Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Submitochondrial Particles”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,297 records · Page 72Linked to original sources
PubMed1990

Anti-mitochondrial antibodies (anti-M7) in heart diseases recognize epitopes on bacterial and mammalian sarcosine dehydrogenase.

The anti-mitochondrial antibody (AMA) anti-M7 has been shown to occur exclusively in sera from patients with acute and chronic myocarditis. Applying different enzymes of the inner mitochondrial membrane to ELISA, anti-M7-positive sera reacted only with sarcosine dehydrogenase (SD) from Pseudomonas aeruginosa. Testing these sera in the Western blot against a commercially available SD as well as against SD prepared from rat liver mitochondria, a determinant at 42 kD and 90 kD, respectively, was visualized. Using submitochondrial particles (SMP) from bovine heart and rat liver another major determinant at 64 kD could be observed with both antigen fractions. Liver SMP also expressed the SD-related, 90-kD epitope. Sera from patients with other AMA-positive and AMA-negative autoimmune diseases were negative with these different determinants. The identity of the 64-kD epitope on heart and liver SMP as well as the 42-kD polypeptide of bacterial SD and the 90-kD epitope on mammalian SD was proven by absorption studies and by elution of antibodies from the antigen bound to the immobilon sheets after immunoblotting. The SD enzyme activity was not affected by anti-64-kD and anti-42-kD antibodies in vitro. It is concluded that anti-M7 antibodies may be stimulated by an antigen expressed on cardiocytes during an infection which shares epitopes with SD, an evolutionary highly conserved protein. SD-sensitized B cell clones could therefore be triggered by the M7-antigen which shows homology to SD.

Animals↗
PubMed2004

'True' antimitochondrial antibody-negative primary biliary cirrhosis, low sensitivity of the routine assays, or both?

Anti-mitochondrial antibody (AMA) is considered the serological hallmark of primary biliary cirrhosis (PBC), but may be missing in a proportion of these patients. We assessed sensitivity and specificity of the currently available techniques for AMA detection in a large series of PBC patients and controls, and analysed their clinical and immunological features according to the AMA status. By indirect immunofluorescence on rat tissue sections and HEp-2 cells, Western immunoblot with bovine submitochondrial particles, and two ELISAs with AMA-specific recombinant proteins, we evaluated the presence of AMA in 127 PBC patients, 166 patients with type 1 autoimmune hepatitis and 100 with non alcoholic fatty liver disease. In PBC patients Western immunoblot detects AMA significantly more often than indirect immunofluorescence on HEp-2 cells (85%versus 72%, P = 0.02) or rodent tissue sections (71%, P = 0.01); both ELISAs are only slightly less sensitive than Western immunoblot (81% and 78%). Ten patients with non alcoholic fatty liver disease were AMA-positive by indirect immunofluorescence, but none recognized AMA-specific epitopes in Western immunoblot or in ELISAs. Twelve patients with type 1 autoimmune hepatitis were AMA-positive by indirect immunofluorescence, but only 6 (3.6%) reacted by Western immunoblot and ELISAs. Western immunoblot or ELISA should be regarded as first-line assay for the detection of AMA. Up to 15% of PBC patients are consistently AMA-negative, yet they share the same clinical, biochemical and histological features of AMA-positive PBC. Detection of AMA in type 1 autoimmune hepatitis might identify a subset of patients at risk of developing a hepatitic/cholestatic syndrome.

Adult↗
PubMed1975

Characterization of the component, which controls the transformation between the kinetic forms of the b cytochromes.

1. In the presence of KCN and a saturating concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA-the active form which was reduced in the rapid phase, and bS-the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, names "y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N1,N1-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107-116] 2. Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "y" and thus permits the observation of the biphasic phenomen, but that it is not essential for the operation of this dynamic control mechanism.

Animals↗
PubMed1975

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.

Adenine Nucleotides↗
PubMed1976

Protein degradation during yeast sporulation. Enzyme and cytochrome patterns.

The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia. The specific activities of ribonuclease and aminopeptidase I raised several-fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase, glucose-6-phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered. The specific activities of NAD-dependent glutamate dehydrogenase, isocitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation. The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism. Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation. None of the enzyme changes observed proved to be 'sporulation-specific' in that it occurred exclusively in sporulating diploid yeast cells. Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism. During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells. Since the relative percentage of total to cycloheximide-insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation. The activity ratio of cytochrome c oxidase to F1-ATPase in submitochondrial particles isolated from vegetative cells and from purified asci was almost identical. This indicates that the loss of membrane-bound mitochondrial cytochromes during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins.

