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:1.6.99.3 (diaphorase)
5,903 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

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.
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PMID:Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase. 137 40

A nitric oxide synthase activity stimulated more than 30-fold by the concurrent presence of Ca2+ and calmodulin (CaM), and inhibited by trifluoperazine (50 microM), has been identified in extracts of GH3 pituitary cells. The CaM-dependent nitric oxide synthase of the crude extract was stimulated more than 9-fold by (6R)-5,6,7,8-tetrahydro-L-biopterin with half-maximal stimulation occurring at a concentration of 300 nM. Fractionation of the extract on DEAE-cellulose enhanced nitric oxide synthase specific activity up to 300-fold and provided a preparation which on Western blot analysis possessed a 152 kDa protein which cross-reacted with antibodies to homogeneous bovine brain nitric oxide synthase. The DEAE-cellulose-purified enzyme exhibited apparent Km values of 4.3 microM, 0.4 microM, 0.3 microM and 4 nM for L-arginine, NADPH, Ca2+ and CaM respectively. The CaM-dependent nitric oxide synthase of GH3 extract bound to 2',5'-ADP-agarose and was eluted by NADPH with a 500-fold increased specific activity. Citrulline formation by the ADP-agarose-purified enzyme was inhibited by NG-nitro-L-arginine, NG-methyl-L-arginine and Nitro Blue Tetrazolium with apparent Ki values of 0.2, 1.8 and 7 microM respectively. The ADP-agarose-purified enzyme displayed cytochrome c reductase activity which was stimulated more than 18-fold by the concurrent presence of Ca2+ and CaM and inhibited by trifluoperazine. NG-Nitro-L-arginine and NG-methyl-L-arginine did not inhibit the cytochrome c reductase activity.
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PMID:Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells. 137 40

Citrulline formation by the interferon-gamma/lipopolysaccharide-inducible murine macrophage nitric oxide synthase is inhibited reversibly by imidazole, 1-phenylimidazole, 4-phenylimidazole, and 2-phenylimidazole with IC50 values of 40 microM, 6 microM, 225 microM, and > 1 mM, respectively. 1-Phenylimidazole inhibited the maximal velocity of citrulline formation but did not alter the concentration of arginine providing half-maximal activity. 1-Phenylimidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase competitively versus (6R)-5,6,7,8-tetrahydro-L-biopterin (THB) with a Ki value of 0.7 microM, but inhibited citrulline formation by Ca(2+)-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells noncompetitively versus THB with a Ki value of 40 microM. Imidazole inhibited citrulline formation by the murine macrophage nitric oxide synthase noncompetitively versus THB with a Ki value of 48 microM. Neither imidazole nor 1-phenylimidazole inhibited the cytochrome c reductase activity of murine macrophage nitric oxide synthase at concentrations 100-fold higher than their IC50 values for inhibiting citrulline formation. The antifungal imidazoles miconazole, ketoconazole, and clotrimazole did not inhibit either citrulline formation or cytochrome c reduction by murine macrophage nitric oxide synthase at concentration as high as 200 microM. Ca(2+)-calmodulin-dependent nitric oxide synthase from GH3 pituitary cells exhibited a Kact for THB of 80 nM, while the inducible murine macrophage nitric oxide synthase exhibited a Kact of 8 microM.
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PMID:Interferon-gamma-inducible murine macrophage nitric oxide synthase: studies on the mechanism of inhibition by imidazole agents. 751 12

Citrulline formation by Ca(2+)-calmodulin (CaM)-dependent nitric oxide synthase from bovine brain is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 2.3 mM, 1.15 mM, 40 microM, and 2.5 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 0.16 microM and was competitive versus both arginine substrate and (6R)-5,6,7,8-tetrahydrobiopterin cofactor. The NADPH oxidase activity of bovine brain CaM-dependent nitric oxide synthase was inhibited by 7-nitroindazole with an IC50 value of 0.6 microM. Citrulline formation by the interferon-gamma/lipopolysaccharide-inducible nitric oxide synthase of murine macrophages (264.7 cell line) is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 470, 240, 56, and 20 microM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a Ki value of 1.6 microM and was noncompetitive versus arginine substrate but competitive versus (6R)-5,6,7,8-tetrahydrobiopterin cofactor. None of the indazoles tested inhibited the cytochrome c reductase activity of either nitric oxide synthase isoform at concentrations up to 1000-fold higher than their IC50 values for inhibition of citrulline formation. These observations are consistent with the proposal that the indazoles exert their inhibitory actions by interaction with the heme-iron of nitric oxide synthase such that oxygen does not bind.
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PMID:The inhibition of the constitutive and inducible nitric oxide synthase isoforms by indazole agents. 751 13

