Exogenous NADPH Research Articles

NADPH-Induced Contractions of Mouse Aorta Do Not Involve NADPH Oxidase: A Role for P2X Receptors

Reactive oxygen species elicit vascular effects ranging from acute dilatation because of hydrogen peroxide-mediated opening of K(+) channels to contraction arising from superoxide-dependent inactivation of endothelium-derived nitric oxide. Given that NADPH oxidases are major sources of superoxide in the vascular wall, this study examined the effects of exogenous NADPH, a substrate of these enzymes, on superoxide generation and isometric tone in mouse isolated aortic rings. NADPH caused concentration-dependent increases in superoxide generation (measured by lucigenin-enhanced chemiluminescence) and vascular tone (isometric tension recordings). However, surprisingly, whereas oxidized NADP(+) was unable to support superoxide production, it was equally as effective as reduced NADPH at stimulating vasocontraction. In addition, an NADPH oxidase inhibitor, diphenyleneiodonium, markedly attenuated NADPH-induced superoxide production, yet had no effect on vasocontractions to NADPH. In contrast, a broad specificity P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, as well as the P2X1 selective antagonist, NF023, markedly attenuated both endothelium-dependent and -independent vasocontractions to NADPH, as did the P2X-desensitizing agent alpha,beta-methylene-ATP. Importantly, alpha,beta-methylene-ATP had no effect on superoxide production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived superoxide in the contractile effects of NADPH in the mouse aorta. Rather, NADPH seems to act as an agonist at two distinct P2X receptor populations; one located on the endothelium and the other on smooth muscle layer, both of which ultimately lead to contraction.

Exogenous NADPH increases cerebral blood flow through NADPH oxidase-dependent and -independent mechanisms.

NADPH, a substrate for the superoxide-producing enzyme NADPH oxidase, produces vasodilation in the cerebral circulation. However, the mechanisms of the effect have not been fully elucidated. We used a peptide inhibitor of NADPH oxidase (gp91ds-tat) and null mice lacking the gp91phox subunit of NADPH oxidase to examine the mechanisms of the cerebrovascular effects of exogenous NADPH. Cerebral blood flow (CBF) was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. Superfusion with NADPH increased CBF (27% at 100 micromol/L) without affecting the EEG. The CBF increase was attenuated by the free-radical scavenger MnTBAP (-54%, P<0.05) but not by the H2O2 scavenger catalase. The response was also attenuated by gp91ds-tat (-64%, P<0.05) and by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (-44%, P<0.05). The increase in CBF produced by NADPH was attenuated in gp91-null mice (-41%, P<0.05). NADPH increased production of reactive oxygen species, assessed by hydroethidine microfluorography, an effect blocked by MnTBAP or gp91ds-tat and not observed in gp91-null mice. These data suggest that the mechanisms of the CBF increases produced by exogenous NADPH are multifactorial and include NADPH oxidase-dependent and -independent factors.

Role of Hydrogen Peroxide in Sperm Capacitation and Acrosome Reaction1

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

Effect of exogenous β-nicotinamide adenine dinucleotide phosphate reduced (NADPH) on Sperm DNA integrity

