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B Kadenbach

Publications and source records attributed to B Kadenbach.

At least 37 records · Page 2Linked to original sources
PubMed1997

The subunit structure of cytochrome-c oxidase from tuna heart and liver.

Cytochrome-c oxidase was isolated from tuna liver and heart, and the subunit composition was analysed by SDS/PAGE by two separation systems. Two additional subunits of the enzyme complex were immunoprecipitated from solubilized mitochondria with an antibody against bovine subunit IV. The N-terminal and internal amino acid sequences of all nuclear-coded subunits were determined after blotting onto poly(vinylidene difluoride) membranes or by tryptic hydrolysis of gel bands and HPLC separation of peptides, respectively. 13 subunits were identified with isoforms for subunits Va, VIc, VIIb and VIII. The isoforms for subunits Va and VIIb are found in liver and heart, isoforms for subunit VIc only in heart, and isoforms for subunit VIII only in liver. Isoforms for subunits Va, VIc and VIIb have not been described in other species. The postulated mechanism of thermogenesis in mammals, based on decreased H+/e- stoichiometry at high ATP/ADP ratios due to binding of ATP to the heart-type subunit VIa [Frank, V. & Kadenbach, B. (1996) FEBS Lett. 382, 121-124], appears not to occur in tuna, because no isoforms of subunit VIa were found.

Amino Acid Sequence↗
PubMed1997

Quantitative analysis of mitochondrial DNA deletion in paraffin embedded muscle tissues from patients with KSS and CPEO.

The percentage of common deletion of mitochondrial DNA (mtDNA) was determined quantitatively by a PCR-based, non-radioactive method in DNA extracted from formalin-fixed, paraffin-embedded skeletal muscle tissues from two patients with Kearns Sayre syndrome (KSS) and one with chronic progressive external ophthalmoplegia (CPEO). The method involved PCR cycle titration of wild-type and deleted mtDNA in parallel, staining of gel bands with the sensitive fluorescence dye SYBR Green I, and quantitation of intensity on a computer screen by the NIH image program. We determined 75% and 71% common deletion of mtDNA in the KSS patients and 35% in the CPEO patient.

Adult↗
PubMed1997

The cDNA sequences of cytochrome c oxidase subunit VIa from carp and rainbow trout suggest the absence of isoforms in fishes.

The cDNAs of subunit VIa of cytochrome c oxidase from rainbow trout liver and carp heart are presented, revealing 82% identity of their deduced amino acid sequences. The two cDNAs are evolutionary equally distant from the livertype (VIaL) and heart-type (VIaH) of mammalian subunit VIa. The data suggest that in ectotherm fishes no isoforms of subunit VIa occur, and that the postulated tissue-specific mechanism of thermogenesis in mammals, based on interaction of ATP with subunit VIaH (Frank, V. and Kadenbach, B. (1996) FEBS Lett. 382, 121-124), is absent.

Amino Acid Sequence↗
PubMed1997

Defects of the respiratory chain in the normal human liver and in cirrhosis during aging.

Defects of the respiratory chain are a typical feature of mitochondrial diseases and occur also during normal aging where they have been described in postmitotic tissues. The present study addresses the question of defect expression in the normal and cirrhotic liver. Randomly distributed defects of complex III (ubiquinone-cytochrome-c-oxidoreductase) and of complex IV (cytochrome-c-oxidase) of the respiratory chain have been detected with age-related increasing frequency both in normal and cirrhotic livers. No defects were present for complex II (succinate-dehydrogenase) and complex V (adenosine triphosphate-synthase) and in liver cell carcinomas. Sixty-one of 107 normal livers (57%) showed defects of the respiratory chain. The defects occurred in advanced age (over 50 years) in 87%. In contrast 50 of 64 cirrhotic livers (78%) had defects and approximately 60% occurred after age 50. The defects were caused by a loss of enzyme protein involving both nuclearly and mitochondrially coded subunits. Ninety-four percent of the defects (n = 275) involved complex IV selectively. In 4% selective defects of complex III were found and combined defects of both complexes occurred in only 2%. In situ hybridization and polymerase chain reaction (PCR) studies for the detection of the common deletion (4.977 bp) and of various point mutations of mitochondrial DNA (mtDNA) revealed no consistent molecular genetic abnormalities in microdissected respiratory chain defective liver cell areas. Single point mutations at nt 3243 and/or 5692 were found only in 7 of 18 microdissected probes from 6 patients. The results show that defects of the respiratory chain occur already in normal livers most probably during cell aging and at a higher rate in cirrhosis. The random defect pattern favors a stochastic process, e.g., free radical damage. However, the role of mutations of mtDNA remains to be established.

Adenosine Triphosphatases↗
PubMed1997

ATP and ADP bind to cytochrome c oxidase and regulate its activity.

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.

Adenosine Diphosphate↗
PubMed1997

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors.

