Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.     

Damage to mitochondrial electron transport and energy coupling by visible light

The effect of treating mitochondria with visible light above 400 nm on electron transport and coupled reactions was examined. The temporal sequence of changes was: stimulation of respiration coupled to ATP synthesis, a decline in ATP synthesis, inactivation of respiration, increased ATPase activity and, later, loss of the membrane potential. Loss of respiration was principally due to inactivation of dehydrogenases. Of the components of dehydrogenase systems, flavins and quinones were most susceptible to illumination, the iron-sulfur centers were remarkably resistant to being damaged. Succinate dehydrogenase was inactivated before choline and NADH dehydrogenase. Redox reactions of cytochromes and cytochrome c oxidase activity were unaffected.Inactivation was O₂-dependent and prevented by anaerobiosis or the presence of substrates for the dehydrogenases. Light in the range 400–500 nm was most effective and the presence of free flavins greatly enhanced inactivation of all of the above mitochondrial activities. This suggests that visible light mediates a flavin-photosensitized reaction that initiates damage involving participation of an activated species of oxygen in the damage propagation.     

Trypsin and calcium ions elicit changes of the membrane potential in pig blood platelets.

The addition of trypsin or thrombin or of Ca²⁺ ions to pig blood platelets was followed by a K⁺- dependent change of the membrane potential similar to that produced by the ionophore valinomycin. The effect of trypsin and of Ca²⁺, but not of valinomycin, was prevented by La³⁺ and by EGTA. It is proposed that upon the modification of the platelet surface by trypsin (and by thrombin under physiological conditions) membrane Ca²⁺ move from the external to the internal side of the platelet surface membrane and open the gates of K⁺ - specific channels.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Evidence for the identity of P430 of Photosystem I and chloroplast-bound iron-sulfur protein

The EPR spectrum of Photosystem-I subchloroplast particles, which had been pre-illuminated in the presence of methyl viologen, showed a large P700⁺ signal whereas bound iron-sulfur proteins were not detected. This observation is consistent with a “one-way” electron discharge by the primary electron acceptor, P430⁻, subsequent to the primary photochemical charge separation, and an accumulation of photooxidized P700⁺. Subsequent illumination of the same sample at 77 °K did not change the EPR spectrum. However, if the pre-illuminated subchloroplast particles were allowed to recover at room temperature by standing in the dark for 10 min or by addition of a chemical reductant, subsequent illumination of the sample at 77 °K yielded an EPR spectrum consisting of signals due to both P700⁺ and reduced iron-sulfur protein.     

Effects of beta-bungarotoxin on mitochondrial respiration are caused by associated phospholipase A activity.

Mitochondria prepared from tissue that had been incubated with β-bungarotoxin exhibited abnormal respiration. The respiratory rate in the presence of substrate only was apparently normal, but it did not increase upon the addition of ADP. This effect could also be obtained by treatment with $\text{V. russelli}$ phospholipase A or oleate. Treatment with lesser amounts of these agents caused the mitochondria to become uncoupled.     

Localized energization of the mitochondrial inner membrane by ATP

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane.The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F₁ · F_o) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211–222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F₁), reflecting the localized energization of the membrane by ATP.     

Possible participation of membrane thiol groups on the mechanism of NAD(P)+-stimulated Ca2+ efflux from mitochondria

The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.     

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of $\text{C57B1}\text{6}$ mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

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 K_m 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 K_m 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 K_d 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.     

Effects of fluorescamine labeling on mitochondrial ATPase activity.

Fluorescamine labeling of rat liver mitochondria enhances the ATPase activity. It reached maximum stimulation when mitochondria were treated with 30–34 nmol fluorescamine per mg of mitochondrial protein. This stimulation is inhibited by N,N′-dicyclohexylcarbodiimide. The maximum stimulation caused by labeling is the same as that obtained from uncoupler with optimum concentration. The chemiosmotic potential (Δμ_H⁺) decreases as the labeling increased. However, Δμ_H⁺ is not abolished completely even when ATPase activity reaches a maximum. The results suggest that primary amino groups may be involved in controlling mitochondrial ATPase activity.     

The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli

Removal of the F₁ ATPase from membrane vesicles of $\text{Escherichia}\text{coli}$ resulted in leakage of protons across the membrane through the F_O portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F₁ ATPase to F₁-stripped everted membrane vesicles. It is concluded that in F₁-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F₁ ATPase.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Progressive loss of mitochondrial creatine phosphokinase activity in muscular dystrophy.

Mitochondria were isolated from the pectoralis and gastrocnemius muscles of chickens with a hereditary muscular dystrophy, and age-matched controls. In the pectoralis, for dystrophic birds aged 0.12, 0.25, 0.55, and 1.55 yr, the creatine phosphokinase activity of the intact mitochondria, expressed in terms of pellet protein, was 69%, 45%, 24%, and 13% as great, respectively, as that of the controls. The corresponding figures for the gastrocnemius were 79%, 46%, 51%, and 28%. The mitochondria from dystrophic muscles exhibited satisfactory respiratory control ratios, P:0 ratios, and state 3 respiratory rates. To check whether their apparent loss of creatine phosphokinase activity was due to the presence of increasing amounts of non-mitochondrial pellet protein, the state 3 respiratory rate was used as a mitochondrial marker; the rates per mg protein were similar in mitochondria from normal and dystrophic muscles of each age group.     

Imipramine and lipid phase transition in inner mitochondrial membrane

As ascertained by freeze-fracture electron microscopy, imipramine prevents lateral phase separation from taking place in inner mitochondrial membranes at sub-zero temperatures. Electron spin resonance (ESR) measurements performed on mitochondrial membranes labeled with the N-oxyl-4′,4′-dimethyloxazolidine derivative of 16-ketostearic acid, show that the spin probe motion is markedly inhibited below 0°C and that 5 mM imipramine attenuates the temperature effect. These results are explained by supposing that imipramine is able to decrease the transition temperature of the inner mitochondrial membrane lipids as it does for simple lipid systems.     

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia–reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.