Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh^? allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids.The following properties of strains carrying the ndh^? allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH.Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh^? strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant.The properties of the group of ndh^? mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.     

Thermostimulation of conidiation and succinic oxidative metabolism of Neurospora crassa

Summary The conidiogenic effect of temperature was investigated for its manifestation on the succinic oxidative system of Neurospora crassa. It was found that the initiation of conidiation was associated with a peak of succinate dehydrogenase activity. The succinate dehydrogenase activity decreased after the peak period of conidial differentiation. The strongest succinate dehydrogenase activity was measured in 37° C cultures.A second wave increase of succinate dehydrogenase was present in femalefertile (25° C) but was absent in the female-sterile (37° and 40° C) cultures.The cytochrome oxidase activity steadily decreased with the aging of the cultures.Malonate (10^-1 m) was found to stimulate conidiation. This stimulation was associated with higher succinate dehydrogenase activity.Riboflavine was found to accumulate with aging and was higher in 37° and 40° C cultures respectively as compared to 25° C cultures.The ultrathin sections of the conidia revealed that increase in conidial size at 37° C was accompanied with the swelling of the mitochondria and prominence of the endoplasmic reticulum. In conidia from 40° C cultures the mitochondria were shrunken and the endoplasmic reticulum broken.Dedicated with gratitude to Prof. Dr. A. Rippel-Baldes.     

Activation of succinate oxidase in the inner membrane of rat liver mitochondria

It is shown that the process of activation of succinate oxidase from inner membranes of the rat liver mitochondria by succinate and malonate is specific for the succinate dehydrogenase component of oxidase. These activation constants are comparable with those found by other authors in activation of succinate dehydrogenase and succinate oxidase from oxaloacetate-preincubated submitochondrial fragments of the bull heart. Probably, the 4-fold activation of succinate oxidase from inner membranes of the liver mitochondria reported in this paper depends on separation of endogenous oxaloacetate from the succinate dehydrogenase component of oxidase.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

Mitochondrial Respiration Associated with Cytoplasmic Male Sterility in Pearl Millet

Respiratory activities in vegetative tissues and the isolated mitochondria of etiolated seedlings and reproductive tissues were studied in two cytoplasmic male sterile (CMS) lines having the same A, cytoplasm with different nuclear backgrounds of pearl millet along with their maintainer, restorer and restored lines. In addition, the assays of cytochrome c oxidase and succinate dehydrogenase were performed in isolated mitochondria. Cyanide-sensitive, cyanide-insensitive and total respirations in vegetative tissues and isolated mitochondria were lower in both the CMS lines than their respective maintainers, restorers and restored hybrids. Except in CMS lines, uptake of 0₂ during anther development increased from premeiotic to postmeiotic stage in all the lines. Consumption of 0₂,however, declined from meiotic to postmeiotic stage in the anthers of CMS lines. Pure mitochondrial preparations were obtained which were 92-94% intact. Enzymes, cytochrome c oxidase (complex IV) and succinate dehydrogenase (complex II) showed lower activities in both the CMS lines than their respective maintainer, restorer and restored hybrids. The CMS lines also displayed lower levels of nuclear encoded enzymes, viz., alternative oxidase and succinate dehydrogenase.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Metabolic differentiation of red and white skeletal muscle in vivo and in monolayer culture

Cytochrome oxidase, succinate oxidase and lactate dehydrogenase were compared in: (a) leg and breast muscle from 11-19-day-old chick embryos; and (b) 2, 6, 10 and 14-day-old primary cell cultures established from myoblasts of embryonic leg and breast muscle. Cytochrome oxidase, succinate oxidase and lactate dehydrogenase activities were higher (48.8, 65.4, 277.6%, respectively) in leg muscle after 19 days in ovo. Cytochrome and succinate oxidase activities were higher (111.3, 48.1%, respectively) in leg muscle cell cultures after 14 days in vitro. The data represent evidence for intrinsic developmental patterns for certain enzymes.     

