Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine 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. 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
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. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The Em vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.
3. With constant haem c concentration (a) 10−4 M and (b) 10−5 M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that Em values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.
4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10−4–7 · 10−6 M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.
5. The results are discussed in comparison with a number of related haem systems. 相似文献
2. The near-ultraviolet difference spectrum of whole cells reveals an absorption peak at 315 mμ with a shoulder around 350 mμ.
3. Both the endogenous respiration and motility of spermatozoa are completely blocked by 0.2 mM CN− and by 0.2 μM antimycin A. 2,4-Dinitrophenol and pentachlorophenol completely inhibit motility at the maximal stimulation of respiration. Rotenone strongly inhibits NADH oxidase of spermatozoa, although it has no effect on the respiration of whole cells.
4. It is concluded that the motility of mussel spermatozoa is tightly coupled to respiration, and the respiratory chain phosphorylating process is the only energy-supplying system for motility. 相似文献
2. It is best formed with an excess of O2 after reduction with a minimum amount of dithionite. It can also be formed at low O2 tension, but then contains some ferric oxidase.
3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.
4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a3 is reduced in the presence of cyanide by dithionite, there is no reaction with O2.
5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a3. The rate of cytochrome a, but not that of cytochrome a3 reduction depends on dithionite concentration.
6. In the presence of dissolved O2, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.
7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.
8. Hydrogen donors known to react with peroxidase-H2O2 complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.
9. These results strengthen the evidence for cytochromes a and a3 but indicate that this difference has disappeared in ‘oxygenated’ oxidase. 相似文献
2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the Km value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The Km value for ADP is 2 μM. The Q10 is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.
3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.
4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent Kd for the binding of [35S]-carboxyatractyloside and [14C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [35S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.
5. Binding of [14C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg2+ at 20 °C. The amount of bound [14C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.
6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa3. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [35S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities. 相似文献
2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O2 was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO2− consumed per mole of O2 consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO2− concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO2 to pH 8.0 at 0.01 M KNO2 with apparent Km's of 0.2 and 1.2 mM NO2−, respectively. Computations of the HNO2 concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO2 which was independent of pH, suggesting the involvement of HNO2 as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN3, NaCN and Na2S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO2− proved to be a competitive inhibitor of the nitrite oxidase system.
3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO3− increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO2 concentration an approximately constant Km of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO3− was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c.
4. The membrane-like composition of the nitrite oxidase system is indicated. 相似文献
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 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
The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a Km of 27 μM for O2. This activity was also inhibited by KCN, NaN3 and NH2OH and by NO2−.
A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO2−. It was inhibited by smaller amounts of KCN, NaN3 and NH2OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a Km for O2 of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO2−.
The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller Km for O2 and would therefore be expected to function more efficiently especially at low concentrations of O2. 相似文献
1. 1. Cyanide inhibits the catalytic activity of cytochrome aa3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·103 M−1·s−1 and a Ki that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.
2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa3−O2 system a biphasic reduction of cytochrome c occurs corresponding to an initial Ki of 0.8 μM and a final Ki of about 0.1 μM for the cytochrome aa3−cyanide reaction.
3. 3. The inhibited species (a2+a33+HCN) is formed when a2+a33+ reacts with HCN, when a2+a32+HCN reacts with oxygen, or when a3+a33+HCN (cyano-cytochrome aa3) is reduced. Cyanide dissociates from a2+a33+HCN at a rate of 2·10−3 s−1 at 22 °C, pH 7.3.
4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a2+a33+ than to a3+a33+.
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
1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.
2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[14C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.
3. 3. High affinity-fuscin binding sites (Kd = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.
4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;
5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (Ki in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (Ki = 5–10 μM); (c) the transport systems of phosphate (Ki ≈ 20 μM) and of glutamate (Ki = 3–5 μM); (d) the ADP transport, indirectly (Ki ≈ 10 μM).
6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH− carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.
7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.
8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.
9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.
Abbreviations: DTNB, 5,5′-dithio-bis-(2-nitrobenzoic acid); PCMB, p-chloromercuribenzoate 相似文献