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)

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).
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PMID:[Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide]. 611 Nov 46

Two components of the histidine permease in Salmonella typhimurium, the membrane-bound P and M proteins, react with the photoaffinity labeling reagent 8-azido-ATP in isolated membranes. The extent of labeling is decreased by the addition of ATP and somewhat less by addition of GTP, CTP, UTP, and ADP. Cyclic AMP, NAD, FAD, and S-adenosylmethionine have little effect. We propose that one or both of these proteins have a site capable of binding an adenine nucleotide and that, therefore, they may be involved in the energy-coupling step in active transport.
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PMID:ATP-binding sites in the membrane components of histidine permease, a periplasmic transport system. 623 89

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.
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PMID:Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography. 648 2

Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b558 (flavocytochrome b558). At least two cytosolic components, 67 kDa protein and a phosphorylated 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2(-)-generating NADPH oxidase system. More recently, the membrane (Raps) and cytosolic (Racs) GTP-binding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system.
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PMID:Activation factors of neutrophil NADPH oxidase complex. 801 44

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
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PMID:NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization. 811 Jan 98

Nitric oxide (NO) was discovered to be a potent vasodilator, inhibitor of platelet aggregation, and active species of nitroglycerin before the discovery of endothelium-derived relaxing factor (EDRF) in 1980. Subsequent studies revealed that EDRF is NO, and is synthesized by mammalian cells from L-arginine through a complex oxidation reaction catalyzed by the flavo-hemoprotein NO synthase (NOS). NOS catalyzes the NADPH- and oxygen-dependent oxygenation of L-arginine to NO plus L-citrulline in a reaction that requires at least six cofactors including NADPH, FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. NO elicits its known physiological actions by activating cytosolic guanylate cyclase, which converts GTP to cyclic GMP. Endothelial NOS and neuronal NOS are constitutively present and activated by increases in intracellular calcium triggered by endogenous chemicals. NO then diffuses into nearby target cells to elevate cyclic GMP levels and thereby trigger cell function. NOS activity can also be regulated by a negative feedback mechanism involving NO itself. Much greater quantities of NO are produced pathophysiologically by a distinct form of NOS that can be induced in vascular endothelium, smooth muscle and macrophages by endotoxin and cytokines. This high-output production of NO is not regulated by calcium and is cytotoxic by mechanisms involving interaction with iron-containing proteins.
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PMID:Physiology and pathophysiology of nitric oxide. 874 1

The superoxide (O-2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The canonical view is that the induction of O-2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67(phox) and Rac1, in the total absence of p47(phox) (turnover = 60 mol O-2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O-2/s/mol cytochrome b559) could only be obtained in the presence of p47(phox). O-2 production, in the absence of p47(phox), was dependent on: high molar ratios of p67(phox) and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67(phox) and Rac1, were incapable of activation. We conclude that p67(phox) and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47(phox) appears to facilitate or stabilize the interaction of p67(phox) and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O-2 under physiological conditions.
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PMID:The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production. 893 91

Adenylylsulfate reductase (EC 1.8.99.2) isolated from Desulfovibrio vulgaris Miyazaki catalyzes electron transfer from dihydroflavin coenzyme (FADH2, FMNH2, or dihydroriboflavin) to adenylyl sulfate (APS), and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS. The reaction with FAD as an electron mediator was markedly stimulated in the presence of menadione. Km of the enzyme was about 0.015 mM for riboflavin and FAD in the presence of menadione. Free flavin coenzyme was found to be the normal cellular constituent. These observations suggested that free flavin coenzyme may be a physiological electron carrier for APS reductase, and the enzyme may be called AMP, sulfite:flavin oxidoreductase. Km (APS) of this enzyme is lower than 1 microM. The enzyme is not inhibited by ATP and GTP, but was inhibited by AMP and sulfite. Its extremely low Km (APS) enables this enzyme to reduce any traces of cytosolic APS which is present only at micromolar concentration, and inhibition by sulfite makes this organism utilize an energetically favorable electron acceptor, sulfite, preferentially over APS which is produced from sulfate at the cost of ATP.
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PMID:Catalytic properties of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki. 911 53

Blue-light stimulation of red light-saturated photosynthetic oxygen evolution in Laminaria saccharina (L.) Lamouroux could be abolished within 5 days by incubation in a solution of 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase I (E.C.3.5.4.16) activity. Photosynthesis in red light was not detectably affected. GTP-cyclohydrolase I, which catalyses the first step in the biosynthetic pathway of pteridines, was shown to be active in Laminaria. Under conditions that lead to complete inhibition of the photosynthetic stimulation, DAHP reduced the content of the pteridines in the tissue considerably. The amount of pterin was about 14%, that of biopterin was about 45% and that of an unidentified pteridine was about 27% of those of the controls. By contrast, the total concentration of flavins (FAD + FMN - riboflavin) was not significantly affected. The results suggest that pterins may be involved in the response of photosynthesis to blue light, possibly participating in photoreception.
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PMID:Inhibition of pteridine biosynthesis eliminates blue-light dependent stimulation of red-light saturated photosynthesis in Laminaria saccharina (L.) Lamouroux. 920 91

p-Hydroxybenzoate hydroxylase, D-amino acid oxidase, cholesterol oxidase and glucose oxidase form a family of structurally related flavoenzymes. Comparison of their three-dimensional structures reveal how the same FAD-binding scaffold has been employed to implement diverse active-site architectures, suited for different types of catalytic reactions. The substrate binding mode differs in each of these enzymes, with the catalytically relevant residues not located on homologous positions. A common feature is provided by the ability of these enzyme to bury their substrates beneath the protein surface. In D-amino acid oxidase and cholesterol oxidase, a loop forms a 'lid' controlling the active site accessibility, whereas in p-hydroxybenzoate hydroxylase is the flavin itself, which swings out to allow substrate binding. The crystallographic analysis has revealed that the GTP-dissociation inhibitor of RAB GTPases has a folding topology remarkably similar to p-hydroxybenzoate hydroxylase. This finding highlights the versatile nature of this folding topology, which in addition to flavin-dependent catalysis, is suited for diverse functions, such as the regulation of GTPases.
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PMID:The PHBH fold: not only flavoenzymes. 954 98


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