The binding of TNP-ATP (2' or 3'-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 microM) and one with lower affinity (Kd = 0.9 microM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 microM). The binding of [35S]ATP alpha S was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 microM) and six at the liver enzyme (Kd = 12 microM). The Paracoccus enzyme had only one binding site (Kd = 16 microM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e-stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature.

Adenosine Diphosphate↗
PubMed1996

A molecular switch in cytochrome C oxidase turns on thermogenesis in heart at low work load.

We describe a new mechanism of respiratory control by ATP on cytochrome c oxidase, which contrasts with the well known respiratory control of the electron transfer chain in mitochondria by ADP/ATP ratios. It is well established that high ADP/ATP ratios stimulate respiration and thus the synthesis of ATP in mitochondria, according to the energy requirements of the cell. Herein we describe the direct stimulation by high ATP/ADP-ratios of bovine heart (but not liver) cytochrome c oxidase activity in reconstituted vesicles. Under these same conditions it has already been shown that ATP decreases the H+/e- stoichiometry of cytochrome oxidase. Thus the observed effect of high ATP on cytochrome c oxidase would be expected to lead to partial uncoupling of energy transduction in mitochondria and to stimulation of thermogenesis.

Adenosine Diphosphate↗
PubMed1996

Biochemical analysis of fibroblasts from patients with cytochrome c oxidase-associated Leigh syndrome.

Cultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18-44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect.

Base Sequence↗
PubMed1996

A quantitative method of determining initial amounts of DNA by polymerase chain reaction cycle titration using digital imaging and a novel DNA stain.

A new nonradioactive method is described for quantitative determination of small amounts of DNA by PCR, examplified with mitochondrial DNA. The method represents a combination of serial dilution PCR and kinetic PCR and avoids the use of radioactivity by applying the fluorescent dye SYBR Green I, allowing visualization of PCR amplified bands on agarose gels in a broad exponential range of PCR cycles. After recording agarose gel images with a video camera in a computer, the band intensities are processed with the NIH image program and analyzed by a new graphical method. This nonradioactive method allows calculation of small original amounts of specific DNA in samples at high accuracy.

Base Sequence↗
PubMed1996

Regulation of the H+/e- stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios.

This paper describes the effect of intramitochondrial ATP/ADP ratios on the H+/e- stoichiometry of reconstituted cytochrome c oxidase (COX) from bovine heart. At 100% intraliposomal ATP the H+/e- stoichiometry of the reconstituted enzyme is decreased to half of the value measured below 98% intraliposomal ATP (above 2% ADP), while it remains constant up to 100% ADP. The decrease is obtained with different COX preparations, independent of the absolute value of the H+/e- stoichiometry. Decrease of H+/e- stoichiometry is prevented by preincubation of the enzyme with a tissue-specific monoclonal antibody to subunit VIa-H (heart type). Tissue-specific regulation of the efficiency of energy transduction in COX of muscle mitochondria could have a physiological function in maintaining the body temperature at rest or sleep, i.e. at low ATP expenditure.

Adenosine Diphosphate↗
PubMed1996

Defects of the respiratory chain in oxyphil and chief cells of the normal parathyroid and in hyperfunction.

Immunohistochemical detection of complex HIII (ubiquinone- cytochrome-c-oxidoreductase) and complex IV (cytochrome-c-oxidase) of the respiratory chain was performed in parathyroids of 164 humans with normal renal function (group I) and in 55 patients with chronic renal insufficiency (group II) obtained at autopsy. In group I, 33 of the 164 cases showed defects of the respiratory chain (20%). Eighty-five percent of the defects occurred in advanced age (> 50 years). In group II, 39 of 55 cases (70%) had defects, and about 70% of the defects occurred after age 50. In both groups, more than 80% of the defects were localized in oxyphil cell nodules. However, not every oxyphil nodule was involved. In group I, selective defects of complex IV predominated and were found in 47 of 86 defects (55%). Combined defects of complexes III and IV were present in 25 of 86 defects (29%). In contrast, in group II combined defects predominated and were found in 45% (107 of 240 defects), whereas single defects of complex IV existed in 38% (93 of 240 defects). The frequency of selective defects of complex III was about 16% to 17% in both groups. In situ hybridization and PCR studies for the detection of the common deletion (4.977 base pairs) and of various point mutations of mitochondrial of (m)DNA revealed no consistent molecular genetic abnormalities. A point mutation in the tRNALeu(UUR) at nucleotide (nt) 3.260 was found in only one probe. The results show that defects of the respiratory chain occur already in normal parathyroids, most probably during cell aging, especially in oxyphil cells and at a higher rate in hyperfunction. The high predominance of respiratory chain defects in oxyphil cells and their random distribution favors mutations of mtDNA as a possible cause of oxyphilic cell transformation and of the respiratory chain defects. However, the mutations of mtDNA in the parathyroids are apparently different from those in other ageing tissues.

