Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.19 (NOS)
7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.
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PMID:Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. 1048 Aug 77

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in pi-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.
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PMID:Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition. 1048 Sep

Nitric oxide (NO) produced in endothelial cells has been implicated in the regulation of blood pressure, regional blood flow, inhibition of platelet aggregation, and endothelial and vascular smooth muscle cell proliferation. In a variety of cardiovascular disease states, such as atherosclerosis, arterial hypertension, and restenosis, expression of endothelial NO synthase (NOS-III) and endothelial NO production appear to be altered. Thus, NOS-III is an attractive target for cardiovascular gene therapy for which adenoviral vectors are one of the most effective vector systems. Therefore, a recombinant adenoviral vector expressing NOS-III (adenovirus type 5 [Ad5] cytomegalovirus [CMV] NOSIII) was constructed and biochemically and pharmacologically characterized both in vitro and in intact cells. Ad5CMVNOSIII-derived recombinant NOS-III was successfully expressed, as shown by immunoprecipitation and immunocytochemistry, and biologically active, as shown by functional assays in human primary umbilical vein and EA.hy926 endothelial cells, as well as 293 human embryonic kidney and Chinese hamster ovary cells. The Km values for NADPH and L-arginine and the Ka for tetrahydrobiopterin as well as the enzyme's dependency on other cofactors were similar to recombinant reference enzyme and literature values. NOS-III expression levels correlated linearly with the multiplicity of infection with Ad5CMVNOSIII and lasted for at least 8 days. NOS-III transfection inhibited endothelial cell proliferation. In conclusion, adenovirus-mediated gene transfer of Ad5CMVNOSIII to vascular and nonvascular cells resulted in the dose-dependent expression of intact, physiologically regulated, and functionally active NOS-III.
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PMID:Biochemical and functional characterization of nitric oxide synthase III gene transfer using a replication-deficient adenoviral vector. 1048 73

Nitric oxide synthase (NOS: EC 1.14.13.39) catalyzes L-arginine oxidation to generate nitric oxide (NO) and L-citrulline. Recently, 7-ethoxyresorufin (7-ER), a specific substrate of cytochrome P-4501A1, was used as a cytochrome P-450 inhibitor to study the mechanism underlying the vasodilatation caused by some drugs, and was suggested to inhibit nitric oxide-mediated relaxation. Herein we demonstrate that 7-ER inhibits NO synthesis by uncoupling neuronal nitric oxide synthase (nNOS). 7-ER is a noncompetitive inhibitor of nNOS with respect to L-arginine with a Ki value of 0.76 +/- 0.06 microM. The decrease in NO formation is inversely correlated with an increase in NADPH oxidation. 7-ER binds to nNOS with a Km value of 0.68 +/- 0.07 microM, as calculated from the nNOS-dependent NADPH oxidation in the absence of L-arginine. nNOS catalyzes the reduction of 7-ER at the expense of NADPH. The flavoprotein inhibitor, diphenyleneiodonium chloride (100 microM), completely inhibited nNOS-dependent 7-ER reduction. While nitro-L-arginine (1 mM) and N(G)-nitro-L-arginine methyl ester (1 mM), specific inhibitors of nNOS, and phenylisocyanide (0.1 mM), a specific heme iron ligand, did not affect the reduction of 7-ER. These results indicate that the reductase domain, but not the oxygenase domain, of nNOS is involved in the reduction of 7-ER. 7-ER uncouples nNOS, shunting electrons from the reductase domain to the oxygenase domain of the enzyme. As a consequence, NO synthesis is inhibited.
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PMID:Neuronal nitric oxide synthase catalyzes the reduction of 7-ethoxyresorufin. 1050 41

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.
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PMID:The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer. 1052 42

The purpose of this study was to determine whether nitric oxide (NO) is present in clinically normal horses under basal conditions and if it increases secondary to naturally acquired small intestinal strangulation obstruction. Thirty-one horses were used; 20 horses with naturally acquired small intestinal strangulation obstruction and 11 clinically normal horses with no signs of gastrointestinal tract disease. Jugular venous blood, abdominal fluid, and urine were collected for NO quantification. Plasma, abdominal fluid, and urine were stored at -70 degrees C until analyzed for NO using a chemiluminescent method. Biopsy specimens collected from the affected jejunal segment, during anesthesia or after immediately after euthanasia, or from the midjejunum of control horses, were divided into subsections for fixation in zinc formalin and cryopreservation in OCT gel. Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) diaphorase histochemical stains were performed on cryopreserved tissues and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunohistochemical stains were performed on formalin-fixed, paraffin-embedded tissues. There were significantly greater plasma and abdominal fluid NO concentrations in affected horses as compared with controls, but there were no significant differences between horses for urine NO concentrations. There was a significant decrease in NADPH diaphorase stain in mucosal epithelium, vasculature, and leukocytes, and in submucosal plexi in affected horses compared with control horses. There was a significant increase in iNOS staining in mucosal and submucosal leukocytes and in mucosal leukocyte nitrotyrosine staining of the affected compared with control horses. Endothelial NOS and neuronal NOS are present under basal conditions in the jejunum of horses and probably mediate physiologic or cytoprotective effects. Plasma and abdominal fluid, but not urine, NO concentrations increase subsequent to small intestinal strangulation obstruction; this may be associated with increased mucosal and submucosal iNOS staining in leukocytes, which was likely due to increased expression subsequent to stimuli associated with ischemia. The increased nitrotyrosine staining in mucosal leukocytes of affected horses likely reflects the presence of peroxynitrite subsequent to increased NO and superoxide production and may reflect a cytotoxic role of NO in small intestinal strangulation obstruction in horses.
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PMID:Detection and comparison of nitric oxide in clinically normal horses and those with naturally acquired small intestinal strangulation obstruction. 1053 1

