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)

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

The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at approximately 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[14C]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in purified murine macrophage NO synthase and have suggested the identity of these reaction centers in both the constitutive (cerebellar) and inducible (macrophage) forms of NO synthase.
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PMID:Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide. 128 Aug 19

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 (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.
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PMID:Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites. 137 33

An endotoxin-induced form of nitric oxide synthase (EC 1.14.23) was purified to homogeneity from rat liver by sequential anion-exchange chromatography and affinity chromatography using 2',5'-ADP-Sepharose. The enzyme has a subunit molecular mass of 135 kDa as determined by SDS/PAGE, a maximum specific activity of 462 nmol of citrulline formed from arginine per min per mg, and a Km for arginine of 11 microM. The enzyme was strongly stimulated by the addition of calmodulin with an EC50 of 2 nM, but removal of free calcium from the assay medium only reduced activity by 15%. Calmodulin inhibitors significantly reduced the enzyme activity. Tetrahydrobiopterin, FAD, and FMN were all required for full enzyme activity. This form of endotoxin-induced nitric oxide synthase from liver differs from the inducible enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium ion concentration.
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PMID:Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver. 137 17

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.
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PMID:Purification of insoluble nitric oxide synthase from rat cerebellum. 137 22

Nitric oxide (NO) is a messenger molecule of macrophages, endothelial cells in blood vessels, and neurons. A neuronal form of NO synthase (NOS) has been previously cloned. We now report the molecular cloning of macrophage NOS. The macrophage enzyme displays 50% sequence identity to the neuronal enzyme. Like neuronal NOS, macrophage NOS has recognition sites for FAD, FMN, and NADPH and also has a consensus calmodulin binding site. Macrophage NOS mRNA is strikingly inducible; it is absent in quiescent macrophages or spleen but is prominent 2-6 hr after endotoxin treatment.
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PMID:Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. 137 16

1. Partially purified soluble nitric oxide (NO) synthase was isolated from the bovine retractor penis muscle (BRP), a tissue in which the inhibitory response to non-adrenergic non-cholinergic nerve (NANC) stimulation appears to be mediated by NO or NO-like material. 2. NO synthase from BRP used L-arginine as a substrate, required NADPH, tetrahydrobiopterin, and FAD as co-factors and was Ca2+/calmodulin-dependent. The activity of NO synthase was inhibited by NG-methyl-L-arginine and NG-nitro-L-arginine, and haemoglobin blocked the effect of NO formed by the enzyme. 3. On reducing SDS polyacrylamide gel electrophoresis the apparent molecular mass of NO synthase from BRP was 160 +/- 2 kDa, which is similar to that of the cerebellar NO synthase. Protein immunoblot and immunoprecipitation showed that NO synthase from BRP cross-reacted with the selective antiserum to neuronal NO synthase from rat cerebellum. 4. Immunohistochemistry using the same antiserum demonstrated that NO synthase in BRP was located exclusively within nerve fibres. Thus, autonomic nerves synthesizing the NANC neurotransmitter seem to contain an isoform of NO synthase which is similar to that from rat cerebellum.
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PMID:Characterization and localization of nitric oxide synthase in non-adrenergic non-cholinergic nerves from bovine retractor penis muscles. 138 87

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.
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PMID:Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. 170 88

Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.
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PMID:Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. 171 77


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