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1.
A versatile time-sharing multichannel spectrophotometer, reflectometer, and fluorometer. 总被引:15,自引:0,他引:15
A simple turbine-driven multi-wavelength time-sharing apparatus affords flexibility and versatility in optical measurements and serves as a fluorometer, reflectometer, and spectrophotometer for four wavelength channels. As a reflectance fluorometer, nearly simultaneous readout of tissue reflectance and NADH or flavoprotein fluorescence can be obtained. As a transmission spectrophotometer and reflectometer, hemoproteins such as myoglobin, catalase, and cytochrome a + a3 with its copper component may be measured. This instrument offers the advantages of high speed of time-sharing, and simplicity, compactness, and flexibility not found in previous designs. In addition, it causes minimal acoustic and electrical disturbance to the experimental system. 相似文献
2.
《Journal of microbiological methods》1987,6(4):185-190
A fluorescamine assay for the detection of a spore-lytic enzyme from Clostridium perfringens is described. The substrate is prepared by treatment of cortical fragments with fluorescamine which reacts with amino terminal groups in the peptidoglycan which are not cross-linked, presumably diaminopimelic acid. Treatment of the labelled substrate with lytic enzymes results in the release of soluble fluorescent products which can be easily measured in a basic fluorometer. The assay is very sensitive, inexpensive and reproducible. As little as 1 μg of lysozyme can be detected by this assay. 相似文献
3.
Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r). 相似文献
4.
In the present study we provide the evidence of different respiration rates and peroxidase activities in maize (Zea mays L.) mitochondria isolated from germinated seeds and roots of 2-week-old seedlings. The negative relationships between mitochondrial
respiration rate measured with NADH as substrate and activities of peroxidases that oxidized NADH in both oxidative and peroxidative
cycles were found. The possible role of peroxidase in the regulation of reactive oxygen species metabolism in expense of NADH
oxidation was hypothesized. 相似文献
5.
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate
(Pi). Apparent K
m values for ADP and Pi were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly:
K
m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient
by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of
NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent
K
m values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously
for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006)
Biochemistry, 45, 14552–14558). 相似文献
6.
Maryam Zarepour Katrin Kaspari Stefan Stagge Ralf Rethmeier Ralf R. Mendel Florian Bittner 《Plant molecular biology》2010,72(3):301-310
Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released
from these substrates are either transferred to NAD+ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable
of oxidizing NADH with concomitant formation of NAD+ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific
activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production.
The observation that NAD+ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD+-dependent xanthine oxidation indicates that both NAD+ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each
sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation
of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase
proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer
of superoxide also in vivo. 相似文献
7.
Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H2O2 applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O2 to 0%, 5%, 21%, or 40% O2, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O2 exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H2O2, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO2, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O2-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat 相似文献
8.
Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria 总被引:2,自引:0,他引:2
Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O2 min?1mg?1, which was reduced to 98 nmol O2 min?1 (mg mitochondrial protein)?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O2 min?1mg?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent Km of 7 μM for NADH, a Vmax of 80 nmol NADH min?1mg?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a Km of 80 μM and a Vmax of 131 nmol NADH min?1mg?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria. 相似文献
9.
Jin Hou Goutham N. Vemuri Xiaoming Bao Lisbeth Olsson 《Applied microbiology and biotechnology》2009,82(5):909-919
During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A
deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation.
However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor
imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the
native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains
was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic
NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol
to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall,
although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of
NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated. 相似文献
10.
Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commerical NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.Abbreviations EDTA =
ethylenediaminetetracetate
- HQNO =
2–n-heptyl-4-hydroxy-quinoline-N-oxide
- DEAE =
diethylaminoethyl 相似文献
11.
This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives
examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation
of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients
can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus.