Aminopeptidases↗
PubMed1976

Electron spin resonance investigations of mitochondrial electron transport in Neurospora crassa. Characterization of paramagnetic intermediates in a standard strain.

1. Submitochondrial particles from Neurospora strain inl-89601 have been analyzed by electron spin resonance spectroscopy (ESR). Numerous signals due to iron-sulfur proteins are observed at low temperatures. Analysis of these ESR signals at various temperatures allows the assignment of resonances to iron-sulfur centers 1-5 that have been described in other organisms. There are no discrepancies between the signals seen in Neurospora and those described in other organisms and it is likely that Neurospora mitochondria contain the same iron-sulfur centers that are observed elsewhere. 2. NADPH and NADH act to reduce the iron-sulfur centers of respiratory complex I. 3. The drug pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorphenyl)pyrrole] is an effective inhibitor of both NADH-supported and succinate-supported electron transport in Neurospora. 4. Analysis of pyrrolnitrin inhibition curves, respiration studies, ESR spectra, and the steady-state level of reduction of cytochrome b in the presence and absence of the drug shows that pyrrolnitrin acts to inhibit electron transport in Neurospora mitochondria at multiple sites in the region between ubiquinone and cytochrome b.

Depression, Chemical↗
PubMed1977

Mitochondrial heredity of resistance to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of cytochrome b oxidation, in Saccharomyces cerevisiae.

3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), an inhibitor of cytochrome b oxidation, has been used for the selection of three resistant mutants (diur) of Saccharomyces cerevisiae. The mutant diur-64 exhibits in vivo cross-resistance to antimycin A while diur-34 and diur-1 are more sensitive to antimycin A than the parental strain. The three mutants exhibit mitochondrial inheritance according to the following criteria: mitotic segregation of diuron-resistant and diuron-sensitive diploids is obtained among the diploid progeny of a cross between diur and dius; non-Mendelian segregation of diuron resistance (4:0) is observed in spores of tetrads issued from diuron-resistant diploid; extensive ethidium bromide treatment leads to the formation of Q- mutants which no longer transmit diur and dius alleles. Evidence for two distinct diuron-resistant loci were obtained by allelism tests. Recombination analysis shows that diuron-resistance is not located in the polar region of the mitochondrial genome. The diur loci are not linked to the erythromycin locus since the upper limit in recombinants frequency (26%) for a non-polar region is obtained between diur and eryr. A low recombinants frequency (3%) is observed in crosses between diur-34 mutation and the two mutants cob1 and cob2 suggesting that diur-34 might be located between these two cytochrome-b-deficient loci. The resistance to diuron is also expressed in vitro since the oxidation rates of succinate by sonicated submitochondrial particles from the mutants are clearly less sensitive to diuron than that of the wild type.

Antimycin A↗
PubMed1977

Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III)

Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.

Animals↗
PubMed1980

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase.

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.

Adenosine Triphosphatases↗
PubMed1981

Spectral properties of cytochrome b-561 and cytochrome b-565 in mucidin-resistant mutants of Saccharomyces cerevisiae.

The oxidation of NADH in submitochondrial particles isolated from MUC1, MUC2 and MUC3 mucidin-resistant mutants of Saccharomyces cerevisiae is specifically resistant to mucidin. Extra reduction of cytochrome b-565 induced by mucidin is demonstrated in all tested mucidin-resistant mutants. Red shift of cytochrome b-561 is induced by mucidin in two independent MUC3 mutants. In MUC1 and MUC2 mutants, the red shift is not induced by mucidin, while that promoted by antimycin A and 2-n-heptyl-4-hydroxyquinoline N-oxide are normal. It is concluded that the extra reduction of cytochrome b-565 and the red shift of cytochrome b-561 elicited by mucidin can be largely dissociated from the overall inhibition of the electron flow by distinct mucidin-resistant mutations in different exons of the split mitochondrial gene of cytochrome b.

Anti-Bacterial Agents↗
PubMed1982

Effects of borohydride-treated oligomycins on processes of energy transduction in mitochondria.

