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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca(2+)-independent conversion of L-arginine to L-citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent Km of 16.5 microM for L-arginine and is dependent on NADPH, FAD, and tetrahydrobiopterin. LPS also induces NOS in C6 glioma cells and microglial cultures but not in cultured cortical neurons. The expression of NOS in astrocytes and microglial cells has been confirmed by immunocytochemical staining using an antibody to the inducible NOS of mouse macrophages and by histochemical staining for NADPH diaphorase activity. We conclude that glial cells of the central nervous system can express an inducible form of NOS similar to the inducible NOS of macrophages. Inducible NOS in glia may, by generating nitric oxide, contribute to the neuronal damage associated with cerebral ischemia and/or demyelinating diseases.
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PMID:Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. 127 98

Brain nitric oxide synthase (NOS), which utilizes NADPH and calcium/calmodulin as cofactors for metabolizing L-arginine to nitric oxide (NO) and L-citrulline, contains recognition sites for the flavins FAD and FMN. Using a spin-trapping technique combined with electron spin resonance spectroscopy, we report that brain NOS generates superoxide O2-. in a calcium/calmodulin-dependent manner. The "specific inhibitors" of NOS, NG-monomethyl L-arginine (L-NMMA), and NG-nitro-L-arginine methyl ester (L-NAME), have different effects on O2-. generation. For L-NMMA, O2-. production is unaffected, while for L-NAME, inhibition of this free radical is concentration-dependent.
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PMID:Generation of superoxide by purified brain nitric oxide synthase. 128 Feb 57

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

Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.
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PMID:L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen. 169 55

We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H L-arginine to 3H L-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mM NADPH, 10 microM tetrahydrobiopterin, 2 microM FAD, 100 microM free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 microM) and FAD inhibitor diphenyliodonium chloride (1 microM) significantly reduced enzyme activity. The EC50 for calcium was 0.1 microM and Km for L-arginine 2.00 +/- 0.49 microM with Vmax 55.8 +/- 28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-L-arginine and ng-monomethyl-L-arginine in a concentration-dependent manner (10(-8)-10(-4) M). A calcium-independent NO synthase activity was also determined, but only constituted between 5-6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform.
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PMID:Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree. 750 55

Nitric oxide synthase (NOS) has been purified over 6,500-fold with a 3.4% yield from rat colorectum with 2',5'-ADP-Sepharose, DEAE cellulose, and gel filtration. The purified enzyme gave a single band corresponding to an apparent molecular mass of 160 Dka on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When assayed in the requisite presence of L-arginine, CaCl2, NADPH, calmodulin, tetrahydro-L-biopterin, and FAD, the purified enzyme exhibited a specific activity of 328 nmol/min/mg L-citrulline formed and an apparent Km for L-arginine of 2.9 microM. Amino acid sequencing of 12 peptides revealed identical sequences to that of the neuronal type enzyme except for two altered amino acid residues. When partial reverse transcription-polymerase chain reaction of RNA from rat colorectum and cerebellum was performed using primers designed according to the amino acid sequences determined, these amino acid changes were found in both cDNA fragments, indicating the identity of the colorectal enzyme to the cerebellar one. A polyclonal antibody raised against NOS purified from rat cerebellum cross-reacted with the NOS from colorectum but not that from IFN-gamma stimulated macrophage-derived cells, RAW 264.7. Immunohistochemical analysis of the colorectum using this specific antibody indicated that Auerbach's plexus is strongly immunoreactive, supporting the hypothesis that NO is an inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum.
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PMID:Nitric oxide synthase from rat colorectum: purification, peptide sequencing, partial PCR cloning, and immunohistochemistry. 752 37

Nitric oxide synthase (EC 1.14.23) was discovered in a Nocardia species. The bacterial nitric oxide synthase was purified as much as 380 fold by affinity chromatography over 2',5'-ADP-agarose. The partially purified enzyme required NADPH, O2, CA++, FAD, FMN, and tetrahydrobiopterin as cofactors in the conversion of L-arginine to L-citrulline and nitric oxide. The apparent Km for L-arginine was determined to be 8.2 microM, and the Vmax was 840 nmole NADPH consumed/min/mg protein. The enzyme was competetively inhibited by NG-nitro-arginine with an apparent Ki of 14.6 microM. The experimental evidence provides confirmation of the first microbial nitric oxide synthase in microorganisms.
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PMID:A bacterial nitric oxide synthase from a Nocardia species. 752 44

Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 microM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 microM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide synthesis in retinal photoreceptor cells. 754 7

Brain NO (nitric oxide) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multi-functional oxidoreductase catalyzing oxidation of L-arginine to NO and L-citrulline, formation of H2O2, and reduction of cytochrome c. We show that substrate analogues and inhibitors interacting with the heme block both the reductive activation of oxygen and the oxidation of L-arginine without affecting cytochrome c reduction. We further demonstrate that N omega-hydroxy-L-arginine is an intermediate in enzymatic NO synthesis. The ratio of L-citrulline to free N omega-hydroxy-L-arginine was > or = 50 under various assay conditions, but could markedly be reduced down to 4 by redox active inhibitors. Brain NO synthase is shown to utilize both L-arginine and N omega-hydroxy-L-arginine for the formation of stoichiometric amounts of NO and L-citrulline. Tetrahydrobiopterin equally enhanced reaction rates from either substrate (approximately 5-fold), but its rate accelerating effects were only observed at NADPH concentrations > or = 3 microM. In the absence of L-arginine or tetrahydrobiopterin, brain NO synthase catalyzes the generation of H2O2. We now show that, in contrast to L-arginine, N omega-hydroxy-L-arginine fully blocked H2O2 formation in the absence of exogenous tetrahydrobiopterin, indicating that N omega-hydroxy-L-arginine is a direct inhibitor of enzymatic oxygen activation. Based on these data, a hypothetical mechanism of enzymatic NO formation is discussed.
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PMID:Multiple catalytic functions of brain nitric oxide synthase. Biochemical characterization, cofactor-requirement, and the role of N omega-hydroxy-L-arginine as an intermediate. 768 5

During the last decade, a multitude of experimental arguments have led to the concept that EDRF is nitric oxide (NO), a messenger not only involved in the control of vasomotor tone but also in vascular homeostasis, neuronal and immunological functions. Regardless of its origin, endogenous NO is produced through the conversion of L-arginine to L-citrulline by NO-synthase (NOS) from which several isoforms have recently been isolated, purified and cloned. NOS-type I (isolated from brain) and type III (isolated from endothelial cells) are termed "constitutive-NOS" and produce picomolar levels of NO from which only a small fraction elicits physiological responses. These isoforms are regulated by Ca(2+)-calmodulin with NADPH, FAD/FMN and tetrahydrobiopterin as co-factors and reveal a high degree of homology with the amino-acid sequence of cytochrome P450 reductase within the C-terminal domain. Functionally, neuronal-NOS type I is important in neurotransmission (modulation of NMDA receptor), the central control of vascular homeostasis and possibly learning and memory. In the peripheral nervous system, NOS appears to be linked to nonadrenergic noncholinergic (NANC) neuronal pathways. Endothelial-NOS type III is essential for the control of vascular tone in response to the release of endogenous mediators, although shear stress is the major trigger of endothelial-NOS activity under physiological conditions. NOS-type III also contributes to the prevention of abnormal platelet aggregation. NOS-types II and IV (isolated from macrophages) are Ca(2+)-calmodulin independent and are termed "inducible-NOS" since their activation is only promoted under pathophysiological situations where macrophages exert cytotoxic effects in response to cytokines. In contrast with NOS-types I and III, activation of NOS-type II in these cells induces the formation of nanomolar levels of NO which act as a defense mechanism of the immune system. Dysfunctions of the L-arginine-NO pathway have been characterized in multiple diseases (atherosclerosis, hypertension, diabetes, sepsis, cerebral ischemia, etc) and the design of more selective activators/inhibitors of NOS isoforms is a new challenge for the understanding of their pathophysiology and treatment.
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PMID:Nitric oxide: an ubiquitous messenger. 829 80


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