These are the parameters Fv, Fm, F0, Fm′, Fv′, NF, and ΔF, the Chl fluorescence ratios Fv/Fm, Fv/F0, ΔF/Fm′, as well as the photochemical (qP) and non-photochemical quenching coefficients (qN, qCN, and NPQ). qN consists of three components (qN = qE + qT + qI), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence
parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the
functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper
adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma
cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information
on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly
reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence
signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, qN, qCN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. qN was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas qCN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended
to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio RFd (Fd/Fs), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (P
N) of leaves. This RFd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating
light (cSL) during 4–5 min. As the RFd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be
determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios. 相似文献
12.
The genes that encode oxygen-insensitive nitroreductases from Clostridium
acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products
NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases
comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins.
The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern
similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference
for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases
were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with
TNT, nitrofurazone and nitrofurantoin compared to the uninduced control. 相似文献
13.
The P/O ratio of Bdellovibrio bacteriovorus, strain Bd 109 Sa, was evaluated by two different methods based on the determination of energy-rich phosphate bonds and either NADH oxidation or oxygen-uptake. P/O values calculated on the basis of NADH oxidation were up to 6, which has to be regarded as being overestimated. P/O values calculated from energy-rich phosphate bonds and oxygen uptake were around 2. The P/O values determined for Escherichia coli B were similar. The loss of phosphorylation efficiency at one site is discussed.The ATP pool turnover rate of Bdellovibrio was 8/min during endogenous respiration and 24/min during substrate respiration. The corresponding values in Escherichia coli B were 3/min and 38/min.This study was performed at the University of Hamburg (Institut für Allgemeine Botanik, Abteilung Mikrobiologic). 相似文献
14.
Tomoko Hamada Takayoshi Wakagi Hirotaka Shiba N. Koyama 《Archives of microbiology》1999,171(4):237-242
A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite.
The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme
catalyzed the oxidation of NADH with the formation of H2O2 and exhibited a specific activity of 46 μmol NADH min–1 (mg protein)–1. NADPH did not serve as a substrate for the enzyme. The K
m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately
9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl.
Received: 3 August 1998 / Accepted: 23 December 1998 相似文献
15.
A.C. Lee X. Xu E. Blachly-Dyson M. Forte M. Colombini 《The Journal of membrane biology》1998,161(2):173-181
In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer
membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the
POR1 gene (Δpor1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase
to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in
the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only
a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that
the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due
to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1
in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability.
The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon
sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria.
Received: 23 May 1997/Revised: 3 October 1997 相似文献
16.
Pierre Germain Fatiou Toukourou Luiz Donaduzzi 《Applied microbiology and biotechnology》1986,24(4):300-305
Summary The enzyme lactate dehydrogenase (LDH) in Clostridium thermohydrosulfuricum is controlled by the type and the concentration of the substrate. In batch fermentations an increase of the initial concentration of glucose leads to an increase in the activity of LDH. This increase in activity is related to the accumulation of fructose 1,6-diphosphate (F 1,6-DP), an intermediate of the Embden-Meyerhof-Parnas (EMP) pathway, which stimulates the enzyme by increasing its affinity for pyruvate and NADH. The K
mvalues of LDH for pyruvate and NADH, which are 2.5×10-3 M and 9.1×10-5 M respectively in absence of F 1,6-DP, fall considerably in the presence of this substrate. In presence of 0.2 mM of F 1,6-DP we observed a K
mof 3.3×10-4 M for pyruvate and 4.1×10-5 M for NADH. 相似文献
17.
