Biomedical subjects
Publications and source records attributed to B Kadenbach.
The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.
Three possible mechanisms by which different levels of thyroid hormones in rats might cause the observed sevenfold change in the apparent proton permeability of the inner membrane of isolated liver mitochondria were investigated. (a) Cytochrome c oxidase was isolated from the livers of hypothyroid, euthyroid and hyperthyroid rats and incorporated into liposomes made with soya phospholipids. There was no difference between the proton current/voltage curves of the three types of vesicles. The hormonal effects, therefore, were not an inherent property of the enzymes, and were not due to different coupling of electron flow through the enzyme to proton transport. (b) The surface area of the mitochondrial inner membrane was shown by three different assays to be greater by a factor of between two and three in mitochondria from hyperthyroid animals than in mitochondria from hypothyroid animals; euthyroid controls were intermediate. This difference in surface area of the inner membrane explains less than half of the difference in apparent proton permeability. (c) The proton permeability of liposomes prepared from phospholipids extracted from mitochondrial inner membranes of hyperthyroid rats was three times greater than the proton permeability of those from hypothyroid rats; euthyroid controls were intermediate. This suggests, first, that the proton permeability of the phospholipid bilayer is an important component of the proton permeability in intact mitochondria and, second, thyroid hormone-induced changes in the bilayer are a major part of the mechanism of increased proton permeability. Such changes may be due to the known differences in fatty acid composition of mitochondrial phospholipids in different thyroid states. Thus we have identified two mechanisms by which thyroid hormone levels in rats change proton flux/mass protein in isolated liver mitochondria: a change in the area of the inner membrane/mass protein and a change in the intrinsic permeability of the phospholipid bilayer.
We have studied cytochrome c oxidase (COX) deficient muscle fibre segments in 6 patients with mitochondrial myopathy and deletions of mitochondrial DNA (mtDNA). The distribution of transcripts of normal and mutated mtDNA in skeletal muscle sections was studied by in situ hybridization. The results were compared with the enzyme histochemical activity of COX and the immunohistochemical distribution of mtDNA encoded and nuclear DNA encoded subunits of COX. In all cases a proportion of the muscle fibres (less than 1-30% of the fibres in cross-sections) had low COX activity and high activity of succinate dehydrogenase (COX deficient muscle fibres). Transcripts of normal and deleted mtDNA showed the same distribution within the tissue as the corresponding mtDNA, indicating that the deleted mtDNA is transcribed. The COX deficient muscle fibres showed accumulation of transcripts of deleted mtDNA, which had a similar distribution as the accumulated mitochondria within these fibres. With few exceptions, there was a low level of transcripts of normal mtDNA in these COX deficient fibres. Immunohistochemical analysis revealed low levels of immunoreactive material using antiserum to the mtDNA encoded subunits II/III as well as the nuclear DNA encoded subunit IV of COX in all COX deficient muscle fibres. The fraction of deleted mtDNA in muscle ranged from 43 to 87%. There was no correlation between the proportion of COX deficient muscle fibres and the fraction of deleted mtDNA. In 2 cases the deletion did not involve any COX gene. One of these cases had 87% deleted mtDNA but less than 1% COX deficient muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous studies have revealed cytochrome-c-oxidase-deficient cardiomyocytes and the 4,977 base pair deletion ("common deletion") of mitochondrial DNA (position 8,482-13,459) in the heart of a patient with dilatative cardiomyopathy and Kearns-Sayre syndrome. In the present investigation the co-localization of the enzymatic and genomic defects was studied. In situ hybridization of mitochondrial DNA (mtDNA) revealed different hybridization patterns in the cytochrome-c-oxidase-deficient cells: (1) a selective reduction of the hybridization signal with an mtDNA probe recognizing the common deletion, indicating predominance of the deleted over the nondeleted mtDNA molecules in the cytochrome-c-oxidase-deficient cells; (2) a reduced hybridization signal with different mtDNA probes, indicating depletion of mtDNA; and (3) normal hybridization signals with different probes in single cytochrome-c-oxidase-deficient cardiomyocytes. These results indicate that different mechanisms may co-exist in Kearns-Sayre syndrome and may lead to defective respiratory chain function. The question of the pathogenetic interrelationship is discussed.
