NADPH Oxidoreductase Research Articles

Neutrophil-mediated phospholipid peroxidation assessed by gas chromatography-mass spectroscopy.

Disease pathophysiology frequently involves manifestations of the systemic inflammatory response syndrome. Oxyradicals represent key inflammatory mediators, and neutrophils are one important source of oxyradicals. This investigation examined neutrophil-mediated peroxidation of dilinoleoyl phosphatidyl-choline (DLPC) liposomes by monitoring the appearance of monohydroxyl linoleic acid with the use of gas chromatography-mass spectroscopy (GC-MS), compared with traditional assessment of thiobarbituric acid-reactive species (TBARS) and phosphatidylcholine-specific conjugated dienes. DLPC was peroxidized in a system using activated neutrophils in balanced salt solution containing chelated iron. 9-Monohydroxyl linoleic acid and 13-monohydroxyl linoleic acid were readily identified in neutrophil-mediated peroxidized DLPC with the use of GC-MS. Neutrophil NADPH oxidoreductase specific activity correlated highly with total ion current or specific ion monitoring of integrated peak areas for peroxidized linoleic acid but correlated poorly with DLPC-derived TBARS or conjugated dienes. These results ascertain that activated neutrophils mediate phosphatidylcholine lipid peroxidation to specific products, which may be precisely monitored with the use of GC-MS. The extent of this peroxidation is highly correlated with the magnitude of the neutrophil respiratory burst.

An hypothesis for preconditioning and heat shock proteins: A model for elucidating damage pathways in the heart?

Evidence suggests that there is enhanced production of heat shock protein (hsp70) during ischemic preconditioning of the mammalian heart. The heart may also be protected from cellular damage in the Ca 2+-paradox protocol by preconditioning with cycles of Ca 2+-depletion. It is suggested that hsp70 provides this cardioprotection by preventing the assembly and/or the activation of a vectorially-organised, transmembrane oxido-reductase which, when fully activated in the normal Ca 2+- and O 2-paradoxes, is responsible for sarcolemma damage and leakage of cytoplasmic proteins. Initial activation is dependent on molecular mobility within the bilayer. It is suggested that events during the activation of the neutrophil NADPH oxido-reductase (which is also inhibited by hsps) may serve as a model for cellular damage and preconditioning in the heart. The Ca 2+-paradox protocol in the heart is well-defined and could be an excellent model for elucidating the mechanisms underlying both the increased synthesis of hsps during preconditioning and their protective effect against cellular damage. Since stress-induced hsps can protect a variety of cells they may act on a common molecular damage system.

A Flavin Reductase Stimulates DszA and DszC Proteins ofRhodococcus erythropolisIGTS8in Vitro

Rhodococcus erythropolisIGTS8 is a gram positive bacterium, which can catabolize dibenzothiophene to 2-hydroxybiphenyl and inorganic sulfur without the cleavage of carbon-carbon bonds. Three structural genes,dszA, dszB,anddszC,have been cloned and shown to be necessary for this phenotype. Here, we demonstrate that a FMN:NADPH oxidoreductase fromVibrio harveyicomplements activities of purified DszA and DszC proteins. Furthermore, we propose that DszA and DszC are oxygenase units that do not use NAD(P)H directly, but instead use FMNH2from a FMN:NADPH oxidoreductase for oxygenation.

Hypothesis: iron chelation plays a vital role in neutrophilic inflammation.

Neutrophil influx into tissues occurs in many diverse diseases and can be associated with both beneficial and injurious effects. We hypothesize that the stimulus for certain neutrophilic inflammatory responses can be reduced to a series of competing reactions for iron, with either a labile or reactive coordination site available, between host chelators and chelators not indigenous to that specific living system. The iron focuses the transport of host phagocytic cells through a metal catalyzed generation of oxidant sensitive mediators including cytokines and eicosanoids. Many of these products are chemotactic for neutrophils. We also postulate that the iron increases the activity of the phagocyte associated NADPH oxidoreductase in the neutrophil. The function of this enzyme is likely to be the generation of superoxide in the host's attempt to chemically reduce and dislodge the iron from its chelate complex. After the reoxidation of Fe2+ in an aerobic environment, Fe3+ will be coordinated by host lactoferrin released by the neutrophil. When complexed by this glycoprotein, the metal does not readily undergo oxidation/reduction and is safely transported to the macrophages of the reticuloendothelial system where it is stored in ferritin. Finally, we propose that the neutrophil will attempt to destroy the chelator not indigenous to the host by releasing granular contents other than lactoferrin. Inability to eliminate the chelator allows this sequence to repeat itself, which can lead to tissue injury. Such persistence of a metal chelate in the host may be associated with biomineralization, fibrosis, and cancer.

