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.     

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.     

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 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.     

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.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0°C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospholipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at ?25°C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

Involvement of hyposialylated immunoglobulins in the inactivation of alpha 1-proteinase inhibitor

The elastase inhibitory capacity of alpha 1-proteinase inhibitor (alpha 1-PI) was measured, using a direct and reproducible method, with phagocytic cells maintained in the tissue culture plate through the assay. The oxidative inactivation of alpha 1-PI is known to be mediated by the action of myeloperoxidase (MPO). The fact that hyposialylated IgG (hs IgG) induce the release of MPO prompted us to investigate the effects of such hs IgG on the inhibitory capacity of alpha 1-PI. The results show that 1-PI inactivation was observed only when phagocytic cells were activated by aggregated hs IgG, and not by unaggregated hs IgG. These observations indicate that hyposialylation should be completed by aggregation to perpetuate the oxidative reactions characteristic of inflammatory diseases.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.