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1.
A phosphatase enzyme hydrolysing NADP+ and NADPH to NAD+ and NADH was found to be present in extracts ofChlorella pyrenoidosa  相似文献   

2.
The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:
  1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
  2. The membrane preparation also catalyzed the reduction of fumarate with H2S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
  3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H2S and fumarate and between NADH and fumarate.
  4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H2S, or NADH, and the oxidation of DMNH2 by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
  5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H2S and NADH oxidation by DMN were not affected by the inhibitor.
It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.  相似文献   

3.
  1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
  2. Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
  3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea 1 and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
  4. 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
  5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
  6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
  7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.
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4.
Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD+ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD+ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD+, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD+-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD+; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.  相似文献   

5.
  1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
  2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a 1 and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
  3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
  4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
  5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
  6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.
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6.
  1. The respiratory chain energy conservation systems of Bacillus megaterium strains D440 and M have been investigated following growth in batch and continuous culture. Respiratory membranes from these strains contained cytochromes b, aa 3 , o and b, c, a, o, respectively; both readily oxidised NADH but neither showed any pyridine nucleotide transhydrogenase activity.
  2. Whole cells of both strains exhibited endogenous →H-/O ratios of approximately 4; when loaded with specific substrates the resultant →H+/O ratios indicated that proton translocating loops 1 and 2 were present in strain D440 and that loops 2 and 3 were present in strain M.
  3. In situ respiratory activities were measured as a function of dilution rate during growth in continuous culture. True molar growth yields with respect to oxygen (Y O 2) of approximately 50 g cells·mole oxygen-1 were obtained for most of the nutrient limitations employed. Average values for Y ATP of 12.7 and 10.8 g cells·mole ATP equivalents-1 were subsequently calculated for strains D440 and M respectively.
  4. Energy requirements for maintenance purposes were low in energy-limited cultures but were substantially increased when growth was limited by nitrogen source (NH 4 + ). Under the latter conditions there is probably a partial uncoupling of energy-conserving and energy-utilising processes leading to energy wastage.
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7.
The experiments presented here were based on the conclusions of our previous results. In order to avoid introduction of expression plasmid and to balance the NADH/NAD ratio, the NADH biosynthetic enzyme, i.e., NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GADPH), was replaced by NADP-dependent GADPH, which was used to biosynthesize NADPH rather than NADH. The results indicated that the NADH/NAD ratio significantly decreased, and glucose consumption and l-lysine production drastically improved. Moreover, increasing the flux through l-lysine biosynthetic pathway and disruption of ilvN and hom, which involve in the branched amino acid and l-methionine biosynthesis, further improved l-lysine production by Corynebacterium glutamicum. Compared to the original strain C. glutamicum Lys5, the l-lysine production and glucose conversion efficiency (α) were enhanced to 81.0 ± 6.59 mM and 36.45 % by the resulting strain C. glutamicum Lys5-8 in shake flask. In addition, the by-products (i.e., l-threonine, l-methionine and l-valine) were significantly decreased as results of genetic modification in homoserine dehydrogenase (HSD) and acetohydroxyacid synthase (AHAS). In fed-batch fermentation, C. glutamicum Lys5-8 began to produce l-lysine at post-exponential growth phase and continuously increased over 36 h to a final titer of 896 ± 33.41 mM. The l-lysine productivity was 2.73 g l?1 h?1 and the α was 47.06 % after 48 h. However, the attenuation of MurE was not beneficial to increase the l-lysine production because of decreasing the cell growth. Based on the above-mentioned results, we get the following conclusions: cofactor NADPH, precursor, the flux through l-lysine biosynthetic pathway and DCW are beneficial to improve l-lysine production in C. glutamicum.  相似文献   

8.
Our previous work revealed proanthocyanidins (PAs) could pose significant enhancement on the activity of H+-ATPase and fermentation efficiency after a transient initial inhibition (Li et al in Am J Enol Vitic 62(4):512–518, 2011). The aim of the present work was to understand the possible mechanism for this regulation. At Day 0.5 the gene expression level of PMA1 in AWRI R2 strain supplemented with 1.0 mg/mL PAs was decreased by around 54 % with a 50 % and a 56.5 % increase in the concentration of intracellular ATP and NADH/NAD+ ratio, respectively, compared to that of control. After the transient adaptation, the gene expression levels of PMA1 and HXT7 in PAs-treated cells were enhanced significantly accompanied by the decrease of ATP contents and NADH/NAD+ ratio, which resulted in the high level of the activities of rate-limiting enzymes. PAs could pose significant effects on the fermentation via glucose transport, the energy and redox homeostasis as well as the activities of rate-limiting enzymes in glycolysis.  相似文献   

9.
  1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
  2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
  3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
  4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
  5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
  6. ATPase activity is apparently unaffected by UFA-depletion, but32Pi-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
  7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.
  相似文献   