IL-1 beta stimulates superoxide and delayed peroxynitrite production by pulmonary vascular smooth muscle cells.

Our previous studies have shown that rat pulmonary microvascular smooth muscle cells (RPMSMC) upregulate inducible nitric oxide synthase (iNOS) and produce nitric oxide (NO) when treated with interleukin-1 beta (IL-1 beta). We now report that an additional effect of IL-1 beta stimulation in RPMSMC is an increase in production of superoxide (O2-) that results in the formation of peroxynitrite (ONOO-). IL-1 beta produced a rapid (within 1 h) concentration-dependent increase in O2-, as detected by ferricytochrome c reduction and lucigenin-enhanced chemiluminescence. O2- production was sensitive to quinacrine and diphenyliodinium, suggesting that NADH and NADPH oxidoreductases were responsible. After induction of iNOS and production of iNOS-derived NO, ONOO- was detected by luminol-enhanced chemiluminescence and was found to cause lipid peroxidation and to form nitrotyrosine in the cytoskeleton, detected by immunostaining. Cell viability, however, appeared to be unaffected. IL-1 beta-mediated induction of RPMSMC-derived ONOO- may have significant effects on pulmonary vascular function in sepsis and inflammatory states.

Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells.

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.

Arabidopsis thaliana NADPH Oxidoreductase Homologs Confer Tolerance of Yeasts toward the Thiol-oxidizing Drug Diamide

To isolate new plant genes involved in the defense against oxidative stress, an Arabidopsis cDNA library in a yeast expression vector was transformed into a yeast strain deficient in the YAP1 gene, which encodes a b-Zip transcription factor and regulates general stress response in yeasts. Cells from approximately 10(5) primary transformants were subjected to a tolerance screen toward the thiol-oxidizing drug diamide, which depletes the reduced glutathione in the cell. Four types of Arabidopsis cDNAs were isolated. Three of these cDNAs (P1, P2, and P4) belong to a plant zeta-crystallin family and P3 is an Arabidopsis homolog of isoflavonoid reductases. As such, all four isolated cDNAs are homologous to NADPH oxidoreductases. P1, P2, and P3 steady-state mRNAs accumulated rapidly in Arabidopsis plants under various oxidative stress conditions, such as treatment with paraquat, t-butylhydroperoxide, diamide, and menadione. The data suggested that proteins encoded by the isolated cDNAs play a distinct role in plant antioxidant defense and are possibly involved in NAD(P)/NAD(P)H homeostasis.

Peroxisomal localization of acyl-coenzyme A reductase (long chain alcohol forming) in guinea pig intestine mucosal cells

Upon differential centrifugation of guinea pig intestine mucosal cells homogenate, fatty acyl-CoA:NADPH oxidoreductase (long chain alcohol forming) was found to be enriched in the light mitochondrial (L) fraction (sedimenting between 66,000 x g min and 500,000 x g min) which contained mainly mitochondria, lysosomes, and peroxisomes. Peroxisomes (marker enzymes: catalase and dihydroxyacetone phosphate acyltransferase) present in the L fraction were separated from other organelles in a Nycodenz density gradient centrifugation employing a vertical rotor. By comparing the distribution of acyl-CoA reductase with different marker enzymes in the gradient, it was concluded that this reductase is primarily localized in the microperoxisomes (microbodies). The topography of the membrane-bound enzyme in the isolated organelles was studied by checking its lability toward trypsin in the absence and presence of the detergent Triton X-100. The results suggested that acyl-CoA reductase is localized on the outer surface (cytosolic side) of microperoxisomal membrane.

