochrome a3 curve was signmoidal, deviating strongly from the theoretical curve.
4. The apparent Km for oxygen uptake of coupled mitochondria in the presence of pyruvate and malate, in the absence of phosphate was found to be 0.5 μM. In the case of uncoupled mitochondria the oxygen tension in the liquid could not be measured with sufficient accuracy to allow comparison with Michaelis-Menten kinetics. The apparent Km for oxygen uptake was less than 0.05 μM. 相似文献
2. As mitochondria lose K+ a striking inhibition of O2 consumption occurs with both succinate and DPN-linked substrates, but not if tetramethyl-p-phenylenediamine + ascorbate serves as substrate. Respiration can be re-activated by adding K+ to the medium.
3. Results are discussed in terms of sensitivity of electron transport or substrate dehydrogenation to intra-mitochondrial K+ content. 相似文献
2. It also inhibits the efflux of intramitochondrial adenine nucleotides induced by this treatment.
3. It is concluded that the inhibitory action of bongkrekic acid on the adenine nucleotide translocator is favoured by the presence of endogenous adenine nucleotides. 相似文献
2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.
3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.
4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.
5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.
6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c1 (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH2/QH• couple and reduction of cytochrome b. 相似文献
2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen.
3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration.
4. The Km for oxygen is less than 0.1 μM at low phosphate potential, irrespective of substrate, and 1–3 μM under reversal conditions.
5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action. 相似文献
1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system.
All the changes in mitochondria described occurred under conditions where cell division was insignificant.
2. Deaeration of the medium decreases the content of cytochromes b and aa3 in the mitochondrial fraction, cytochrome aa3 “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions.
3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure.
4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria.
5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole. 相似文献
2. When mitochondria are preincubated at 25 °C under energized conditions in the presence of respiratory inhibitors with the substrates of glutamate dehydrogenase, an oxidation of NADPH, but not of NADH, is brought about by decreasing the reaction temperature. Both the rate of NADPH oxidation and the final steady-state mass-action ratio of nicotinamide nucleotides are dependent on the concentration of ammonia and on the final reaction temperature. A similar effect is observed when rhein is added to the reaction medium at 25 °C in order to inhibit the energy-linked transhydrogenase reaction.
3. In the presence of the substrates of glutamate dehydrogenase, intact ratliver mitochondria catalyse an ATPase reaction due to the simultaneous activity of the energy-linked transhydrogenase and the non-energy-linked transhydrogenation catalysed by glutamate dehydrogenase.
4. These findings are discussed in relation to the nicotinamide nucleotide specificity of glutamate dehydrogenase and to a possible compartmentation of nicotinamide nucleotides in intact rat-liver mitochondria. 相似文献
2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous 45Ca. A positive correlation was observed between fluorescence and energized 45Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous 45Ca.
3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with 45Ca. Endogenous 45Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the 45Ca content of the mitochondria or the fluorescence of the probe.
4. Mitochondria treated with antimycin A lost most of their endogenous 45Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released 45Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the 45Ca previously released.
5. Following this energized uptake of 45Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the 45Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane. 相似文献