Cytochrome c oxidase (complex IV of the respiratory chain) was studied histochemically in autoptic human extraocular muscles (n = 135), revealing randomly distributed single fibers without enzyme activity. The enzyme defect was expressed in all the mitochondria of an involved fiber as evidenced by ultracytochemistry. Succinate dehydrogenase showed normal histochemical reactivity. The defects occurred already in the second decade and were regularly seen from the third decade on. The defect density (defects/mm2) increased from approx. 1/mm2 below the fifth decade to about 4/mm2 in advanced age (P = 0.000). The highest defect density was observed in the levator palpebrae muscle. On the whole, the defect density was about 5-6 times higher in the extraocular muscles than in the limb muscle, diaphragm and heart (Müller-Höcker, 1989, 1990). Immunocytochemical detection of cytochrome c oxidase showed that loss of cytochrome c oxidase activity was due to an almost complete absence of both nuclear and mitochondria subunits of the enzyme. The results document different organ and heterogenic cellular sensitivity to the age-related loss of cytochrome c oxidase. The loss of both mitochondrial and nuclear subunits indicates that nuclear factors are most probably involved in the decline of the respiratory chain function in senescence.
Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody-producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC-purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa-h (heart-type), after amplification by the polymerase chain reaction, with a probe of VIa-l (liver-type).
Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.
We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.
The cytochrome c oxidase enzyme complex of eukaryotes is made up of three mitochondrial-coded subunits and a variable number of nuclear-coded subunits. Some nuclear-coded subunits are present in multiple forms and probably perform a tissue- or development-specific function. A detailed evolutionary analysis of the cytochrome c oxidase subunits that have been sequenced to date is reported here. We have found that gene duplication events from which the liver and heart isoforms of rat subunits VIa and subunit VIII originated can both be dated at about 240 +/- 90 million years ago, long before the radiation of mammalian lineages. Sequence divergence between the processed-type pseudogenes for the subunits IV, VIc and VIII have been estimated. Our results indicate that they arose fairly recently, thus suggesting that retroposition is a continuing process. We show that the rate of silent substitution in mitochondrial-coded subunits is 5-10 times higher than in nuclear-coded subunits; on the other hand replacement rates, although differing from gene to gene, are roughly of the same order of magnitude in both nuclear and mitochondrial genes. In the case of most of the nuclear-coded proteins we observed a slightly greater similarity between rats and cow, which agrees with the data obtained for mitochondrial-coded subunits.
The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I-III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mitochondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g. VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochrome c oxidation by the reconstituted enzyme from bovine heart but not from P. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochrome c oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochrome c oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.
A 5-month-old boy died of progressive heart failure that started at the age of 3 months. Autopsy revealed a mitochondrial cardiomyopathy and a mitochondrial myopathy of the limb muscle and diaphragm. Cytochemically random defects of cytochrome c oxidase were visualized by light and electron microscopy in the diaphragm and especially the heart muscle, the limb muscle showing a diffuse attenuation whereas the liver and kidneys reacted normally. The activities of NADH-dehydrogenase (complex I) and cytochrome c oxidase (complex IV) were severely diminished (20% residual activity of controls) in the skeletal and heart muscle. In the heart, succinate cytochrome c reductase (complex II/III) was additionally decreased to the same degree. Loss of cytochrome c oxidase activity was based on a reduction of both mitochondrial and nuclear derived subunits in the heart and diaphragm as revealed by immunohistochemical analysis, whereas the limb muscle showed a normal immunoreactive protein content. The results illustrate heterogeneous tissue expression of respiratory chain enzyme defects and demonstrate that a cardiomyopathy may be the leading presentation of a mitochondrial disorder in early infancy.
