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)

The cytokine-induced nitric oxide synthase (NOS) of macrophages is a homodimeric enzyme that contains iron protoporphorin IX (heme), FAD, FMN, tetrahydrobiopterin, and calmodulin. To investigate how the enzyme's quaternary structure relates to its catalytic activity and binding of prosthetic groups, dimeric NOS and its subunits were purified separately and their composition and catalytic properties compared. In contrast to dimeric NOS, purified subunits did not synthesize NO or contain bound heme or tetrahydrobiopterin. However, the subunits did contain FAD, FMN, and calmodulin in amounts comparable with dimeric NOS, displayed the light absorbance spectrum of an FAD- and FMN-containing flavoprotein, and generated an air-stable flavin semiquinone radical upon reduction of their ferricyanide-oxidized form. Dimeric NOS and NOS subunits were equivalent in catalyzing electron transfer from NADPH to cytochrome c, dichlorophenolindophenol, or ferricyanide at rates that were 8-30-fold faster than the maximal rate of NO synthesis by dimeric NOS. Reconstitution of subunit NO synthesis required their incubation with L-arginine, tetrahydrobiopterin, and stoichiometric amounts of heme and correlated with formation of a proportional amount of dimeric NOS in all cases. The dimeric NOS reconstituted from its subunits contained 0.9 heme and 0.44 tetrahydrobiopterin bound per subunit and had the spectral and catalytic properties of native dimeric NOS. Thus, NOS subunits are NADPH-dependent reductases that acquire the capacity to synthesize NO only through their dimerization and binding of heme and tetrahydrobiopterin. The ability of heme, tetrahydrobiopterin, and L-arginine to promote subunit dimerization is unprecedented and suggests novel roles for these molecules in forming and stabilizing the active dimeric NOS.
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PMID:Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. 769 6

Nitric oxide is a recently discovered biomolecule with a broad range of actions and synthesized by nitric oxide synthases. We investigated effects of lipids on particulate nitric oxide synthase purified from cultured bovine aortic endothelial cells by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline. Phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine dose-dependently enhanced the enzyme activity up to 3 fold in the presence of Ca2+, calmodulin, NADPH, FAD, and (6R)-5,6,7,8-tetrahydrobiopterin. These phospholipids increased the Vmax of nitric oxide synthase without altering the Km for L-arginine and the affinities for Ca2+ and calmodulin. These findings suggest that phospholipids play an important role in modulating endothelial nitric oxide synthase activity.
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PMID:Activation of nitric oxide synthase from cultured aortic endothelial cells by phospholipids. 769 45

Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8-kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open-reading frame of 3441 bases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that the protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulin-binding site, consisting of a strongly hydrophobic region surrounded by basic amino acids, is present. The full-length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin-dependent nitric-oxide synthase. A structural comparison suggested that the liver nitric-oxide synthase is the same as the macrophage enzyme. Northern-blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P. acnes and lipopolysaccharide.
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PMID:Molecular cloning of a cDNA encoding an inducible calmodulin-dependent nitric-oxide synthase from rat liver and its expression in COS 1 cells. 769 62

A 2618-bp cDNA that encodes the human mitochondrial glycerol-3-phosphate dehydrogenase has been isolated from a HeLa cell cDNA library and the nucleotide sequence determined. An open reading frame encodes a protein of 727 amino acids that is 96% similar to the rat protein and, like the rat protein, contains sites homologous to the Ca(2+)-binding sites of calmodulin, as well as FAD- and putative glycerol-phosphate-binding sites.
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PMID:The sequence of a human mitochondrial glycerol-3-phosphate dehydrogenase-encoding cDNA. 782 23

The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) is an essential component of the glycerol phosphate shuttle and is abundant in the pancreatic insulin cell, skeletal muscle, and brain. Although DNA clones for this enzyme and its homologues have been isolated from bacteria and yeast, it has never been cloned from a higher eukaryote. We have cloned and sequenced cDNAs encoding the rat mitochondrial glycerol-3-phosphate dehydrogenase. The longest cDNA (2337 base pairs) encodes a deduced protein of 727 amino acids that shows strong homology to the yeast and bacterial FAD-dependent glycerol phosphate dehydrogenases. The amino terminus of the purified mature protein was sequenced and shows identity with the deduced amino acid sequence beginning with residue 43. The 42 preceding amino acids are consistent with a mitochondrial leader peptide. A highly conserved FAD-binding domain and conserved regions possibly involved with glycerol phosphate binding are present. An unexpected finding was the homology of the deduced protein to calmodulin. Analysis of the deduced protein sequence shows a region near the carboxyl terminus containing two sequences homologous to "EF-hand" calcium-binding domains that are not present in the shorter yeast and bacterial homologues. The second of these domains appears to have features compatible with considerable affinity for calcium, whereas the first does not. The finding of a potential calcium-binding region is consistent with the known enhancement by calcium of the mammalian enzyme activity at low substrate concentrations and the lack of a requirement for calmodulin. This is the first report of EF-hands in a metabolic enzyme or in a mitochondrial protein.
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PMID:Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA. Evidence for EF-hand calcium-binding domains. 818 39