Objective: Human spermatozoa have been shown to generate reactive oxygen species (ROS) via oxidation of endogenous NADPH that is involved in sperm capacitation. However, excessive ROS generation is also known to cause sperm DNA damage. The objective of our study was to detect the ability of mature and immature spermatozoa to generate ROS in response to exogenous NADPH and to measure the extent of the subsequent DNA damage.Design: Prospective-controlled study.Materials and Methods: Semen samples were collected from five donors with normal standard semen parameters. Samples were divided into mature and immature fractions using Isolate double density gradient (90%, 47%). Each fraction was further subdivided into 2 subsets; the first was divided into 3 aliquots and incubated with 5 mM NADPH for 0, 3 and 24 hours. The second subset was similarly divided and incubated without NADPH to serve as control. Sperm DNA damage and ROS levels were assessed using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry and chemiluminescence assay (x 106 counted photons per minute) using lucigenin as a probe.Results: The median and interquartile values (25thand 75th percentiles) of DNA damage are shown in the table. Significantly higher DNA damage (P <0.05) was seen in both NADPH treated sample fractions compared to controls only after 24 hours incubation. Levels of ROS in the mature fraction (90%) were significantly higher than controls (16.4 ± 9.6 vs. 2.36 ± 3.8, P = 0.032) only after 24 hours of incubation, while in the immature fraction (47%) ROS levels were significantly higher at both 3 (1.45 ± 1.15 vs. 0.4 ± 0.3, P = 0.032) and 24 hours (18.38 ± 13.46 vs. 3.2 ± 6.03, P = 0.032). DNA fragmented sperm showed a positive correlation with ROS levels (r = 0.54, P = 0.002) in samples incubated with NADPH for 24 hours. Tabled 1∗P value <0.05 considered significant in samples compared to controls. †P value <0.05 considered significant after incubation for 24 hours compared to 0 and 3 hours.Conclusions: Our results indicate that NADPH may play a role in sperm ROS production and DNA fragmentation. ROS generation is triggered earlier in the immature fraction but DNA damage takes longer time to develop.Supported by: Funds from The Cleveland Clinic Foundation. Objective: Human spermatozoa have been shown to generate reactive oxygen species (ROS) via oxidation of endogenous NADPH that is involved in sperm capacitation. However, excessive ROS generation is also known to cause sperm DNA damage. The objective of our study was to detect the ability of mature and immature spermatozoa to generate ROS in response to exogenous NADPH and to measure the extent of the subsequent DNA damage. Design: Prospective-controlled study. Materials and Methods: Semen samples were collected from five donors with normal standard semen parameters. Samples were divided into mature and immature fractions using Isolate double density gradient (90%, 47%). Each fraction was further subdivided into 2 subsets; the first was divided into 3 aliquots and incubated with 5 mM NADPH for 0, 3 and 24 hours. The second subset was similarly divided and incubated without NADPH to serve as control. Sperm DNA damage and ROS levels were assessed using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupled flow cytometry and chemiluminescence assay (x 106 counted photons per minute) using lucigenin as a probe. Results: The median and interquartile values (25thand 75th percentiles) of DNA damage are shown in the table. Significantly higher DNA damage (P <0.05) was seen in both NADPH treated sample fractions compared to controls only after 24 hours incubation. Levels of ROS in the mature fraction (90%) were significantly higher than controls (16.4 ± 9.6 vs. 2.36 ± 3.8, P = 0.032) only after 24 hours of incubation, while in the immature fraction (47%) ROS levels were significantly higher at both 3 (1.45 ± 1.15 vs. 0.4 ± 0.3, P = 0.032) and 24 hours (18.38 ± 13.46 vs. 3.2 ± 6.03, P = 0.032). DNA fragmented sperm showed a positive correlation with ROS levels (r = 0.54, P = 0.002) in samples incubated with NADPH for 24 hours. Tabled 1 ∗P value <0.05 considered significant in samples compared to controls. †P value <0.05 considered significant after incubation for 24 hours compared to 0 and 3 hours. Conclusions: Our results indicate that NADPH may play a role in sperm ROS production and DNA fragmentation. ROS generation is triggered earlier in the immature fraction but DNA damage takes longer time to develop. Supported by: Funds from The Cleveland Clinic Foundation.

Kinetics of NADPH-Induced Non-Photochemical Reduction of the Plastoquinone Pool in Spinach Chloroplasts

Kinetics of non-photochemical reduction of the photosynthetic intersystem electron transport chain by exogenous NADPH was examined in osmotically lysed spinach chloroplasts by chlorophyll (Chl) fluorescence measurements under anaerobic condition. Upon the addition of NADPH, the apparent F0 increased sigmoidally, and the value of the maximal slope was calculated to give the reduction rate of plastoquinone (PQ) pool. Application of 5 µM antimycin A lowered significantly both the ceiling and the rate of the NADPH-induced Chl fluorescence increase, while the suppressive effect of 10 µM rotenone was slighter. This indicated that dark reduction of the PQ pool by NADPH in spinach chloroplasts under O2-limitation condition could be attributed mainly to the pathway catalysed sequentially by ferredoxin-NADP+ oxidoreductase (FNR) and ferredoxin-plastoquinone reductase (FQR), rather than that mediated by NAD(P)H dehydro- genase (NDH).

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts.

Chlorophyll a fluorescence rise kinetics (from 50 mus to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O(2)-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts.

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat.

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.

Role of NADPH:cytochrome P450 reductase in the hypoxic accumulation and metabolism of BRU59-21, a technetium-99m-nitroimidazole for imaging tumor hypoxia

Nitroimidazoles labeled with technetium-99m are being investigated as non-invasive markers of tumor hypoxia. They are bioreductive compounds that require enzymatic reduction for retention in hypoxic cells, but little is known about the cellular factors affecting their accumulation in hypoxic cells. If the absolute accumulation of hypoxia markers is affected by enzyme levels, an inaccurate assessment of the hypoxic cell fraction in tumors may occur. BRU59-21, 99mTc-oxo[[3,3,9,9-tetramethyl-6-[(2-nitro-1H-imidazol-1-yl)methyl]5-oxa-4,8-diazadioximato]-(3-)- N, N′, N″, N‴] technetium (V), a technetium-99m-nitroimidazole that is being studied as a potential marker of tumor hypoxia, was used in the present study to evaluate the effect of NADPH:cytochrome P450 reductase (EC 1.6.2.4) levels on BRU59-21 accumulation and metabolism. Metabolism of BRU59-21 in hypoxic cellular lysates derived from Chinese hamster ovary cells overexpressing NADPH:cytochrome P450 reductase was 8-fold greater than in control cells. This effect required the presence of exogenous NADPH. The increased metabolism of BRU59-21 in lysates overexpressing NADPH:cytochrome P450 reductase was inhibited at 4° and by the addition of NADPH:cytochrome P450 reductase inhibitors. The addition of inhibitors of other nitroreductase enzymes had no effect on BRU59-21 metabolism in these lysates. When the accumulation and metabolism of BRU59-21 were studied in stirred suspension cultures, it was found that cells overexpressing NADPH:cytochrome P450 reductase exhibited about a 3-fold increase in both the hypoxic metabolism and the accumulation of BRU59-21. These findings suggest that NADPH:cytochrome P450 reductase is an important enzyme in BRU59-21 metabolism in model systems of tumor hypoxia.

