Involvement of the Reductase Domain of Neuronal Nitric Oxide Synthase in Superoxide Anion Production†

Biochemistry ◽  
1997 ◽  
Vol 36 (49) ◽  
pp. 15277-15284 ◽  
Author(s):  
R. Timothy Miller ◽  
Pavel Martásek ◽  
Linda J. Roman ◽  
Jonathan S. Nishimura ◽  
Bettie Sue Siler Masters
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Superoxide Anion ◽  
Neuronal Nitric Oxide Synthase ◽  
Superoxide Anion Production ◽  
Anion Production ◽  
Oxide Synthase ◽  
Reductase Domain

Endothelial Dysfunction Coincides With an Enhanced Nitric Oxide Synthase Expression and Superoxide Anion Production

Hypertension ◽  
1997 ◽  
Vol 30 (4) ◽  
pp. 934-941 ◽  
Author(s):  
Anne Bouloumié ◽  
Johann Bauersachs ◽  
Wolfgang Linz ◽  
Bernward A. Schölkens ◽  
Gabriele Wiemer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Superoxide Anion ◽  
Superoxide Anion Production ◽  
Anion Production ◽  
Oxide Synthase

scholarly journals New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

2011 ◽  
Vol 11 ◽  
pp. 2443-2457 ◽  
Author(s):  
Michaela Pekarova ◽  
Antonin Lojek ◽  
Hana Martiskova ◽  
Ondrej Vasicek ◽  
Lucia Bino ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Superoxide Anion ◽  
Macrophage Activation ◽  
Inos Expression ◽  
Superoxide Anion Production ◽  
Anion Production ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increasedO2∙-formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controllingO2∙-and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels ofO2∙-soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependentO2∙-formation in inflammatory macrophages.

scholarly journals Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

2002 ◽  
Vol 367 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Kirsty KNIGHT ◽  
Nigel S. SCRUTTON
Keyword(s):  
Nitric Oxide ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Stopped Flow ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
Oxide Synthase ◽  
Reductase Domain

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. The second and third steps represent further reduction of the enzyme flavins and NADP+ release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP+ release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+ is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.

scholarly journals Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

1999 ◽  
Vol 274 (32) ◽  
pp. 22313-22320 ◽  
Author(s):  
Subrata Adak ◽  
Sanjay Ghosh ◽  
Husam M. Abu-Soud ◽  
Dennis J. Stuehr
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Acidic Residues ◽  
Oxide Synthase ◽  
Reductase Domain
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