Membrane preparations, capable of high rates of respiration-linked ATP synthesis, have been obtained from a gram-positive methylotrophic bacterium Bacillus sp. MGA3. NADH, succinate, reduced TMPD and methanol were shown to be suitable substrates for the oxidative phosphorylation. Esterification of orthophosphate was dependent on electron transfer, as evidenced by the requirement for both substrate and oxygen. Phosphorylation was also dependent on ADP and was destroyed by boiling the membrane preparation. The phosphorylation was markedly uncoupled by carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone (CCCP) and was inhibited by N,N-dicyclohexylcarbodiimide (DCCD). KCN caused strong inhibition of substrate oxidation as well as phosphorylation for all substrates tested. Rotenone, amytal and antimycin A caused inhibition when NADH or methanol were used as substrates. Antimycin A inhibited respiration and ATP synthesis with succinate as substrate and had no effect on ascorbate —N,N,N,N-tetramethyl-p-phenylenediimide (TMPD) oxidation by membrane preparations of Bacillus sp. MGA3. P/O ratios determined were 2.4 with NADH, 1.7 with succinate and 0.8 with reduced TMPD. The measured P/O ratio with methanol-oxidizing system was similar to that with NADH (about 2.4).Abbreviations CCCP
Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone
- DCCD
N,N-dicyclohexylcarbodiimide
- TMPD
N,N,N,N-tetramethyl-p-phenylenediimide
- Q
ubiquinone Q 相似文献
18.
Glenwyn D. Kemp F. Mark Dickinson Colin Ratledge 《Applied microbiology and biotechnology》1988,29(4):370-374
Summary The oxidation of primary aliphatic alcohols by microsomal membrane fractions of alkane grown Candida tropicalis was shown to be due to the action of an inducible alcohol oxidase with a wide substrate specificity towards aliphatic alcohols. Stoichiometric studies showed that NADH production, in the presence of fatty alcohols, was due to the activity of an inducible fatty aldehyde dehydrogenase. The oxidase activity could be measured directly by hydrogen peroxide production via a peroxidase and a chromogenic redox indicator. 相似文献
19.
Type‐II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction
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Filipa V. Sena Ana P. Batista Teresa Catarino José A. Brito Margarida Archer Martin Viertler Tobias Madl Eurico J. Cabrita Manuela M. Pereira 《Molecular microbiology》2015,98(2):272-288
A prerequisite for any rational drug design strategy is understanding the mode of protein–ligand interaction. This motivated us to explore protein–substrate interaction in Type‐II NADH:quinone oxidoreductase (NDH‐2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs‐2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH‐2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge‐transfer complex formed between NAD+ and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half‐reaction affected by the presence of HQNO, an inhibitor. We analyzed protein–substrate interactions by fluorescence and STD‐NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD+‐protein complex, which is released by the quinone. 相似文献
20.
Yasuo Nakayama Seiichi Nonomura Chuji Tatsumi 《Bioscience, biotechnology, and biochemistry》2013,77(6):1423-1437
The metabolism of coumarin by a strain of Pseudomonas isolated from soil which utilizes coumarin as a sole carbon source was studied. The metabolic pathway was shown to be coumarin→dihydrocoumarin→melilotic acid→2,3-dihydroxyphenylpropionic acid, based on the results of (1) isolation and identification of metabolic products, (2) survey on the utilization of the postulated intermeidates and (3) examination of enzymatic reaction. An alternative pathway involving o-coumaric acid and 2,3-dihydroxycinnamic acid as intermediates at the metabolism of coumarin was also discussed.Coumarin reducing enzyme (dihydrocoumarin : NAD[NADP] oxydo-reductase) which catalizes the reduction of coumarin to dihydrocoumarin was partially purified from the extracts of the above strain of Pseudomonas and some properties of the enzyme were investigated. The optimum pH of the reaction was 5.25. The enzyme is highly specific with respect to coumarin, and Km values for coumarin and NADH were 6.6 × 10?6 m and 4.1 × 10?5 m, respectively. The enzyme activity was extremely sensitive to sulfhydryl reagents particularly to p-chloromercuribenzoate. 2-Mercaptoethanol or dithiothreitol protected the enzyme from inactivation by low temperature storage. The molecular weight of enzyme was estimated to be about 140,000 by gel permiation Chromatographic method. The enzyme showed a substrate inhibition at higher concentrations of coumarin. This inhibition was noncompetitive with respect to NADH. The enzyme was also inhibited by many coumarin analogues. 3-Hydroxycoumarin showed noncompetitive inhibition with both coumarin and NADH. The mechanism of inhibition for the enzyme is discussed. It is concluded that enzyme protein contains zinc atom and that NADH is attached to zinc in the enzyme reaction. 相似文献