Adult↗
PubMed1995

The number of nucleotide binding sites in cytochrome C oxidase.

The binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [35S]ATP alpha S and 8-azido-[gamma-32P]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus dentrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants Kd of about 0.2 and 0.9 microM. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a Kd of 0.3 microM. The two-subunit enzyme of Paracoccus dentrificans had only one binding site with a Kd of 3.6 microM. The binding of [35S]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent Kd's of 7.5 and 12 microM, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a Kd of 20 microM. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-32P]ATP.

Adenine Nucleotides↗
PubMed1995

Effect of ionic strength on membrane potential generation in reconstituted cytochrome c oxidase.

In this study we applied the potential-sensitive fluorescent dye indocyanine to measure the initial rate of membrane potential development in liposomes containing reconstituted cytochrome c oxidase from bovine heart. A linear relationship was found between the initial rate of membrane potential development, related to the H+/e(-)-stoichiometry, and the rate of electron flow at various substrate concentrations. By increasing the salt concentration a decrease of the initial rate of membrane potential development was found. A similar dependence on the salt concentration was also found for the membrane potential under steady state conditions. The data further demonstrate variation of the H+/e(-)-stoichiometry in cytochrome c oxidase depending on the salt concentration in the buffer.

Animals↗
PubMed1995

Regulation of mitochondrial energy generation in health and disease.

In mammalian cytochrome c oxidase (COX) three of the ten nuclear coded subunits (VIa, VIIa, VIII) occur in tissue-specific isoforms. The isoform distribution, however, varies in liver and heart of different species. Subunit VIII is different in liver and heart of bovine, dog, rat and chicken, but identical in human (liver-type) on one hand, and sheep, rabbit and rainbow trout (heart-type) on the other hand, as determined by N-terminal sequencing. Two moles of trinitrophenyl-ATP bind to monomeric COX from bovine heart and one to COX from bovine liver with dissociation equilibrium constant (Kd) values of about 3 microM. One binding site at the heart enzyme is blocked by a monoclonal antibody to subunit VIa-H. ATP (and/or ADP) interact with COX at two or three high-affinity binding sites, as shown by titration of the spectral changes of COX. Isolated COX from bovine heart was reconstituted with variable intraliposomal ATP/ADP ratios. By measuring the RCR (respiratory control ratio) and RCRVal (related to the valinomycin-respiration), which is a direct measure of the H+/e(-)-stoichiometry (Wilson and Prochaska, Arch. Biochem. Biophys. 282 (1990) 413-420), almost complete inhibition of the proton pump activity of COX by high intraliposomal ATP concentrations was found. The vectorial of protons for the formation of water, however, appears to be unaffected by nucleotides. This regulatory mechanism is assumed to have physiological significance for thermogenesis in muscle at rest. COX of fibroblasts from patients suffering from Leigh's syndrome, which is associated with a decreased COX activity, are suggested to have an incompletely assembled enzyme complex. This suggestion is further corroborated by the higher temperature-sensitivity of the enzyme when compared with COX from normal control fibroblasts. Defective regulation of COX via nuclear coded subunits is also proposed to cause mitochondrial diseases.

Amino Acid Sequence↗
PubMed1995

Immunohistochemical analysis of muscle cytochrome c oxidase deficiency in children.

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochrome c oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic investigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seen with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.

Animals↗
PubMed1995

Species-specific expression of cytochrome c oxidase isozymes.

Cytochrome c oxidase was isolated from livers and hearts of sheep, dog and rabbit, and the polypeptide composition was analyzed by two different SDS-PAGE separation systems. The gels were blotted on PVDF-membranes and the N-terminal amino acid sequences of the tissue-specific subunits VIa, VIIa and VIII were determined in a protein sequencer. Except for subunit VIIa from rat, subunits VIa and VIIa from all investigated mammals are tissue-specific expressed in liver and heart. In contrast, subunit VIII is clearly different in liver and heart of bovine, dog and rat, but identical in liver and heart of human (liver-type), sheep, rabbit and also in rainbow trout (heart-type). The data suggest a strong species-specific variation of the regulatory properties of cytochrome c oxidase in different tissues.

Amino Acid Sequence↗
PubMed1995

Human aging is associated with stochastic somatic mutations of mitochondrial DNA.

Deletions and point mutations of mitochondrial DNA (mtDNA), which are characteristic of various human mitochondrial diseases, have been identified mainly in postmitotic tissues like brain, heart and skeletal muscle of healthy humans of advanced age but not in young people. An exponential increase with age was described for deletions of mtDNA. This paper reviews the molecular basis and experimental results on mutations of mtDNA in patients with mitochondrial diseases and in aged individuals. In addition new data on the exponential increase of point mutations of mtDNA, characteristic for MERRF and MELAS disease, in extraocular muscle from elderly humans are shown. Finally the 'mitochondrial hypothesis on aging' based on stochastic somatic mutations of mtDNA is presented.

Aging↗