10.
In the absence of direct evidence concerning the nature of the early Earth environments, it is acceptable under the uniformitarian principle to attempt to define primitive habitats from modern procaryotic physiology. Combining the rock and fossil record with present phylogenetic reconstuctions, application of this paleoecological approach to the evolutionary biochemistry and physiology of nitrogen fixation and photosynthesis leads to several inferences about the nature of Archean environments:
  1. To stimulate nitrogenase evolution and avoid its repression, the activity of the NH 4 + ion was less than 10?3, and probably lower.
  2. To be consistent with a moderately protective ozone screen, while not also repressing nitrogenase activity, incursions of abiotic dissolved oxygen at levels in the range 10?1.2?10?3.5 PAL would have been acceptable.
  3. To induce the formation and activity of RuBP carboxylase, the pCO2 was less than 100 PAL.
  4. To support Photosystem I activity, sulfide concentrations of at least 10?4 M were present in the photic zone.
  5. To avoid a too-rapid oxidation of sulfide, the pH was probably between 6–7, where H2S exceeds HS?.
Evolutionary ‘pressure’ to stimulate the later development of oxygenic photosynthesis (Photosystem II), would require several subsequent habitat modifications:
  1. Lowering the sulfide to < 10?4 M to inhibit Photosystem I.
  2. Raising the pH above neutral (HS? > H2S), to mediate more rapid oxidation of HS?.
  3. Maintaining either an illumination below 300–400 lux (to avoid photosynthetic O2 self-repression of nitrogen fixation), or an adequate local source of combined nitrogen (aNH 4 + > 10?4) to repress nitrogen fixation entirely.
  相似文献   

11.
The coenzyme-non-specific glutamate dehydrogenase (EC 1.4.1.3) from Scenedesmus acutus in inhibited by p-hydroxymercuribenzoate only in the deamination reaction. From this result and from its stability in the presence of urea it is concluded that this enzyme exhibits and equilibrium between three conformations: aminating and deaminating conformations induced by NADH-2-oxoglutarate and NAD+-glutamate, respectively, and the “native” conformation in the absence of substrates.  相似文献   

12.
  1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration.
  2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO2 assimilation began at this point.
  3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions.
  4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO2 assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.
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13.
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique Na+ pumping respiratory complex found only in prokaryotes, that plays a key role in the metabolism of marine and pathogenic bacteria, including Vibrio cholerae and other human pathogens. Na+-NQR is the main entrance for reducing equivalents into the respiratory chain of these bacteria, catalyzing the oxidation of NADH and the reduction of quinone, the free energy of this redox reaction drives the selective translocation of Na+ across the cell membrane, which energizes key cellular processes. In this review we summarize the unique properties of Na+-NQR in terms of its redox cofactor composition, electron transfer reactions and a possible mechanism of coupling and pumping.  相似文献   

14.
  • 1.1. Purified thyroidal NAD+ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action.
  • 2.2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD+ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine.
  • 3.3. NAD+ and ADP-ribose partially protect against inactivation with similar efficiencies.
  • 4.4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD+ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione.
  • 5.5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains.
  • 6.6. The mechanistic implications of these results are discussed.
  相似文献   

15.
Flow cytometric and light/fluorescence microscopic analysis of murine erythroleukemic cells (MELC) and electron microscopic investigation of porcine microsomal membrane preparations suggest that tributyltin (TBT) toxicity is mediated through fixation processes (protein denaturation, crosslinking, and so on) within the plasma membrane/cytoplasm complex. This hypothesis was derived from the following observations:
  1. Exposure of the MELC to micromolar concentrations of TBT results in increased resistance to detergent-mediated cytolysis;
  2. Exposure of porcine renal microsomal membrane preparations to similar concentrations results in inhibition of vanadate-mediated crystallization of Na+,K+-ATPase, a process requiring protein mobility within the membrane;
  3. Flow cytometric and fluorescence microscopic analyses indicate that MELC exposed to submicromolar concentrations of TBT exhibit increased cellular carboxyfluorescein retention; and
  4. Nuclei prepared from TBT-treated cells by detergent-mediated cytolysis exhibit increased axial light loss, 90° light scatter, fluorescein isothiocyanate fluorescence, and the presence of adherent protein-aceous tags. The DNA distribution histogram of such nuclei also is perturbed.
  相似文献   

16.
The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD+. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K m) of 5.87 μM, a turnover rate (k cat) of 0.24/sec, and a catalytic efficiency (k cat/K m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k cat/K m, 107.75/mM/sec).  相似文献   

17.
  1. Out of 20 exogeneous substrates only ethanol and, to a much lesser extent, lactate and pyruvate were shown to be capable of stimulating the respiration of Acholeplasma laidlawii cells. However, none of these substrates changed the initial rate of active transport of 3-O-methyl-d-glucose (3-O-MG).
  2. From inhibitory analyses and spectroscopic data, it is apparent that the respiratory chain of A. laidlawii has no cytochromes and is probably not responsible for oxidative phosphorylation.
  3. Valinomycin and nigericin stimulated cell respiration only in the presence of K+-ions, while monensin stimulated it in the presence of Na+-ions.
  4. 3-O-MG transport was shown to be sensitive to uncouplers, ATPase inhibitors and arsenate are resistant to a majority of respiratory inhibitors tested. This suggested that there was no relationship between respiration and carbohydrate transport in the A. laidlawii cells. Further evidence was provided by the absence of respiratory stimulation during the transport of non-metabolizing carbohydrates.
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18.
The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD+ and NADPH/NADP+ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.  相似文献   

19.
The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe2+ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H2O2 was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H2O2 was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction  相似文献   

20.
Lysosome-solubilized pig liver NADH-cytochrome b5 reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK 2=0.22 min?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[14C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b5 reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.  相似文献   

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