In vitro modulation of human neutrophil superoxide anion generation by various calcium channel antagonists used in ischemia-reperfusion resuscitation

Generation of toxic oxygen species by activated polymorphonuclear leukocytes (PMNs) may represent an important mechanism of ischemia-reperfusion injury. Concentration-response data concerning inhibition of Superoxide anion (O 2 −) generation by NADPH oxidoreductase (NADPH OR) from isolated human PMN were generated for five calcium channel antagonists commonly utilized in ischemia-reperfusion investigational therapeutics. Regression analysis derived IC 50 values for verapamil, nimodipine, nicardipine and lidoflazine were 45, 20, 12 and 7 μM respectively. Inhibition of the extent of reaction at 5 min paralleled inhibition of initial velocity. No inhibition by flunarizine was noted at concentrations ≤ 25 μM (where it did not alter reaction mixture composition). Only nicardipine demonstrated a significant concentration-response effect relative to prolonging lag time preceding O 2 − synthesis. Inhibition appeared at least partially reversible for all five agents. Neither PMN activation/ desensitization, free-radical scavenging, nor PMN cytotoxicity appeared to be involved in the inhibition of PMN O 2 − synthesis by these agents. Ca 2+ antagonist inhibition of PMN NADPH OR appears to involve more than simple inhibition of Ca 2+ flux across the PMN plasma membrane. Direct inhibition of the intracellular events involved in the activation and/or activity of NADPH OR may be operative.

Spectrophotometric identification of 8-hydroxy-5-deazaflavin: NADPH oxidoreductase activity in streptomycetes producing tetracyclines

Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimosus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF420) using NADPH as a dnor of hydrogen and electrons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) also was a substrate, although with a lower reaction rate than that for SF420. NADH could not substitute for NADPH. The F420-reductase activity was also observed in homogenates of S. aureofaciens spores.

Spectrophotometric identification of 8-hydroxy-5-deazaflavin: NADPH oxidoreductase activity in streptomycetes producing tetracyclines

Cell-free extracts of vegetative mycelia of Streptomyces aureofaciens and Streptomyces rimosus were found to reduce streptomycete-origin 8-hydroxy-5-deazaisoalloxazine derivatives (SF420) using NADPH as a dnor of hydrogen and electrons. 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) also was a substrate, although with a lower reaction rate than that for SF420. NADH could not substitute for NADPH. The F420-reductase activity was also observed in homogenates of S. aureofaciens spores.

Characterization of an 8-hydroxy-5-deazaflavin: NADPH oxidoreductase from Streptomyces griseus

An 8-hydroxy-5-deazaflavin-dependent oxidoreductase was isolated from the actinomycete Streptomyces griseus and purified 590-fold with 72% overall yield. The enzyme catalyzes electron transfer between 8-hydroxy-5-deazaflavins and NADPH. It seems to be more specific than methanogenic oxidoreductase as it has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. A molecular weight of 42,000 was found with gel permeation chromatography, while SDS gel electrophoresis indicated the presence of two identical subunits. Maximal enzymatic activity was found at 0.32 M NaCl and pH 5.9 for reduction of 8-hydroxy-5-deazaflavin and pH 7.9 for the reverse reaction. From the kinetic constants it was estimated that the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions. These results indicate the occurrence of 8-hydroxy-5-deazaflavin-dependent electron transfer in microorganisms not belonging to the archaebacteria.

8-Hydroxy-5-deazaflavin-dependent electron transfer in the extreme halophile Halobacterium cutirubrum

Cell extracts of the extreme halophile Halobacterium cutirubrum were found to contain 8-hydroxy-5-deazaflavin as well as 8-OH-5-deazaflavin: NADPH oxidoreductase activity. The oxidoreductase was partially purified and showed maximum activity at pH 5.4, which is unusually low for halobacteria, and 5.3 M NaCl, close to the intracellular salt concentration. The results indicate the presence of an 8-OH-5-deazaflavin-dependent electron transfer system in a nonmethanogenic organism.

Partial Characterization of an Enzyme from the Fungus Ascochyta rabiei for the Reductive Cleavage of Pterocarpan Phytoalexins to 2′-Hydroxyisoflavans

Crude protein extracts from the chickpea (Cicer arietinum L.) pathogenic fungus Ascochyta rabiei catalyze the NADPH dependent conversion of the pterocarpan phytoalexins medicarpin and maackiain to 1) 2′-hydroxyisoflavans, 2) 1 a-hydroxypterocarp-1,4-diene-3-one, and 3) 10,2′-dihydroxyisoflav-8-ene-7-one derivatives. A pterocarpan : NADPH oxidoreductase which cleaves the benzylphenyl ether bond of the phytoalexins was purified some 100-fold and partially characterized with regard to its kinetic properties. The oxidoreductase was shown to be specific for NADPH and 3-hydroxypterocarpans. The enzyme appears to be constitutively expressed by A. rabiei.

Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli.

When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent Km of 280 microM.