A water-soluble iron complex with N-dithiocarboxysarcosine (Fe-DTCS) has been developed as an ESR spin-trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe-DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 +/- 0.7 microM in the citrulline-formation assay. DTCS alone did not inhibit the activity. An iron complex with N-methyl-D-glucamine dithiocarbamate, a similar spin-trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 +/- 2.9 microM. Fe-DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS-mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin-trapping agent Fe-DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.
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PMID:Reaction of neuronal nitric oxide synthase with the nitric oxide spin-trapping agent, iron complexed with N-dithiocarboxysarcosine. 1058 70

Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-free ferric neuronal NOS (NNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)()-methyl-L-Arg, N(G)()-nitro-L-Arg) and BH(4), together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (gamma(MAX): 419, 538, 568 NM) and incubation for 2-3 days at 4 degrees C converted the domain to a native enzyme-type, predominantly high-spin state (gamma(MAX): approximately 395, approximately 512, approximately 650 NM). 7,8-Dihydrobiopterin and other thiols (E.G., beta-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH(4) and DTT, respectively. the UV-visible absorption spectrum of L-Arg-bound ferric full length NNOS, which exhibits a relatively intense band at approximately 650 NM (epsilon equals 7.5-8 MM(-)(1) CM(-)(1)) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 H) incubation of the oxygenase domain with BH(4) and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH(4) in its reduced form, In restoring the pterin- and/or substrate-binding capability of the E. coli-expressed, BH(4) free, dimeric NNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.
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PMID:Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase. 1062 50

Nitric oxide (NO) may subserve different functions in different central neurons subjected to axotomy. The difference may depend on whether the neurons basally express neuronal nitric oxide synthase (nNOS), a biosynthetic enzyme of NO. This is supported by our previous finding that suggests the differential role of NO in neurons of nucleus dorsalis (ND) and red nucleus (RN) which have different basal expression of nNOS. This study aimed to establish firmly the functions of NO, as revealed by nNOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, by the administration of endogenous NO donor, l-arginine (l-arg), and NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME). To relate the role of NO to glutamate receptors (GluR), the distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) in the two nuclei were revealed by immunohistochemical techniques. nNOS immunoreactivity was void in ND neurons, but expressed weakly in the RN normally. It was induced in ipsilateral ND neurons and upregulated on both sides of RN after spinal cord hemisection. Neuronal loss in the ipsilateral ND was augmented by l-arg, but reduced by l-NAME. In the contralateral RN, l-arg attenuated neuronal loss. NMDAR1 was present in most neurons in ND. After axotomy, some NMDAR1 immunoreactive neurons of the ipsilateral ND were induced to express NOS, whereas RN neurons showed strong staining for NMDAR1 and all the AMPA subunits. Most of the NOS-positive neurons in the RN were coexistent with GluR2 in normal rats and those subjected to axotomy. The present data demonstrated that NO exerted neurodestructive function in the non-NOS-containing ND neurons characterized by NMDAR as the predominant glutamate receptor. NO might be beneficial to the NOS-containing RN neurons. This could be attributed to the presence of GluR2. Possible diverse synthesizing pathways of NO in two different central nuclei were suggested from the observation that NOS was colocalized with NADPH-d in ND neurons, but not in RN neurons.
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PMID:Neuroprotective and neurodestructive functions of nitric oxide after spinal cord hemisection. 1068 69

Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O(2)) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor alpha (TNFalpha) through a mechanism dependent on the production of O(2) and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFalpha promoter activity and TNFalpha production. N(omega)-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFalpha up-regulation. Likewise, Gln(361)eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFalpha. Similar to the effect of eNOS, a O(2) donor dose-dependently increased TNFalpha production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFalpha up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O(2) production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O(2), interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O(2) production.
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PMID:Superoxide production and reactive oxygen species signaling by endothelial nitric-oxide synthase. 1074 95


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