Citrulline formation by the Ca2+ CaM-dependent nitric oxide synthase of bovine endothelium is inhibited reversibly by 7-nitroindazole, 1-phenylimidazole, and imidazole. As measured at 0.67 microM (6R)-5,6,7,8-tetrahydrobiopterin (BH4), IC50 values of 0.8, 200, and 50 microM were determined for 7-nitroindazole, 1-phenylimidazole, and imidazole, respectively. Increasing concentrations of added BH4 cofactor increased the IC50 values for 7-nitroindazole and 1-phenylimidazole but did not alter the IC50 value for imidazole. 7-nitroindazole inhibited citrulline formation by the endothelial cNOS noncompetitively versus arginine substrate but competitively versus BH4 with a Ki value of 0.8 microM. 1-Phenylimidazole inhibited citrulline formation by the endothelial cNOS competitively versus both arginine substrate and BH4 with a Ki value of 50 microM. Imidazole inhibited citrulline formation competitively versus arginine substrate but noncompetitively versus BH4 with a Ki value of 50 microM. Neither 7-nitroindazole, 1-phenylimidazole, nor imidazole inhibited the cytochrome c reductase activity of endothelial cNOS at concentrations up to 5000-fold higher than their Ki values for inhibition of citrulline formation. By comparison with the previously determined kinetic properties of the other nitric oxide synthase isoforms, these observations establish that 1-phenylimidazole displays marked specificity for inhibiting the inducible nitric oxide synthase isoform and, since 7-nitroindazole has been reported not to elevate blood pressure (McCall et al., 1991, Br. J. Pharmacol. 102, 234-238), fails to confirm the expected insensitivity of the constitutive endothelial nitric oxide synthase to inhibition by 7-nitroindazole.
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PMID:The inhibition of the constitutive bovine endothelial nitric oxide synthase by imidazole and indazole agents. 752 99

Nitric oxide, a simple gas which serves as a neurotransmitter in the CNS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfaction has been questioned by experiments in which nitric oxide synthase, the enzyme that generates nitric oxide, could not be localized to the olfactory epithelium. We have localized nitric oxide synthase to the olfactory neurons in adult rat and catfish olfactory epithelia using a modified nicotinamide adenine dinucleotide phosphate diaphorase technique. In the rat, staining was also found in cells with morphology reminiscent of microvillar olfactory cells. In contrast, the respiratory epithelium and the sustentacular cells in the olfactory epithelium displayed no staining. The nicotinamide adenine dinucleotide phosphate diaphorase reaction, which has been shown to co-localize with immunohistochemical staining for nitric oxide synthase in the brain, was stimulated by addition of the nitric oxide synthase substrate L-arginine, and was inhibited by the nitric oxide synthase inhibitor L-NG-nitro arginine, indicating that staining was specific for nitric oxide synthase. Unilateral bulbectomy, which causes degeneration of mature olfactory neurons on the bulbectomized size, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were substantiated by biochemical assays for nitric oxide synthase by monitoring the production of [3H]-L-citrulline from [3H]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of mature olfactory neurons in rat and catfish olfactory epithelial suggesting the presence of nitric oxide synthase in these cells. Our histological and biochemical findings, in conjunction with data from other research, are supportive of a role for nitric oxide synthase in olfactory function.
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PMID:NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons. 754 62

Neurons that synthesize nitric oxide from arginine produce stoichiometric amounts of citrulline. We investigated whether nitric oxide-releasing enteric neurons have the capacity to recycle citrulline to arginine and thereby sustain nitrergic neurotransmission. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity, enzymes capable of citrulline to arginine conversion, were both localized in discrete populations of myenteric and submucosal neurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity co-localized with neuronal beta-nicotinamide adenine dinucleotide phosphate diaphorase staining, a marker for nitric oxide synthase. The functional significance of argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimulation, which were assessed by measuring contractions, inhibitory junction potentials and electrical slow waves. As shown previously, arginine analogues (L-nitroarginine methyl ester or L-nitroarginine; 100 microM) inhibited nitric oxide-dependent responses, and excess L-arginine restored inhibitory responses. Citrulline alone (0.1-2 mM) had no effect on nitrergic transmission under control conditions, but in the presence of L-nitroarginine methyl ester or L-nitroarginine, citrulline (0.1-2 mM) restored nitrergic transmission in a concentration-dependent manner. Other neutral amino acids (L-serine, L-leucine) did not mimic the effects of citrulline. Taken together, these data suggest that enteric nitrergic neurons have the enzymatic apparatus and functional capability of recycling citrulline to arginine.
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PMID:Recycling of L-citrulline to sustain nitric oxide-dependent enteric neurotransmission. 854 1

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.
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PMID:Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin. 863 54

Copper complexes with superoxide dismutase (SOD) activity show a wide range of pharmacological activities. We have investigated the effect of ([N,N'-bis(2-pyridylmethylene)-1,4-butanediamine]-(N,N',N", N"')]-Cu(II)-chloride (Cu-PuPy) and ([N,N'-bis(2-pyridyl-phenyl)methylene-1,4-butanediamine]-(N,N',N", N"'))-Cu(II)-chloride (Cu-PuPhePy) on the multiple catalytic functions of rat brain NO synthase (NOS). Both drugs inhibited the formation of L-citrulline as well as the enzymatic reduction of cytochrome c. The uncoupled oxidation of NADPH, catalyzed by neuronal NOS in the absence of L-arginine, was inhibited by Cu-PuPy but stimulated by Cu-PuPhePy, suggesting that the phenyl-substituted compound acts as a parasitic electron acceptor. Our data identify copper complexes with SOD mimicking activity as a novel class of neuronal NOS inhibitors blocking the reductase (diaphorase) activity of the enzyme.
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PMID:Structural and functional analogs of CuZn superoxide dismutase inhibit rat brain nitric oxide synthase by interference with the reductase (diaphorase) domain. 873 37


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