The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

Towards specific NADPH oxidase inhibition by small synthetic peptides

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase is essential for host defenses against pathogens. ROS are very reactive with biological molecules such as lipids, proteins and DNA, potentially resulting in cell dysfunction and tissue insult. Excessive NADPH oxidase activation and ROS overproduction are believed to participate in disorders such as joint, lung, vascular and intestinal inflammation. NADPH oxidase is a complex enzyme composed of six proteins: gp91phox (renamed NOX2), p22phox, p47phox, p67phox, p40phox and Rac1/2. Inhibitors of this enzyme could be beneficial, by limiting ROS production and inappropriate inflammation. A few small non-peptide inhibitors of NADPH oxidase are currently used to inhibit ROS production, but they lack specificity as they inhibit NADPH oxidase homologues or other unrelated enzymes. Peptide inhibitors that target a specific sequence of NADPH oxidase components could be more specific than small molecules. Here we review peptide-based inhibitors, with particular focus on a molecule derived from gp91phox/NOX2 and p47phox, and discuss their possible use as specific phagocyte NADPH oxidase inhibitors.     

NADPH oxidase inhibitors: a decade of discovery from Nox2ds to HTS

NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns.     

Xanthine oxidase activity in the brain

Résumé La xanthine oxydase a été déterminée dans la partie surnageante des homogenéisats centrifugés de cerveau et cervelle de divers animaux (souris, rat, cobaye, lapin, vache) par spectrophotométrie de l'acide urique produit après incubation de 2 h avec l'hypoxanthine. Le cerveau du rat et du cobaye sont les plus riches en cette enzyme, mais la cervelle de la souris est dépourvue d'activité. La xanthine déhydrase s'est montrée plus active après incubation avec le NAD.     

Monoacylcadaverines as substrates for both monoamine oxidase and diamine oxidase; low rates of activity

Summary Monoacetylcadaverine and monopropionylcadaverine were found to be substrates for both rat liver monoamine oxidase and hog kidney diamine oxidase, but all the K_m-values for the oxidases were very high. The amines were common substrates for type A and type B monoamine oxidase.     

D-aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

A mutant mouse strain ddY/DAO- lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.     

D-Aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

Summary A mutant mouse strain ddY/DAO^– lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.     

Monoamine oxidase activity in Helix pomatia.

The distribution and characterization of MAO in various tissues of the snail were analyzed. Only low amounts of the enzyme exist in the different tissues and data suggest that there is more than one type of MAO.     

Mineralocorticoid treatment and the adrenalectomy-induced increase in monoamine oxidase activity

Summary Bilateral adrenalectomy in the rat results in an increase in heart monoamine oxidase activity in animals drinking water and in animals drinking 0.9% saline. Daily administration of deoxycorticosterone acetate prevented the increased monoamine oxidase activity in the animals drinking saline but not in those drinking water.Acknowledgment. This work was supported in part by a grant from the American Heart Association — Louisiana, Inc.     

Distribution of polyamine oxidase activity in rat tissues and subcellular fractions

The activity of polyamine oxidase (PAO) in rat tissues, and its subcellular distribution, were assayed using a simple polarographic method. The highest PAO activity was measured in the liver and the lowest in the skeletal muscle. In liver, kidney and uterus the highest specific PAO activity was found in the light mitochondrial fraction. PAO was not found in the microsomal fraction except in the kidney.     

Monoamine oxidase activity in tissues of spontaneously hypertensive rats

Summary Monoamine oxidase (MAO) activity was assayed both in central and peripheral blood vessels of spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKR). The activity of MAO in the brain and peripheral vasculature was essentially the same in both SHR and WKR. It can therefore be concluded that central and peripheral vascular MAO activity is not altered in the genetically hypertensive animals.     

Regulation of cyclin-Cdk activity in mammalian cells

Cell cycle progression is driven by the coordinated regulation of the activities of cyclin-dependent kinases (Cdks). Of the several mechanisms known to regulate Cdk activity in response to external signals, regulation of cyclin gene expression, post-translational modification of Cdks by phosphorylation-dephosphorylation cascades, and the interaction of cyclin/Cdk complexes with protein inhibitors have been thoroughly studied. During recent years, much attention has also been given to mechanisms that regulate protein degradation by the ubiquitin/proteasome pathway, as well as to the regulation of subcellular localization of the proteins that comprise the intrinsic cell cycle clock. The purpose of the present review is to summarize the most important aspects of the various mechanisms implicated in cell cycle regulation.     

Distribution of polyamine oxidase activity in rat tissues and subcellular fractions.

The activity of polyamine oxidase (PAO) in rat tissues, and its subcellular distribution, were assayed using a simple polarographic method. The highest PAO activity was measured in the liver and the lowest in the skeletal muscle. In liver, kidney and uterus the highest specific PAO activity was found in the light mitochondrial fraction. PAO was not found in the microsomal fraction except in the kidney.     

Correlation betweenpara-phenylene diamine oxidase activity and the copper content of rat serum

Résumé Il y a une corrélation très forte entre l'activité de lapara-phénylène diamine oxydase du sérum sanguin du rat et de son taux en cuivre.     

Monoamine oxidase activity in membrane structures of rat liver cell

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