NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O2-−) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O2-− scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O2-− production by BH4 autoxidation. Because the N-5-methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation-resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H2O2 production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC50 approx. 0.2 μM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 μM). Concomitantly with inhibition, the NADP+/L-citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC50 approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high-affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O2-− production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O2-− scavengers are present, a physiological role for NO-induced autoinhibition is unlikely.

Download Full-text

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.6.h2020 ◽

2000 ◽

Vol 278 (6) ◽

pp. H2020-H2027 ◽

Cited By ~ 10

Author(s):

Roberto R. Giraldez ◽

Alexander Panda ◽

Jay L. Zweier

Keyword(s):

Nitric Oxide ◽

Endothelial Dysfunction ◽

Nitric Oxide Synthase ◽

Immunoelectron Microscopy ◽

Endothelial Nitric Oxide Synthase ◽

Western Blots ◽

No Formation ◽

Triton X ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Whereas altered nitric oxide (NO ⋅) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO ⋅ formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO ⋅ synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO ⋅ formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by thel-[14C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO ⋅ generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO ⋅ synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.

Download Full-text

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

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.5.7512584 ◽

1994 ◽

Vol 42 (5) ◽

pp. 569-575 ◽

Cited By ~ 57

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.

Download Full-text

The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study

Biochemical Journal ◽

10.1042/bj3620733 ◽

2002 ◽

Vol 362 (3) ◽

pp. 733-739 ◽

Cited By ~ 124

Author(s):

Jeannette VÁSQUEZ-VIVAR ◽

Pavel MARTÁSEK ◽

Jennifer WHITSETT ◽

Joy JOSEPH ◽

Balaraman KALYANARAMAN

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Spin Trapping ◽

No Formation ◽

Superoxide Formation ◽

Oxide Synthase ◽

Epr Spin Trapping ◽

Endothelial Nitric Oxide ◽

Superoxide Release

Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH4). Thus alterations in endothelial BH4 levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH4, and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH4 diminished superoxide release from eNOS, with efficiency BH4>6-methyl-BH4>5-methyl-BH4. In contrast, partially oxidized BH4 analogues, 7,8-dihydrobiopterin (7,8-BH2) and sepiapterin had no effect. Neither l-arginine nor NG-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH4 and l-arginine stimulated ˙NO production at maximal rates of 148nmol/min per mg of protein. These results indicate that BH4 acts as a ‘redox switch’, decreasing superoxide release and enhancing ˙NO formation. This role was verified by adding 7,8-BH2 or sepiapterin to fully active eNOS. Both 7,8-BH2 and sepiapterin enhanced superoxide release while inhibiting ˙NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH4 metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.

Download Full-text

Localization of endothelial nitric oxide synthase in the normal and failing human atrial myocardium

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166397 ◽

1996 ◽

Vol 54 ◽

pp. 786-787

Author(s):

Chi-Ming Wei ◽

Margarita Bracamonte ◽

Shi-Wen Jiang ◽

Richard C. Daly ◽

Christopher G.A. McGregor ◽

...

Keyword(s):

Nitric Oxide ◽

Heart Failure ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Cardiac Tissues ◽

Mammalian Tissues ◽

End Stage Heart Failure ◽

End Stage ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Nitric oxide (NO) is a potent endothelium-derived relaxing factor which also may modulate cardiomyocyte inotropism and growth via increasing cGMP. While endothelial nitric oxide synthase (eNOS) isoforms have been detected in non-human mammalian tissues, expression and localization of eNOS in the normal and failing human myocardium are poorly defined. Therefore, the present study was designed to investigate eNOS in human cardiac tissues in the presence and absence of congestive heart failure (CHF).Normal and failing atrial tissue were obtained from six cardiac donors and six end-stage heart failure patients undergoing primary cardiac transplantation. ENOS protein expression and localization was investigated utilizing Western blot analysis and immunohistochemical staining with the polyclonal rabbit antibody to eNOS (Transduction Laboratories, Lexington, Kentucky).

Download Full-text