scholarly journals NADPH-diaphorase activity of nitric oxide synthase in the olfactory bulb: co-factor specificity and characterization regarding the interrelation to NO formation.

1994 ◽  
Vol 42 (5) ◽  
pp. 569-575 ◽  
Author(s):  
R Spessert ◽  
C Wohlgemuth ◽  
S Reuss ◽  
E Layes
Keyword(s):  
Nitric Oxide ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Olfactory Bulb ◽  
Reductase Activity ◽  
Nadph Diaphorase ◽  
Cytochrome P450 Reductase ◽  
No Formation ◽  
Olfactory Glomeruli ◽  
Oxide Synthase

The neuronal form of the enzyme nitric oxide synthase (nNOS) synthesizes the messenger molecule nitric oxide (NO). In addition to NO formation, nNOS exhibits a so-called NADPH-diaphorase (NADPH-d) activity. This study focused on the characterization of NADPH-d activity with regard to NO formation in the rat olfactory bulb. In this area of the brain pronounced staining is localized in discrete populations of neuronal somata and in olfactory glomeruli. Diaphorase staining combined with demonstration of nNOS by polyclonal antibodies revealed that NADPH-d activity of neuron somata is associated with nNOS immunoreactivity. It is concluded that neuron somata exhibit NADPH-d activity of nNOS. NADPH-d activity of nNOS did not utilize beta-NADH or alpha-NADPH. Moreover, NADPH-d activity was inhibited in the presence of alpha-NADPH. Dichlorophenolindophenol (DPIP), an artificial electron acceptor and an inhibitor of NO formation, totally suppressed NADPH-d staining of neurons, supporting the concept that the NADPH-d of neuron somata is due to nNOS. Cytochrome C, miconazole, EGTA, and trifluoperazine, which have been reported to inhibit cytochrome P450 reductase activity of NOS, did not affect NADPH-d staining. Hence, NADPH-d activity of NOS does not involve cytochrome P450 reductase activity as required for NO formation. Contrary to NADPH-d activity of neuron somata, staining of olfactory glomeruli was not co-localized with nNOS immunoreactivity. Glomerular staining was also observed in the presence of beta-NADH and alpha-NADPH. Further, it was unchanged in the presence of the NO formation inhibitor DPIP. Hence, the glomerular staining in the presence of NADPH is not due to the NADPH-d activity of NOS. We conclude that staining of neuronal structures in the presence of NADPH does not necessarily represent NADPH-d activity of NOS.

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.

NADPH-diaphorase/nitric oxide synthase-positive elements in the human olfactory bulb

Neuroreport ◽  
1998 ◽  
Vol 9 (14) ◽  
pp. 3141-3146 ◽  
Author(s):  
J G. Briñón ◽  
C Crespo ◽  
E Weruaga ◽  
J Alonso ◽  
T Sobreviela ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Olfactory Bulb ◽  
Nadph Diaphorase ◽  
Positive Elements ◽  
Oxide Synthase

scholarly journals Fixation conditions affect the intensity but not the pattern of NADPH-diaphorase staining as a marker for neuronal nitric oxide synthase in rat olfactory bulb.

1994 ◽  
Vol 42 (10) ◽  
pp. 1309-1315 ◽  
Author(s):  
R Spessert ◽  
E Layes
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Olfactory Bulb ◽  
Biochemical Study ◽  
Polyclonal Antibodies ◽  
Staining Intensity ◽  
Nadph Diaphorase ◽  
Practical Significance ◽  
Histochemical Marker ◽  
Oxide Synthase

NADPH-diaphorase (NADPH-d) is commonly used as a histochemical marker for the neuronal form of the enzyme nitric oxide synthase (NOS). A recent biochemical study showed that in broken-cell preparations NADPH-d activity did not fully represent NOS and that NOS-unrelated NADPH-d activity was suppressed during fixation. Because it is unknown whether fixation also affects NOS-associated NADPH-d activity, we investigated the effects of various widely used fixatives on NADPH-d staining in relation to NOS immunoreactivity, obtained with polyclonal antibodies, in rat olfactory bulb. We found that the intensity of NADPH-d staining associated with NOS, as well as that unrelated to NOS, depends on fixation conditions. Addition of glutaraldehyde or lysine/sodium periodate to the fixative decreased intensity of NADPH-d staining. Fixative-dependence of NADPH-d staining was observed not only in the presence of the "normal" co-substrate beta-NADPH but also in the presence of the stereoisomer alpha-NADPH. Unlike the staining intensity, the staining pattern of NOS-associated as well as NOS-unrelated NADPH-d did not change after treatment with various fixatives. Our findings are of considerable practical significance because it has become clear that fixation conditions affect the sensitivity but not the selectively of the NADPH-d reaction as a marker for the presence of NOS.

scholarly journals Nitric oxide, vascular function and exercise

2012 ◽  
Vol 34 (3) ◽  
pp. 28-32
Author(s):  
Lisardo Boscá ◽  
Antonio Castrillo
Keyword(s):  
Nitric Oxide ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Vascular Function ◽  
Oxidation Process ◽  
Mammalian Species ◽  
Cytochrome P450 Reductase ◽  
Consecutive Reactions ◽  
Gas Molecule ◽  
Oxide Synthase

NO (nitric oxide) is a diffusible gas molecule produced intracellularly by three distinct nitric oxide synthase (NOS) enzymes that are highly conserved in mammalian species. The NOS enzymes are haemcontaining proteins that catalyse a fiveelectron oxidation process of the guanidino nitrogen of arginine and in the presence of O2. NOS activity is, in fact, a tandem of two consecutive reactions: a reductase step catalysed by a moiety of NOS homologous with the cytochrome P450 reductase, including the NADPH and flavin nucleotide cofactorbinding domains, and an oxygenase step coupling the reduction of Fe3+ to Fe2+ in the haem to the release of NO and citrulline from arginine and molecular oxygen. The reaction requires several cofactors, including tetrahydrobiopterin and calcium/calmodulin.

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