Low-level chemiluminescence of bovine heart submitochondrial particles

Submitochondrial particles from bovine heart mitochondria showed low-level chemiluminescence when supplemented with organic hydroperoxides. Chemiluminescence seems to measure integratively radical reactions involved in lipid peroxidation and related processes. Maximal light-emission was about 1500 counts/s and was reached 2-10min after addition of hydroperoxides. Ethyl hydroperoxide, cumene hydroperoxide and t-butyl hydroperoxide were effective in that order. Antimycin and rotenone increased chemiluminescence by 50-60%; addition of substrates, NADH and succinate did not produce marked changes in the observed chemiluminescence. Cyanide inhibited chemiluminescence; half-maximal inhibitory effect was obtained with 0.03mm-cyanide and the inhibition was competitive with respect to t-butyl hydroperoxide. Externally added cytochrome c (10-20mum) had a marked stimulatory effect on chemiluminescence, namely a 12-fold increase in light-emission of antimycin-inhibited submitochondrial particles. Stimulation of hydroperoxide-induced chemiluminescence of submitochondrial particles by cytochrome c was matched by a burst of O(2) consumption. O(2) is believed to participate in the chain radical reactions that lead to lipid peroxidation. Superoxide anion seems to be involved in the chemiluminescence reactions as long as light-emission was 50-60% inhibitible by superoxide dismutase. Singlet-oxygen quenchers, e.g. beta-carotene and 1,4-diazabicyclo[2,2,2]-octane, affected light-emission. beta-Carotene was effective either when incorporated into the membranes or added to the cuvette. The present paper suggests that singlet molecular oxygen is mainly responsible for the light-emission in the hydroperoxide-supplemented submitochondrial particles.     

Prevention of lipid peroxidation by NAD(P)H in rat liver submitochondrial particles

In rat liver submitochondrial particles both NADH and NADPH inhibit lipid peroxidation induced by cumene hydroperoxide. Concomitantly with the inhibition of lipid peroxidation, NADH and NADPH strongly stimulate the peroxidase activity of rat liver submitochondrial particles. Rotenone slightly prevents both the protective effect on malondialdehyde formation and peroxidase activity. The peroxidase activity of rat liver submitochondrial particles was attributed to the NAD(P)H-mediated reduction of mitochondrial cytochrome P-450 which can act upon hydroperoxides, by decomposing them to alcohols.     

Self-reduction of the iron(III)-doxorubicin complex

The Fe³⁺-doxorubicin complex undergoes reactions that suggest that the complex self-reduces to a ferrous oxidized-doxorubicin free radical species. The Fe³⁺-doxorubicin system is observed to reduce ferricytochrome c, consume O₂ and react with 2,2′-bipyridine. Bipyridine acts as a “ferrous ion scavenger” as it reacts with the ferrous ion produced by Fe³⁺-doxorubicin self-reduction. In the absence of O₂, a ferrous doxorubicin complex accumulates. In the presence of oxygen, Fe²⁺ recycles back to Fe³⁺. The rates of these reactions were measured and the Fe³⁺-doxorubicin self-reduction was determined to be the rate-determining step. The Fe³⁺-doxorubicin induced inactivation of cytochrome c oxidase and NADH cytochrome c reductase on beef heart submitochondrial particles occurs at a rate similar to Fe³⁺-doxorubicin self-reduction. Thus the rate at which damage to these mitochondrial enzymes occurs may be controlled by a nonezymatic Fe³⁺-doxorubicin self-reduction.     

Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain

Myxothiazol inhibited oxygen consumption of beef heart mitochondria in the presence and absence of 2,4-dinitrophenol, as well as NADH oxidation by submitochondrial particles. The doses required for 50% inhibition were 0.58 mol myxothiazol/mol cytochrome b for oxygen consumption of beef heart mitochondria, and 0.45 mol/mol cytochrome b for NADH oxidation by submitochondrial particles. Difference spectra with beef heart mitochondria and with cell suspensions of Saccharomyces cerevisiae revealed that myxothiazol blocked the electron transport within the cytochrome b-c₁ segment of the respiratory chain. Myxothiazol induced a spectral change in cytochrome b which was different from and independent of the shift induced by antimycin. Myxothiazol did not give the extra reduction of cytochrome b typical for antimycin. Studies on the effect of mixtures of myxothiazol and antimycin on the inhibition of NADH oxidation indicated that the binding sites of the two inhibitors are not identical.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

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.     