The effect of phenolic compounds on the activity of respiratory chain enzymes and on respiration and phosphorylation activities of potato tuber mitochondria

The effect of derivatives of benzoic and cinnamic acids, quereetin,p-benzoquinone, and 2,5-dimethylbenzoquinone on oxygen consumption mitoehondrial suspensions and on the activity of some respiratory chain enzymes was studied. Benzoquinone and 2,5-dimethylbenzoquinone highly significantly inhibited the respiration and phosphorylation rates and malate- and succinate dehydrogenase activities. Chlorogenic acid, similarly as the quinones, very significantly inhibited the activities of the studied dehydrogenases but did not affect cytochrome oxidase. Oxygen consumption by intact mitochondria was not inhibited, only the oxidativo phosphorylation was significantly uncoupled. Quereetin significantly enhanced dehydrogenase activities and completely inhibited cytochrome oxidase activity. The respiration and phosphorylation activities of the mitochondria were significantly inhibited by quereetin. The effect of the other phenolic compounds studied on respiration and phosphorylation activities was not significant. Succinate dehydrogenase activity was the most affected enzyme among the respiratory chain enzymes. It was significantly inhibited by all the above phenolic compounds at 1^-4M or 5 10^-5M concentrations with the exception of gallic acid.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

The Action of the Notch Locus in DROSOPHILA MELANOGASTER. II. Biochemical Effects of Recessive Lethals on Mitochondrial Enzymes

We show that six mapped recessive lethal point mutations of the Notch locus affect mitochondrial enzyme activities: NADH oxidase, NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. The mutant N^264-40, which has the same morphological and embryological effects as the Notch⁸ deletion, demonstrates the same biochemical effects and dosage relations as Notch⁸. The other five mapped recessive lethals also affect four enzymic activities. They show specific patterns of activity that depend in several cases on the wild-type chromosome in the heterozygous females. That effect occurs with mutants located in the extreme right part of the Notch locus where some mutations, according to other authors, show temperature-sensitive expression.     

Oxidative and phosphorylative activity of mitochondria from pea cotyledons during maturation of the seed

Summary The respiration rate and the activity of some mitochondrial enzymes from pea cotyledons have been followed during the final phases of seed development, when the relative water content of the cotyledons dropped from 65 to 13%. Succinate, malate and -ketoglutarate oxidase activity, and succinate and malate dehydrogenase activity per cotyledon increased when the relative water content dropped from 65 to about 55%. A further drop of the relative water content was accompanied by a strong decrease of the activity of the succinate and malate oxidase system, but only a slight decrease of succinate and malate dehydrogenase activity. Mitochondrial fractions from air-dry, mature cotyledons showed a low activity of the succinate and malate oxidase system but their dehydrogenase activity was relatively high. The phosphorylation efficiency and respiratory control gradually decreased during maturation. These results indicate that during maturation of the pea seed certain mitochondrial enzymes partly lose their activity.     

Regulation of succinate oxidation by NAD+ in mitochondria purified from potato tubers

NAD⁺ supplied to purified Solanum tuberosum mitochondria caused progressive inhibition of succinate oxidation in State 3. This inhibition was especially pronounced at alkaline pH and at low succinate concentrations. Glutamate counteracted the inhibition. NAD⁺ promoted oxaloacetate accumulation in State 3; supplied oxaloacetate inhibited O₂ uptake in the presence of succinate much more severely in State 3 than in State 4. NAD reduction linked to succinate oxidation by ATP-dependent reverse electron transport was likewise inhibited by oxaloacetate. We conclude that NAD⁺-induced inhibition of succinate oxidation is due to an inhibition of succinate dehydrogenase resulting from increased accumulation of oxaloacetate generated from malate oxidation via malate dehydrogenase. The results are discussed in the context of the known regulatory characteristics of plant succinate dehydrogenase.     

Maternal Hypermethioninemia Affects Neurons Number,Neurotrophins Levels,Energy Metabolism,and Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Expression/Content in Brain of Rat Offspring

In the current study, we verified the effects of maternal hypermethioninemia on the number of neurons, apoptosis, nerve growth factor, and brain-derived neurotrophic factor levels, energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase), expression and immunocontent of Na⁺,K⁺-ATPase, edema formation, inflammatory markers (tumor necrosis factor-alpha and interleukin-6), and mitochondrial hydrogen peroxide levels in the encephalon from the offspring. Pregnant Wistar rats were divided into two groups: the first one received saline (control) and the second group received 2.68 μmol methionine/g body weight by subcutaneous injections twice a day during gestation (approximately 21 days). After parturition, pups were killed at the 21st day of life for removal of encephalon. Neuronal staining (anti-NeuN) revealed a reduction in number of neurons, which was associated to decreased nerve growth factor and brain-derived neurotrophic factor levels. Maternal hypermethioninemia also reduced succinate dehydrogenase and complex II activities and increased expression and immunocontent of Na⁺,K⁺-ATPase alpha subunits. These results indicate that maternal hypermethioninemia may be a predisposing factor for damage to the brain during the intrauterine life.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Repression of oxalic acid-mediated mineral phosphate solubilization in rhizospheric isolates of Klebsiella pneumoniae by succinate