To differentiate the 2 major myopathies of infancy due to cytochrome c oxidase (COX) deficiency, we studied muscle biopsies from 4 patients with fatal myopathy and 4 with benign myopathy using biochemical, histochemical, and immunohistochemical techniques. Immunohistochemistry with antibodies directed against individual subunits of COX differentiated the 2 phenotypes: the fatal infantile myopathy was characterized by absence of the nuclear DNA (nDNA)-encoded subunit VIIa,b of COX, while in the benign myopathy both VIIa,b and the mitochondrial DNA (mtDNA)-encoded subunit II were absent. Early differential diagnosis between fatal and benign COX-deficient myopathies is of critical importance for prognosis and management of these infants, because the benign form is initially life-threatening but ultimately reversible.
We studied three patients with Leigh's syndrome (LS) and cytochrome c oxidase (COX) deficiency. Biochemical studies in brain, muscle, heart, liver, kidney, and fibroblasts disclosed a generalized COX deficiency. Kinetic studies of COX activity in brain mitochondria showed a low Vmax and a normal Km for reduced cytochrome c. Immunologic studies showed decrease of all COX subunits studied, without a specific defect of any one of them. Southern blot analysis excluded large deletions of mitochondrial DNA (mtDNA) but revealed a generalized increase in mtDNA quantity. Although Northern blot analysis showed no alteration in the 12 COX subunit mRNAs studied, two of three patients showed a decreased steady state rate of COX transcription in brain. COX deficiency in LS thus appears to be related to a decreased amount of otherwise normal COX holoenzyme.
Mammalian mitochondrial DNA codes for 13 proteins, which are all components of energy transducing enzyme complexes of the respiratory chain, i.e. the complexes which translocate protons across the inner mitochondrial membrane. The number of subunits of these enzyme complexes increase with increasing evolutionary stage of the organism. The additional nuclear coded subunits of the enzyme complexes from higher organisms are involved in the regulation of respiration, as demonstrated by the influence of intraliposomal ATP and ADP on the reconstituted cytochrome c oxidase (COX) from bovine heart. This regulation is not found with the reconstituted enzyme from P. denitrificans, which lacks the nuclear coded subunits. Some of the nuclear coded subunits occur in tissue-specific isoforms, as reported for COX and NADH dehydrogenase. Tissue-specific regulation of COX activity is also demonstrated by the differential effects of intraliposomal ADP on the kinetics of reconstituted COX from bovine liver and heart, which differ in subunits VIa, VIIa and VIII. At least 3 different COX isozymes occur in bovine liver, heart or skeletal muscle and smooth muscle. An evolutionary relationship between COX subunits VIa and VIc and between VIIa and VIIb is suggested based on the crossreactivity of monoclonal antibodies, amino acid sequence homology and hybridization at low stringency of PCR-amplified cDNAs for subunits VIa-1, VIa-h and VIc from the rat.
Cytochrome c oxidase (COX) was isolated from bovine smooth muscle (rumen), and compared with the enzyme from bovine liver, heart and skeletal muscle. A new isozyme of COX was found to be expressed in smooth muscle, which differs from the isozyme in liver and heart or skeletal muscle. SDS-PAGE as well as N-terminal amino acid sequencing of separated subunits from gel bands revealed the expression of the liver isoforms for subunits VIa and VIII and of the heart isoform for subunits VIIa in COX from smooth muscle.
The point mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA) from patients with myoclonic epilepsy and ragged red fibers (MERRF) was quantitatively analyzed after digestion with the restriction endonuclease Nae I of the PCR amplified DNA. Since the point mutation is not part of a restriction site for a commonly available restriction endonuclease, the Nae I restriction site was introduced by PCR using a mispairing primer. The percentage of mutated mtDNA was determined in a few hairs of five members of an affected family by counting the radioactivity of the fragments after PCR amplification with labelled dATP.
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A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.