Channeling efficiency in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain: the effects of site-directed mutagenesis of NADP binding residues

The three-dimensional structure of the dehydrogenase-cyclohydrolase bifunctional domain of the human trifunctional enzyme indicates that Arg-173 and Ser-197 are within 3 Å of the 2′-phosphate of bound NADP. Site-directed mutagenesis confirms that Arg-173 is essential for efficient binding and cannot be substituted by lysine. R173A and R173K have detectable dehydrogenase activity, but the K m values for NADP are increased by at least 500-fold. The S197A mutant has a K m for NADP that is only 20-fold higher than wild-type, indicating that it plays a supporting role. Forward and reverse cyclohydrolase activities of all the mutants were unchanged, except that the reverse cyclohydrolase activity of mutants that bind NADP poorly, or lack Ser-197, cannot be stimulated by 2′,5′-ADP. The 50% channeling efficiency in the forward direction is not improved by the addition of exogenous NADPH and cannot be explained by premature dissociation of the dinucleotide from the ternary complex. As well, channeling is unaffected in mutants that exhibit a wide range of dinucleotide binding. Given that dinucleotide binding is unrelated to substrate channeling efficiency in the D/C domain, we propose that the difference in forward and reverse channeling efficiencies can be explained solely by the movement of the methenylH 4folate between two overlapping subsites to which it has different binding affinities.

NADPH Oxidase-like Activity in Hemocytes of the Pacific Oyster Crassostrea gigas.

To test whether NADPH oxidase-like activity in hemocytes of the Pacific oyster, Crassostrea gigas, occurs, we investigated molecular oxygen (O2) consumption and superoxide anion (O2-) generation during the respiratory burst. When oyster hemocytes were stimulated with phorbol myristate acetate (PMA), a rapid increase in O2 consumption was recorded accompanying a cyanide-independent respiratory burst using a Clark-type oxygen electrode. The O2 consumption was almost completely inhibited by the addition of 2μM diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. Oyster hemocytes, on stimulation with PMA, exhibited a relatively strong O2--dependent chemiluminescent (CL) response at the peak photocount of 4.67×105CPM/2 × 106hemocytes. The CL response induced by PMA-stimulated hemocytes was markedly reduced by 88% in the presence of 2μM DPI. Furthermore, O2-generation was measured by the reduction of acetylated ferricytochrome c. When the primary O2-generation by PMA-stimulated hemocytes was terminated by detergent mediated cell lysis, and reconstituted by the addition of exogenous NADPH, O2-generation in the hemocyte lysate was restored. Findings obtained in our experiments suggest that an NADPH oxidase-like activity, which is associated with a specific O2--forming system similar to that in mammalian phagocytes, exists in oyster hemocytes.

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross‐correlation Phase Fluorometry

AbstractThe fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple‐exponential model at 14°C and a double‐exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady‐state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide‐F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide‐F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide‐F688 (the Chlide‐NADPH: protochlorophyllide oxidoreductase [PORJ‐NADP+complex) and an additional 1.6 ns component when the formation of Chlide‐F696 (the Chlide‐POR‐NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide‐F696 as being 70%.

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H2O2 production by roots of young seedlings was monitored using a non‐destructive in vivo assay at pH 5.0. A particularly high rate of H2O2 production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H2O2 production. In the soybean root H2O2 production can be stimulated 10‐fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase‐catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O2 to H2O2. Comparative measurements showed that the NADH‐induced H2O2 production of the roots resembles the H2O2‐forming activity of horseradish peroxidase with respect to NADH and O2 concentration requirements and sensitivity to inhibition by KCN, NaN3, superoxide dismutase and catalase. NADH‐induced H2O2 production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H2O2 production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH‐induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O2. It is concluded that the basal H2O2 production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O2−‐producing oxidase.