Two strains of Klebsiella (SM6 and SM11) were isolated from rhizospheric soil that solubilized mineral phosphate by secretion of oxalic acid from glucose. Activities of enzymes for periplasmic glucose oxidation (glucose dehydrogenase) and glyoxylate shunt (isocitrate lyase and glyoxylate oxidase) responsible for oxalic acid production were estimated. In presence of succinate, phosphate solubilization was completely inhibited, and the enzymes glucose dehydrogenase and glyoxylate oxidase were repressed. Significant activity of isocitrate lyase, the key enzyme for carbon flux through glyoxylate shunt and oxalic acid production during growth on glucose suggested that it could be inducible in nature, and its inhibition by succinate appeared to be similar to catabolite repression.     

Inhibition of mitochondrial function in isolated rat liver mitochondria by azole antifungals

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related drug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I₅₀ was 32, 300, and 110 μM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I₅₀ of 350 μM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I₅₀ of 74 μM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action. © 1997 John Wiley & Sons, Inc.     

Is the ubiquinone pool in the respiratory chain of the bacterium Paracoccus denitrificans really unhomogeneous?

We have established the participation of a mobile redox pool in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans. In testing the kinetical homogeneity of the pool it was found that the ratio of fluxes of electron transport toward the terminal acceptors oxygen and nitrate was coincident for the respiratory substrates NADH and succinate; this provides evidence against the preferential link of one dehydrogenase with a distinct terminal enzyme through the separate pool of ubiquinone. The deviation from the expected behavior observed in comparing the titration of NADH oxidase and succinate oxidase with respiratory inhibitors such as mucidin (inhibitor in the bc₁ region) or cyanide can be accounted for by the activation of succinate dehydrogenase upon the increase in the reduced state of respiratory components during the titration.     

Decreased Succinate Dehydrogenase Activity of Gamma and Alpha Motoneurons in Mouse Spinal Cords Following 13 Weeks of Exposure to Microgravity

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm²) gamma and medium-sized (300–700 μm²) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm²) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.     

NADPH Oxidase System as a Superoxide-generating Cyanide-Resistant Pathway in the Respiratory Chain of Corynebacterium glutamicum

The respiratory chain of Corynebacterium glutamicum was investigated, especially with respect to a cyanide-resistant respiratory chain bypass oxidase. The membranes of C. glutamicum had NADH, succinate, lactate, and NADPH oxidase activities, and menaquinone, and cytochromes a ₅₉₈, b _562(558), and c ₅₅₀ as respiratory components. The NADH, succinate, lactate, and NADPH oxidase systems, all of which were more cyanide-resistant than N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity (cytochrome aa ₃ terminal oxidase), had different sensitivities to cyanide; the cyanide sensitivity of these oxidase systems increased in the order, NADPH, lactate, NADH, and succinate. Taken together with the analysis of redox kinetics in the cytochromes and the effects of respiratory inhibitors, the results suggested that there is a cyanide-resistant bypass oxidase branching at the menaquinone site, besides cyanide-sensitive cytochrome oxidase in the respiratory chain. H⁺/O measurements with resting cells suggested that the cyanide-sensitive respiratory chain has two or three coupling sites, of which one is in NADH dehydrogenase and the others between menaquinone and cytochrome oxidase, but the cyanide-resistant bypass oxidase may not have any proton coupling site. NADPH and lactate oxidase systems were more resistant to UV irradiation than other systems and the UV insensitivity was highest in the NADPH oxidase system, suggesting that a specific quinone resistant to UV or no such a quinone works in at least NADPH oxidase system while the UV-sensitive menaquinone pool does in other oxidase systems. Furthermore, superoxide was generated in well-washed membranes, most strongly in the NADPH oxidase system. Thus, it was suggested that the cyanide-resistant bypass oxidase system of C. glutamicum is related to the NADPH oxidase system, which may be involved in generation of superoxide anions and probably functions together with superoxide dismutase and catalase.