Alteration of inner-membrane components and damage to electron-transfer activities of bovine heart submitochondrial particles induced by NADPH-dependent lipid peroxidation.

We investigated the changes of the inner-membrane components and the electron-transfer activities of bovine heart submitochondrial particles induced by the lipid peroxidation supported by NADPH in the presence of ADP-Fe3+. Most of the polyunsaturated fatty acids were lost as a result of the peroxidation, and phospholipids were changed to polar species. Ubiquinone was also modified to polar substances as the peroxidation proceeded. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the disappearance of 27000-Mr and 30000-Mr proteins and the appearance of highly polymerized substances. Flavins and cytochromes were not diminished, but the respiratory activity was lost. The reactions of NADH oxidase and NADH-cytochrome c reductase were most sensitive to the peroxidation, followed by those of succinate oxidase and succinate-cytochrome c reductase. Succinate dehydrogenase and duroquinol-cytochrome c reductase were inactivated by more extensive peroxidation, but cytochrome c oxidase was only partially inactivated. NADH-ferricyanide reductase was not inactivated. The pattern of the inactivation indicated that the lipid peroxidation affected the electron transport intensively between NADH dehydrogenase and ubiquinone, and moderately at the succinate dehydrogenase step and between ubiquinone and cytochrome c.     

Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoreductase by rotenone, piericidin A, and MPP+.

The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.     

Mitochondrial respiratory chain of Tetrahymena pyriformis The properties of submitochondrial particles and the soluble b and c type pigments

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 °C there are two b-type pigments with half-reduction potentials of −0.04 and −0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a₂ with E_m7.2 of 0.245 and 0.345 V.

EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.

Cytochrome c₅₅₃ is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c₅₅₃ is a negatively charged protein at neutral pH with an E_m7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a K_D of 0.8 · 10⁻⁶ M. Reduced cytochrome c₅₅₃ serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c₅₅₃ shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.

A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and E_m7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6.     

Polyunsaturated fatty acids promote 8-hydroxy-2-deoxyguanosine formation through lipid peroxidation under the glutamate-induced GSH depletion in rat glioma cells

It has been reported that glutamate decreased the intracellular glutathione (GSH) concentration and thereby induced cell death in C6 rat glioma cells. Polyunsaturated fatty acids such as arachidonic acid, gamma-linolenic acid, and linoleic acid enhanced lipid peroxidation promoting 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation under the glutamate-induced GSH-depletion. The enhancement of lipid peroxidation by polyunsaturated fatty acids was species-dependent. Some antioxidants capable of scavenging oxygen and lipid radicals and some iron or copper scavengers inhibited both the lipid peroxidation and the 8-OH-dG formation, consequently protecting against cell death induced by glutamate-induced GSH depletion. These results suggest that GSH depletion caused by glutamate induces lipid peroxidation and consequently 8-OH-dG formation and that polyunsaturated fatty acids enhance lipid peroxidation associated with mediated 8-OH-dG formation through a chain reaction.     