Microassay for rapid measurement of 7-ethoxyresorufin- O-deethylase activity in intact fish hepatocytes

In vitro assays using isolated fish cells are increasingly used to measure the induction of cytochrome P4501A (CYP1A) and its associated enzyme activity, 7-ethoxyresorufin- O-deethylase (EROD). Compared to permanent cell lines, primary hepatocyte cultures represent an in vitro system that is closer to the in vivo situation, and, therefore, may be a particularly suitable model to study physiological and xenobiotic regulation of CYP1A expression. The application of conventional EROD assays to primary hepatocyte cultures, however, suffers from some disadvantages such as the labour-intensity and the need for high cell numbers per sample. In this report, we present an approach for assessment of EROD activity in teleost hepatocytes cultured in 48-well plates without the need for preparation of microsomes. The assay is based on the method of (Kennedy, S. W., Lorenzen, A., James, C. A. and Collins, B. T. (1993) Anal. Biochem. 211, 102–112.) but employs live instead of dead cells. For EROD detection, 7-ethoxyresorufin is added directly to the live cells and its metabolism to resorufin is observed by repeated measurements over 20 min in a fluorescence plate reader. The assay does not require the addition of exogenous NADPH. Resorufin utilization through cytoplasmic DT-diaphorase has to be inhibited by addition of dicoumarol. Disturbance by conjugate formation accounts to not more than 20% of total resorufin formed. After measurement, the cells can be washed and, after continued incubation, can be used for further assays.

Biochemical responses to environmental contaminants in chub ( Leuciscus cephalus) caught in the morava river

In vitro assays using isolated fish cells are increasingly used to measure the induction of cytochrome P4501A (CYP1A) and its associated enzyme activity, 7-ethoxyresorufin-O-deethylase (EROD). Compared to permanent cell lines, primary hepatocyte cultures represent an in vitro system that is closer to the in vivo situation, and, therefore, may be a particularly suitable model to study physiological and xenobiotic regulation of CYP1A expression. The application of conventional EROD assays to primary hepatocyte cultures, however, suffers from some disadvantages such as the labour-intensity and the need for high cell numbers per sample. In this report, we present an approach for assessment of EROD activity in teleost hepatocytes cultured in 48-well plates without the need for preparation of microsomes. The assay is based on the method of (Kennedy, S. W., Lorenzen, A., James, C. A. and Collins, B. T. (1993) Anal. Biochem. 211, 102–112.) but employs live instead of dead cells. For EROD detection, 7-ethoxyresorufin is added directly to the live cells and its metabolism to resorufin is observed by repeated measurements over 20 min in a fluorescence plate reader. The assay does not require the addition of exogenous NADPH. Resorufin utilization through cytoplasmic DT-diaphorase has to be inhibited by addition of dicoumarol. Disturbance by conjugate formation accounts to not more than 20% of total resorufin formed. After measurement, the cells can be washed and, after continued incubation, can be used for further assays.

Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.

Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts: Characterization of a NAD(P)H–plastoquinone oxidoreductase activity

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching F max, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H–PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin–NADP +-reductase (FNR), we infer that FNR is not involved in the NAD(P)H–PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H–PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H–PQ oxidoreductase is estimated to about 160 nmol O 2 min −1 mg −1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes.

Nitric Oxide Inactivates NADPH Oxidase in Pig Neutrophils by Inhibiting Its Assembling Process

The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation.

Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine

Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine

Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine.

Human spermatozoa possess a specialized capacity to generate reactive oxygen species (ROS) that is thought to be of significance in the redox regulation of sperm capacitation (De Lamirande and Gagnon, 1993; Aitken et al., 1995). However, the mechanisms by which ROS are generated by these cells are not understood. In this study we have examined the possible significance of NADPH as a substrate for ROS production by human spermatozoa. Addition of NADPH to viable populations of motile spermatozoa induced a sudden dose-dependent increase in the rate of superoxide generation via mechanisms that could not be disrupted by inhibitors of the mitochondrial electron transport chain (antimycin A, rotenone, carbonyl cyanide m-chlorophenylhydrazone [CCCP], and sodium azide), diaphorase (dicoumarol) xanthine oxidase (allopurinol), or lactic acid dehydrogenase (sodium oxamate). However, NADPH-induced ROS generation could be stimulated by permeabilization and was negatively correlated with sperm function. Both NADH and NADPH were active electron donors in this system, while NAD+ and NADP+ exhibited little activity. Stereo-specificity was evident in the response in that only the beta-isomer of NADPH supported superoxide production. The involvement of a flavoprotein in the electron transfer process was indicated by the high sensitivity of the oxidase to inhibition by diphenylene iodonium and quinacrine. These results indicate that NAD(P)H can serve as an electron donor for superoxide generation by human spermatozoa and present a simple strategy for the production of motile populations of free radical generating cells with which to study the significance of these molecules in the control of normal and pathological sperm function.

Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO2 via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO2 via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.