Cinchoninone: Nadph oxidoreductases i and ii-novel enzymes in the biosynthesis of quinoline alkaloids in Cinchona ledgeriana

An enzyme (cinchoninone :NADPH oxidoreductase) which catalyses the reduction of cinchoninone to an unequal mixture of cinchonine and cinchonidine has been isolated from cells of Cinchona ledgeriana in suspension culture and its properties have been studied. It is present in two isoenzymic forms which can be separated by ionexchange chromatography on DEAE-cellulose. Both forms of the enzyme are cytosolic and have an absolute requirement for NADPH. They both catalyse reversible reactions. Isoenzyme I acts specifically on cinchoninone in the forward direction and cinchonidine, cinchonine, cupreine and cupreidine in the reverse direction, while isoenzyme II has a broad specificity acting on all the quinoline alkaloids of Cinchona. The kinetic properties of these two isoenzymes are presented. This is the first description of an enzyme wholly committed to the biosynthesis of Cinchona quinoline alkaloids.

An evaluation of the tautomerism of cinchoninone and quinidinone made using a combination of 1H NMR and 13C NMR spectroscopy

The Cinchona alkaloids cinchoninone and qiunidinone are the substrates for the cinchoninone: NADPH oxidoreductases I and II described previously. The in vitro assay of this activity is complicated by the tendency of the substrates in neutral aqueous solution to undergo mutarotation owing to tautomerism. The composition of such solutions at equilibrium has been determined using a combination of 1H NMR and 13C NMR spectroscopy and shown to contain the keto, enol and diol forms of both the 8 S and 8 R steric series. All these species exist at different concentrations. Furthermore, the position of the equilibrium favours the 8 S-isomers rather than the 8 R-form found in both the crystalline solid and the natural biosynthetic intermediate, cinchoninone.

Contribution of the Pentose Phosphate Pathway and Glycolytic Pathway to Dormancy Breakage and Germination of Peanut (Arachis hypogaeaL.) Seeds

Levels of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate : NADPH oxidoreductase, E.C. 1.1.1.49) 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate : NADP+ oxidoreduc tase, E.C. 1.1.1.44) and aldolase (fructose 1, 6-diphosphate, D-glyceraldehyde, 3-phosphate lyase, E.C. 4.1.2.13) were assayed in the seeds of genetically dormant and non-dormant pure lines of groundnut. In dormant lines cotyledons showed increased levels of activity of G-6-PDH and 6-PGDH during dry storage after-ripening. While the embryonic axis did not exhibit detectable levels of enzyme activities immediately after harvest, the activity started after a lapse of time during dry storage. When seeds of dormant lines were incubated with kinetin (6-furfurylaminopurine) a distinct increase in the levels of both the enzymes was observed. The levels of aldolase activity gradually decreased in the cotyledons and increased in the embryonic axis of both control and kinetin treated seeds during the period of after-ripening. Compared to control, kinetin treatment increased the aldolase activity in the embryonic axis and decreased it in the cotyledons. In non-dormant lines the activity of both the enzymes of PP pathway increased sharply both in the cotyledons and embryonic axis while aldolase activity decreased in the cotyledons and increased in the embryonic axis during germination i.e., from 24 h to 96 h of germination. Abscisic acid caused inhibition of enzyme activities to a large extent.

Nuclear magnetic resonance studies of the old yellow enzyme. 1. 15N NMR of the enzyme recombined with 15N-labeled flavin mononucleotides.

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with specifically 15N-labeled flavin mononucleotide and investigated by 15N NMR spectroscopy in the oxidized and reduced state. The results indicate that in the oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition, hydrogen bonds exist between the N(1) and N(3) atoms of FMN and the apoprotein. The resonance position of N(10) indicates that this atom is somewhat sp3-hybridized, i.e. lifted out of the molecular plane of the isoalloxazine ring system. In the reduced state the N(1) atom is negatively charged and the N(3) atom forms a hydrogen bond with the apoprotein. The N(10) atom in protein-bound FMN exhibits about the same hybridization state as in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp3-hybridized. This interpretation can also be derived from the 15N(5)-1H coupling constant. Among the flavoproteins thus far studied by NMR techniques, old yellow enzyme is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lifted out of the molecular plane. The isoelectric focussing properties of old yellow enzyme and a new easy method for the preparation of the apoprotein are also reported.

Nuclear magnetic resonance studies of the old yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides.

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively 13C-enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized. From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins. The 13C and 15N NMR data on the old-yellow-enzyme--phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme--phenolate complexes.