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

cDNAs which encode the rat testis and pancreatic islet mitochondrial glycerol phosphate dehydrogenase (mGPD) (EC 1.1.99.5), the key enzyme of the glycerol phosphate shuttle, were recently cloned and sequenced and found to contain calmodulin-like calcium-binding sequences, thus explaining the widely observed calcium activation of the enzyme from many tissues of higher eukaryotes. mGPD activity and protein, as judged from Western analysis, appear to be most abundant in testis and pancreatic islets in the rat. mGPD is known to be located within the inner mitochondrial membrane. At a physiologic concentration of glycerol phosphate (75 microM), half maximal activity of Triton X-100-solubilized testis mGPD was seen in the presence of 0.1-0.25 microM free calcium. Calcium (10(-6)-10(-5) M) lowered the Km of mGPD from 2.5 mM glycerol phosphate (islet mGPD) and 3.2 mM glycerol phosphate (testis mGPD) to 0.4 mM glycerol phosphate. Calcium activation of mGPD from both testis and islets was not prevented by calmodulin inhibitors, which is consistent with mGPD possessing regions that can mediate its own activation by calcium. 45Calcium overlay experiments, in which proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed with 45Ca, showed that mGPD is a major calcium-binding protein in testis mitochondrial membranes. A hydropathy plot suggested that the mature mGPD protein has three transmembrane helices. The first membrane-spanning region coincides with the FAD site and thus this site is placed within the membrane. The hydropathy analysis indicated that the calcium-binding region and the putative glycerol phosphate-binding site lie outside the membrane exposed to the cytosolic environment. This is consistent with earlier biochemical evidence which indicated that these sites are situated outside the membrane (M. Klingenberg, Eur. J. Biochem. 13, 247-252, 1970). This suggests that cytosolic calcium can regulate mGPD activity. Since simultaneous oscillations in electrical activity, cytosolic calcium, glycolysis, and insulin release occur in the pancreatic beta cell, mGPD activity might also fluctuate and allow the glycerol phosphate shuttle to participate in glycolytic oscillations.
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PMID:Calcium activation of mitochondrial glycerol phosphate dehydrogenase restudied. 857 75

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exits as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 HZ at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH., was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D=5.2cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.
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PMID:EPR spectroscopic characterization of neuronal NO synthase. 861 87

Inducible nitric oxide (NO) synthase (iNOS) is comprised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one full-length subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2',5'-ADP affinity chromatography. The heterodimer catalyzed NADPH-dependent NO synthesis from L-arginine at a rate of 52 +/- 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.
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PMID:Heme iron reduction and catalysis by a nitric oxide synthase heterodimer containing one reductase and two oxygenase domains. 863 49

We have isolated and sequenced clones of an inducible nitric oxide synthase (iNOS) from cDNA library of interleukin-1 beta-treated rat aortic endothelial cells (EC) completely free from other cell types. The cloned cDNA contains an ORF consisting of 3441 bp, which encodes 1147 amino acid residues. The deduced amino acid sequence contains putative binding sites for NADPH, FMN, FAD, calmodulin and heme. By comparison with amino acid sequences of other isoforms, rat EC iNOS is very similar (92% similarity) to mouse macrophage iNOS. There are four AUUUA motifs, potentially responsible for the instability of the mRNA, in 3'non-coding region of rat EC iNOS cDNA. Transient transfection of cultured rat vascular smooth-muscle cells with a full-length rat EC iNOS cDNA/SR alpha 296 construct by electroporation resulted in massive NO production in proportion to the doses of cDNA used. Northern blot analysis using rat EC iNOS cDNA as a probe revealed that cycloheximide treatment led to a marked accumulation of iNOS mRNA in the presence and absence of interleukin-1 beta. No appreciable decay in the cycloheximide-induced iNOS mRNA accumulation was observed, suggesting that blockade of de novo protein synthesis stabilizes mRNA. These results demonstrate that rat EC iNOS is identical (or very similar) to macrophage iNOS, and suggest that the EC iNOS gene is also regulated at the post-transcriptional level.
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PMID:Molecular cloning of endothelial, inducible nitric oxide synthase gene from rat aortic endothelial cell. 864 11


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