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)

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. 137 27

A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L-citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system.
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PMID:Purification and characterization of a human NO synthase. 172 2

Nitric oxide synthase produces NO, citrulline, water, and NADP at the expense of arginine, NADPH, and dioxygen. While citrulline has been considered to be an inert by-product of the high output inducible isoform of NO synthase (iNOS), we show here that immunostimulants induce a metabolic pathway in vascular smooth muscle cells, which enables them to regenerate arginine from citrulline. Regeneration of arginine from citrulline is accomplished by two urea cycle enzymes: arginino-succinate synthetase (AS) and argininosuccinate lyase (AL). Whereas AL is constitutive to vascular smooth muscle cells, AS mRNA and enzyme activity is markedly induced in cells by treatment with bacterial lipopolysaccharide (LPS). The induction of AS mRNA and activity by LPS follows a time course which mirrors that for iNOS but lags 1-2 h behind. As shown for iNOS, interferon-gamma does not itself induce AS but is synergistic with LPS. AS induction is suppressed by glucocorticoids, actinomycin D, and, to a lesser extent, cycloheximide. On the other hand, AS induction is unaffected by an excess of citrulline or the inhibitor of iNOS, N omega-methyl-L-arginine. Our results show the urea cycle enzymes AS and AL confer cells with the capacity to produce NO without a need for exogenous arginine. In conjunction with NOS, citric acid cycle enzymes that covert fumarate to oxaloacetate (fumarase and malate dehydrogenase) and oxaloacetate to aspartate (aspartate transaminase), AS and AL form a novel arginine-citrulline cycle that enables high output NO production by cells.
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PMID:Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for nitric oxide synthesis. 751 85

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.
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PMID:NADPH-diaphorase activity of nitric oxide synthase in the olfactory bulb: co-factor specificity and characterization regarding the interrelation to NO formation. 751 84

We are reporting a distinct constitutive isoform of nitric oxide synthase that has been purified to homogeneity from human platelet cytosolic fractions. Purification involved ultra centrifugation at 100,000 x g followed by two sequential affinity chromatography procedures: adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin Sepharose 4B. Purified enzyme appeared as a single band (approximately 80 kDa) under denaturing condition (SDS-PAGE). The native enzyme appears to be dimeric, since its molecular weight estimated by gel filtration was approximately 150 kDa. Enzyme activity was dependent on L-arginine, NADPH and (6R)-5,6,7,8-tetrahydro-L-biopterine. Partially purified platelet NOS (100,000 x g supernatant) activity was sensitive to calmodulin antagonists and to the N omega-Monomethyl-L-arginine, a substrate analog of L-arginine.
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PMID:Isolation of nitric oxide synthase from human platelets. 751 42

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.
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PMID:Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells. 751 46

The objective of these investigations was to study the regulatory properties of brain constitutive NO synthase. NOS activity was determined in 18,000 X g supernatant by conversion of 3H-L-arginine to 3H-L-citrulline in the presence of NADPH. The expression of catalytic activity of NOS required the presence of calcium ion and calmodulin. The preincubation of enzyme preparations at 37 degrees C in standard reaction mixture led to time-dependent inhibition of L-citrulline formation. This inhibition also required the presence of calcium ion during preincubation phase, and the enzyme remained calmodulin-dependent as exhibited by sensitivity to calmodulin antagonists trifluoperazine (TFP) and calcineurin. The modified enzyme showed significant decrease in the Vmax with NADPH and L-arginine without any change in apparent Km. Inclusion of protease inhibitors, leupeptin, pepstatin A, PMSF and soyabean trypsin inhibitor to the preparations did not alter preincubation-dependent inhibition of NO synthase. Thus, the calcium-dependent inhibitory phenomenon was not due to either the denaturation or proteolysis or the loss of calmodulin sensitivity of NO synthase. These observations indicate that cytosolic isoform of constitutive NO synthase undergoes dual regulation by physiological concentrations of calcium ion.
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PMID:Calcium-dependent inhibition of constitutive nitric oxide synthase. 752 Nov 66

Potent and selective inhibition of neuronal nitric oxide synthase (nNOS) compared to endothelial NOS (eNOS) and inducible NOS (iNOS) may be useful to treat cerebral ischemia (stroke) and other neurodegenerative diseases. S-Methyl-L-thiocitrulline (Me-TC) and S-ethyl-L-thiocitrulline (Et-TC) inhibited the oxidation of L-arginine and the L-arginine-independent oxidation of NADPH by nNOS from human brain. Me-TC and Et-TC were slow, tight binding inhibitors of nNOS with second-order association rate constants (kon) of 2.6 x 10(5) M-1 s-1 and 1.3 x 10(5) M-1 s-1, respectively. The respective dissociation rate constants (koff) were 3 x 10(-4) s-1 and 0.7 x 10(-4) s-1. Thus, the Kd values calculated from koff/kon were 1.2 and 0.5 nM, respectively. L-Arginine was a competitive inhibitor of Me-TC and Et-TC binding with competition constant (Ks) values of 2.2 and 2.7 microM, respectively. The Km of nNOS for L-arginine was 1.6 microM. The active site concentration of nNOS was estimated by titration with Et-TC. Based on this active site concentration, a kcat of 0.4 s-1 for the oxidation of L-arginine, was calculated. Me-TC and Et-TC were less potent inhibitors of human iNOS (Ki values of 34 and 17 nM, respectively) and human eNOS (Ki values of 11 and 24 nM). Thus, Me-TC and Et-TC were 10- and 50-fold, respectively, more potent inhibitors of nNOS than eNOS. Furthermore, Me-TC was also 17-fold selective for rat nNOS in neuronal tissue compared to rat eNOS in vascular endothelium, suggesting that Me-TC may be selective for nNOS in vivo and therefore, may be therapeutically useful to treat neurodegenerative diseases.
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PMID:Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. 752 10

Although nitric oxide (NO) appears to be one of the oxidation products of L-arginine catalyzed by NO synthase (NOS; EC 1.14.13.39), past studies on the measurement of NO in cell-free enzymatic assays have not been based on the direct detection of the free NO molecule. Instead, assays have relied on indirect measurements of the stable NO oxidation products nitrite and nitrate and on indirect actions of NO such as guanylate cyclase activation and oxyhemoglobin oxidation. Utilizing a specific chemiluminescence assay, we report here that the gaseous product of L-arginine oxidation, catalyzed by both inducible macrophage and constitutive neuronal NOS, is indistinguishable from authentic NO on the basis of their physicochemical properties. NO gas formation by NOS was dependent on L-arginine, NADPH, and oxygen and inhibited by NG-methyl-L-arginine and cyanide anion. Superoxide dismutase (SOD) caused a marked, concentration-dependent increase in the production of free NO by mechanisms that were unrelated to the dismutation of superoxide anion or activation of NOS. These observations indicate that free NO is formed as a result of NOS-catalyzed L-arginine oxidation and that SOD enhances the generation of NO without directly affecting NO itself. SOD appears to elicit a novel biological action, perhaps accelerating the conversion of an intermediate in the L-arginine-NO pathway such as nitroxyl (HNO) to NO.
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PMID:Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase. 752 87

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.
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PMID:Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord. 752 98


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