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-Fe3+-induced lipid peroxidation were investigated by measuring oxygen consumption and malondialdehyde formation in beef heart submitochondrial particles. In the native particles, lipid peroxidation showed an initial lag phase, which was prolonged by increasing concentrations of ascorbate. Lipid peroxidation in these particles was almost completely inhibited by conditions leading to a reduction of endogenous ubiquinone, such as the addition of succinate or NADH in the presence of antimycin. Lyophilization of the particles followed by three or four consecutive extractions with pentane resulted in a complete removal of vitamin E and a virtually complete removal of ubiquinone, as revealed by reversed-phase high pressure liquid chromatography. In these particles, lipid peroxidation showed no significant lag phase and was not inhibited by either increasing concentrations of ascorbate or conditions leading to ubiquinone reduction. Treatment of the particles with a pentane solution of vitamin E (alpha-tocopherol) restored the lag phase and its prolongation by increasing ascorbate concentrations. Treatment of the extracted particles with pentane containing ubiquinone-10 resulted in a restoration of the inhibition of lipid peroxidation by succinate or NADH in the presence of antimycin, but not the initial lag phase or its prolongation by increasing concentrations of ascorbate. Malonate and rotenone, which prevent the reduction of ubiquinone by succinate and NADH, respectively, abolished, as expected, the inhibition of the initiation of lipid peroxidation in both native and ubiquinone-10-supplemented particles. Reincorporation of both vitamin E and ubiquinone-10 restored both effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimerization of hydroxylated species of m-aminophenol by cytochrome c with hydrogen peroxide

The cytochrome c and hydrogen peroxide-dependent oxidation of m-aminophenol was investigated by electrochemistry and spectrophotometry. The results indicated that the hydroxylated species of m-aminophenol have at least two conjugated substituted groups on the ring system (most possibly, its oxidized form 2-hydroxy-4-iminoquinone), and that the degradation of cytochrome c by hydrogen peroxide can also be prevented in the presence of m-aminophenol. The hydroxyl radical scavengers, mannitol and sodium benzoate, almost completely eliminate the hydroxylation of m-aminophenol. But oxo-heme species scavenger, uric acid, does not inhibit the hydroxylation. Combining the results of mass spectrum, nuclear magnetic resonance and element analysis with that of spectrophotometry, electrochemistry and chemical scavengers, it is suggested that cytochrome c may act as a peroxidase, which facilitates the hydroxylation and subsequent dimerization of m-aminophenol.     

The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.     

Further characterization of gradient-fractionated sub-mitochondrial membrane fragments from beef heart mitochondria

We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455–473). Further characterization of these two membrane fragments is reported. (1) Potassium chloride at the concentration of 0.15 M extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6–8-fold in both X- and Y-fragments. Subsequently KCl extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KCl extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K⁺ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycin-supplemented X- and Y-fragments.     

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes.

Dihydroxyfumaric acid induced lipid peroxidation in rat liver microsomes. This reaction was heat-insensitive contrary to the mitochondrial peroxidation reported in the previous paper, and was enhanced by p-chloromercuribenzoate. Additions of Fe²⁺ and Fe³⁺ stimulated both the lipid peroxidation and the disappearance of dihydroxyfumaric acid. On the other hand, addition of Mn²⁺ or Cu²⁺, which stimulated the disappearance of dihydroxyfumaric acid, inhibited the lipid peroxidation. Hydroxyl radical scavengers, superoxide dismutase and catalase had no effect on this lipid peroxidation and dihydroxyfumaric acid disappearance. The cytochrome p-450 content decreased about 70 % in parallel with the lipid peroxidation.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Lipid peroxidation and the reduction of ADP-Fe3+ chelate by NADH-ubiquinone reductase preparation from bovine heart mitochondria.

The NADH-ubiquinone reductase preparation (Complex I) of bovine hart mitochondria catalysed in the presence of reduced coenzymes and ADP-Fe3+ the lipid peroxidation of liposomes prepared from mitochondrial lipids. The apparent Km values for the coenzymes and the optimal pH of the reactions agreed well with those of the lipid peroxidation of the submitochondrial particles treated with rotenone. On assay of the reduction of ADP-Fe3+ chelate by the reduction of cytochrome c in the presence of superoxide dismutase and antimycin A or by the oxidation of reduced coenzymes, the reactions were not affected by rotenone but were inhibited by thiol-group inhibitors. The properties of the ADP-Fe3+ reductase activity were highly consistent with those of the lipid-peroxidation reaction. These observations suggest that electrons from reduced coenzymes are transferred to ADP-Fe3+ chelate from a component between a mercurial-sensitive site and the rotenone-sensitive one of the NADH dehydrogenase and that the reduction of ADP-Fe3+ chelate by the NADH dehydrogenase is an essential step in the lipid peroxidation.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. 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.