Increase in hepatic ubiquinone on administration of diethylhexyl phthalate to the rat

It is shown for the first time that the content of ubiquinone of liver increases (2.5 fold) on dietary administration of the widely-used industrial Plasticizer diethylhexyl Phthalate to the rat. The increase is localized almost entirely in mitochondria in which the concentration of the quinone Per mg Protein is 1.7 times the control. IncorPoration of the radioactive Precursor (acetate) reveals that the biosynthesis of ubiquinone is increased in the livers of Plasticizer-administered animals. The rate of degradation is not altered.     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

The semiquinone cycle. A hypothesis of electron transfer and proton translocation in cytochromebc-type complexes

The Q cycle and theb cycle are the main current models of action of the cytochromebc-type complexes of mitochondria, bacteria, and chloroplasts. Both are based on the concept, proposed in 1972, of two sequential one-electron oxidations of (ubi)quinol along two discrete pathways which operate at different redox potentials, and with bound semiubiquinone as an intermediate. The models differ in two respects, viz. in the pathway of electron transfer and the principle of linkage of electron transfer to proton translocation. In this article we outline a new model, called the semiquinone or, simply, SQ cycle, which is based on the electron transfer principles of theb cycle but which incorporates the Q cycle concept of direct coupling between electron transfer and proton translocation through action of ubiquinone.This paper is dedicated to the memory of Bob Casey, who died in Helsinki on the 2nd of August 1985.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

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.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).