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1.
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 N264-40, which has the same morphological and embryological effects as the Notch 8 deletion, demonstrates the same biochemical effects and dosage relations as Notch 8. 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. 相似文献
2.
A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein -1/min -1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa 3 type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase. 相似文献
3.
In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO 2 production and lipid synthesis from [U- 14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO 2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases. 相似文献
4.
The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H +/O ratio. However, in the strain that lacked NDH-2, membrane l-lactate oxidase activity increased, while NDH-2 over-expression led to decreased l-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, l-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane l-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum. 相似文献
6.
NADPH oxidase activity, in addition to NADH oxidase activity, has been shown to be present in the respiratory chain of Corynebacterium glutamicum. In this study, we tried to purify NADPH oxidase and NADH dehydrogenase activities from the membranes of C. glutamicum. Both the enzyme activities were simultaneously purified in the same fraction, and the purified enzyme was shown to be a single polypeptide of 55 kDa. The N-terminal sequence of the enzyme was consistent with the sequence deduced from the NADH dehydrogenase gene of C. glutamicum, which has been sequenced and shown to be a homolog of NADH dehydrogenase II. In addition to high NADH-ubiquinone-1 oxidoreductase activity at neutral pH, the purified enzyme showed relatively high NADPH oxidase and NADPH-ubiquinone-1 oxidoreductase activities at acidic pH. Thus, NADH dehydrogenase of C. glutamicum was shown to be rather unique in having a relatively high reactivity toward NADPH. 相似文献
7.
Only one type (membrane-bound form) of NADH dehydrogenase could be detected in the log-phase cells of Bacillus megaterium. By sonification this enzyme could be effectively solubilized, while NADH oxidase remained bound to the membrane. A molecular weight of about 40 Kd was estimated for the dehydrogenase by gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) with an activity stain. Mercuric chloride and 2- n-heptyl-4-hydroxyquinoline- N-oxide (HQNO) were inhibitors for both the NADH dehydrogenase and oxidase in B. megaterium. The inhibition studies of NADH oxidation suggested that NADH dehydrogenase provided the primary electron source for NADH oxidase in this organism in vitro. NADH dehydrogenase was highly specific for NADH, and K m was estimated to be 28.2 M. The enzyme was subjected to end-product inhibition of a competitive type. 相似文献
8.
The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mol/min·mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mol/min·mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metablolism.This work was supported by the Aquatic Biology Research Unit of the University of Manitoba from a Federal Fisheries Research Grant. 相似文献
9.
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. 相似文献
10.
The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na +, K +-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na +, K +-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na +, K +-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na +, K +-ATPase activity. Tested compounds did not affect Na +, K +-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na +, K +-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na +, K +-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome. 相似文献
11.
This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181T). 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 18181T. 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. 相似文献
12.
We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria
observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes I (NADH dehydrogenase),
II (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling
protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA
content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude
that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency
in enzyme complexes of the mitochondrial respiratory chain. 相似文献
13.
We report a functional and molecular analysis of nine oncocytic tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was accompanied by an increase in enzymatic activities of respiratory complexes 1 (NADH dehydrogenase), 11 (succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes of the mitochondrial respiratory chain. 相似文献
14.
Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence. 相似文献
15.
The energy-dependent and independent transhydrogenase activities and the NADH oxidase of membrane particles of Escherichia coli WS1 were inactivated by phospholipase A from Crotalus terrificus. Ca 2+-activated ATPase was stimulated by this treatment. Although these results suggest that phospholipid is involved in the transhydrogenase systems, trypsin treatment produced similar results. Proteolytic activity was not detected in the phospholipase preparation but its presence could not be ruled out. Membranes containing different unsaturated fatty-acid components were obtained by growing the fatty-acid auxotroph, E. coli K1060, on linoleic, oleic, or elaidic acids. Discontinuities in the Arrhenius plots of the activities of NADH oxidase, Ca 2+-activated ATPase, energy-dependent and independent transhydrogenases, were observed at definite temperatures (transition temperatures). With the exception of NADH oxidase, the transition temperatures could not be correlated with those expected for phase changes in the phospholipids of the membranes. Transition temperatures were also found when a lipid-free, purified ATPase was used. It is concluded that phase changes in the bulk of the phospholipids do not effect transhydrogenase and ATPase activities, and that there is no evidence that the bulk of the phospholipid is involved in the activity of these enzymes. However, we cannot exclude the possibility that a limited amount of lipid in immediate contact with the enzyme protein is essential for its activity. 相似文献
16.
Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q 0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K
m-values for both pyridine nucleotides were in the molar range ( K
m[NADH]=9.8 M, K
m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP + reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP + reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme
Q 0-2,3-dimethoxy-5-methyl-1,4-benzoquinone
- FNR
ferredoxin: NADP + reductase
- MD
menadione
- MV
methylviologen
- NDH
NAD(P)H dehydrogenase
- PQ
plastoquinone
- PQ 10
decylplastoquinone
- SDH
succinate dehydrogenase
- UQ 10
decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) 相似文献
17.
The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis. 相似文献
18.
In spite of previous reports, the activities of respiratory oxygen uptake by whole cells are higher with chemotrophically than with phototrophically grown cells of Rhodospirillum rubrum and Rhodospirillum tenue. The same applies to NADH dependent respiratory reactions as determined with isolated crede membrane preparations. This is largely, but not only, due to an outstandingly high increase in activity of cytochrome c-oxidase measurable upon adaptation of phototrophically grown cells to chemotrophic conditions. In R. rubrum the dependency of the total respiratory chain on the activities of different sections of this chain becomes confused by the presence of differently composed membranes (i.e. cytoplasmic and intracytoplasmic membranes) which under the experimental conditions become functionally differentiated to different extents. But in R. tenue, which does not produce intracytoplasmic membranes, respiration at low activities parallels clearly cytochrome c oxidase activities while high respiratory activities parallel the activities of NADH dehydrogenase. The data are interpreted to indicate that, in cells of facultative phototrophic bacteria, the formation of the respiratory chain, up to certain stages, depends on the formation of the terminal oxidase. At least in R. tenue this is comparable to the role of bacteriochlorophyll in the formation of the photosynthetic apparatus.Abbreviation Bchl
bacteriochlorophyll 相似文献
19.
The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway. 相似文献
20.
Rhodospirillum rubrum CAF10, a spontaneous cytochrome oxidase defective mutant, was isolated from strain S1 and used to analyze the aerobic respiratory system of this bacterium. In spite of its lack of cytochrome oxidase activity, strain CAF10 grew aerobically in the dark although at a decreased rate and with a reduced final yield. Furthermore, aerobically grown mutant cells took up O 2 at high rates and membranes isolated from those cells exhibited levels of NADH and succinate oxidase activities which were similar to those of wild type membranes. It was observed also that whereas in both strains O 2 uptake (intact cells) and NADH and succinate oxidase activities (isolated membranes) were not affected by 0.2 mM KCN, the cytochrome oxidase activity of the wild type strain was inhibited about 90% by 0.2 mM KCN. These data indicate the simultaneous presence of two terminal oxidases in the respiratory system of R. rubrum, a cytochrome oxidase and an alternate oxidase, and suggest that the rate of respiratory electron transfer is not limited at the level of the terminal oxidases. It was also found that the aerobic oxidation of cellular cytochrome c
2 required the presence of a functional cytochrome oxidase activity. Therefore it seems that this electron carrier, which only had been shown to participate in photosynthetic electron transfer, is also a constituent of the respiratory cytochrome oxidase pathway.Abbreviations DCIP
2,6-dichlorophenolindophenol
- DMPD
N,N-dimethyl- p-phenylenediamine
- TMPD
N,N,N,N-tetramethyl- p-phenylenediamine
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]-glycine 相似文献
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