Sodium borohydride in ethanol solution under mild conditions brings about the stepwise reduction of the 7-keto and the 11-keto groups of rutamycin and the oligomycins to the corresponding hydroxyl groups without further alterations of the macrocyclic lactone structure or other features of the molecule. The reduced compounds, as well as the parent antibiotics, inhibit the ADP-dependent (state 3) respiration, and the Pi formation and proton extrusion that are linked to ATP hydrolysis, but have no effect on other respiration-linked activities in intact rat liver mitochondria. Analogous inhibitory effects of borohydride-treated antibiotics are also observed in rat-liver submitochondrial particles. The reduced compounds are less potent inhibitors than the parent antibiotics. The reduced compounds are more efficient as inhibitors of Pi formation stimulated by conventional uncouplers (e.g. 2,4-dinitrophenol), than of Pi formation stimulated by certain amine-fluorescamine modifiers (e.g.) the benzylamine-fluorescamine compound. In contrast, the parent antibiotics are unable to discriminate between uncoupler-stimulated and modifier-stimulated Pi formation. It is suggested that rutamycin and the oligomycins bind to H+-ATPase as a result of hydrogen bonding to, at least, the 7-keto and/or the 11-keto groups of the antibiotics. When these keto groups are reduced to hydroxyl groups the hydrogen-bonding is less efficient due to the pronounced directional characteristic of hydrogen-bonding to keto groups.

Adenosine Triphosphatases↗
PubMed1982

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes.

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.

Animals↗
PubMed1982

Reduction of respiratory-chain cytochrome b by lactate in Saccharomyces cerevisiae.

Cytochrome b of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers. 1. The respiration of D-lactate and L-lactate is 30-40% inhibited by antimycin A. 2. Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of D-lactate and L-lactate (half-optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bc1 inhibitors. 3. Reduction of cytochrome b and c1 occurs in purified complex III of yeast in the presence of L-lactate and added L-lactate: NAD+ oxidoreductase. 4. In the particles obtained from yeast grown in lactate the oxidation of L-lactate involves the reduction of a pigment absorbing at 558 nm.

Chemical Phenomena↗
PubMed1982

Effect of thiol reagents on the proton conductivity of the H+-ATPase of mitochondria.

The role of thiol groups in the proton conduction by the H+-ATPase of mitochondria is examined. A detailed kinetic analysis of the effect of arsenite and N-ethylmaleimide on the anaerobic relaxation of the proton gradient set up by respiration in 'inside-out' submitochondrial particles from beef-heart has been carried out. Arsenite, which reacts with vicinal dithiols, is shown to enhance the proton conductivity of the H+-ATPase. This effect is exerted on the F0 moiety of the complex and apparently mimics and is, in fact, favoured by a state of high proton conductivity induced in the complex by the respiratory delta mu H+. N-Ethylmaleimide (MalNEt), which is a permeant monothiol blocking reagent, appears to attack critical -SH groups in a reaction leading to inhibition of the proton conductivity of the H+-ATPase. Also the inhibitory action of MalNEt on proton conduction is exerted on the F0 moiety of the H+-ATPase. Whilst the stimulatory effect of arsenite develops rapidly, the inhibitory action of MalNEt is sluggish and takes more than 10 min to fully develop. This and other kinetic characteristics, as well a partial additivity of the inhibition by MalNEt with that by oligomycin, indicate that the inhibitory action of MalNEt is associated to a substantial conformational transition in F0. Differences in the mechanism of inhibition of proton conduction by MalNEt and triphenyltin are also presented.

Adenosine Triphosphatases↗
PubMed1986

The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate.

The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a Ki = 0.4 mM. Binding of [3H]carboxyatracylate and [3H]bongkrekate is largely inhibited by pyridoxal-P treatment with Ki approximately 1 mM. [3H]Carboxyatractylate is not and [3H]bongkrekate weakly removed by pyridoxal-P, whereas [3H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [3H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [3H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysine-containing peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier.

Animals↗
PubMed1987

Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides.

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein.

Animals↗
PubMed1989

The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification.

1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an antimycin- or myxothiazol-sensitive manner. However, nitric oxide was not detected by the electrode during the reduction of nitrate. Nitric-oxide synthesis from nitrate could be detected with cells in the presence of very low concentrations of Triton X-100 which selectively inhibits nitric-oxide reductase activity. 5. Nitric oxide was detected as an intermediate in denitrification by including haemoglobin with an anaerobic suspension of cells that was reducing nitrate. The characteristic spectrum of the nitric oxide derivative of haemoglobin was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoplasm↗
PubMed1990

Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein.

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.

Amino Acid Sequence↗