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)

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.
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PMID:Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. 3 75

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.
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PMID:Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques. 10 96

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.
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PMID:Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania). 11 98

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.
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PMID:Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis. 16 15

Steroidogenesis by Y-1 adrenal tumor cells in culture is stimulated by ATP, adenyl-5'-yl imidodiphosphate (App(NH)), adenosine 5'(beta, alpha-methylene)triphosphate (App(CH2)p), ADP, AMP, NAD, FAD, and adenosine but not by adenine or other nucleoside triphosphates. ATP, App(NH)p, App(CH2)p, and adenosine are active in the micromolar range. Like adrenocorticotropic hormone (ACTH), the onset of stimulation is immediate and occurs to the same extent. Also active are 2'- and 5'-deoxyadenosine and 2-chloroadenosine whereas adenine xyloside, L-riboside, or arabinoside have very low activity. Stimulation is accompanied by rounding of the cells. Dipyridamole, an inhibitor of adenosine transport, increased the response to low concentrations of adenosine, suggesting that adenosine acts externally. Stimulation of steroidogenesis by adenosine or phosphorylated adenosine compounds fails to occur in the presence of crystalline adenosine deaminase, and the effect of the enzyme on adenosine, ATP, or NAD stimulation is reversed by the competitive inhibitor erythro-9-[3-(nonane-2-ol)]adenine. This suggests that the enzyme acts specifically on adenosine and a requirement for the conversion of the above compounds to adenosine seems probable. The inhibition of cAMP effects by adenosine deaminase suggests that some of its effects are also mediated by conversion to adenosine. Similar stimulation is seen in I-10 Leydig tumor cells, but an ACTH-resistant mutant of Y-1 cells, called OS-3, is relatively resistant to adenosine. Adenosine and 2-chloroadenosine stimulate adenylate cyclase in membranes from Y-1 and I-10 cells at concentrations slightly greater than are effective for steroidogenesis. Other nucleosides are ineffective. Like the NH2-terminal 24 residues of adrenocorticotropic hormone (1-24 ACTH), the adenosine effect in Y-1 membranes is rapid and is on the Vmax intercept (versus ATP) and not on the Km. In contrast to steroidogenesis, adenosine is only a partial agonist for adenylate cyclase. It effect occurs in the presence of ITP, GTP, or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Theophylline inhibits adenosine-stimulated steroidogenesis. Inhibition of adenylate cyclase occurs in the same concentration range but is of the mixed type.
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PMID:Activation of steroidogenesis and adenylate cyclase by adenosine in adrenal and Leydig tumor cells. 18 24

Adenylyl sulfate reductase has been purified from the anaerobic sulfate-reducing bacterium, Desulfovibrio vulgaris, and judged to be homogenous by several criteria. Different forms of the enzyme could be visualized in polyacrylamide gels after electrophoresis and these polymeric species have been studied by a combination of absorption spectra, polyacrylamide gel electrophoresis, and sedimentation velocity experiments. A dimeric species of molecular weight 440,000 is stable in potassium phosphate buffer but can be dissociated to a 220,000 molecular weight species by either changing the buffer system to Tris-maleate or addition of AMP, DAMP, or adenylyl sulfate. Other catalytically active nucleotides are not capable of effecting this dissociation. The enzyme was determined to contain 12 non-heme irons, 12 acid-labile sulfides, and 1 FAD per molecule when calculated on the basis of a monomeric molecular weight of 220,000. ;el electrophoresis in the presence of sodium dodecyl sulfate indicated subunits of molecular weight 72,000 and 20,000. The extinction coefficient when determined in phosphate buffer at 372 nm is 108,000 M-1 cm-a. Steady state kinetic experiments employing ferricyanide, cytochrome c, and reduced methyl viologen as artificial electron transfer reagents were performed and the kinetic constants obtained under various conditions. Several nucleotide substrates were employed and compared in each assay with respect to Km and Vmax. The reduction of cytochrome c was found to be sensitive to both anaerobiosis and superoxide dismutase, suggesting the involvement of superoxide anions with this electron acceptor.
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PMID:Some physical and kinetic properties of adenylyl sulfate reductase from Desulfovibrio vulgaris. 23 33

The kinetic properties and regulation of activity of GTP-cyclohydrolase, the enzyme of the first step of flavinogenesis in the Pichia guilliermondii yests, partially purified by gel-filtration were studied. It was found that the curve of the dependence of reaction rate on substrate concentration is non-hyperbolic. FAD inhibited the enzyme activity, while riboflavin and FMN had no such effect. In addition to FAD, 5'-AMP, 3',5'-AMP, ADP, ATP, NAD and NADP inhibited the enzyme activity. Under combined action of FAD and AMP on GTP-cyclohydrolase no synergetic or antagonistic effects of the inhibitors on the enzyme activity were observed. The enzyme appreciably lost its sensitivity to FAD and AMP after thermal treatment. The data obtained suggest that GTP-cyclohydrolase from P. guilliermondii is an allosteric enzyme, which is inhibited by the end product of flavinogenesis FAD, as well as by other 5'-AMP-containing nucleotides.
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PMID:[Regulation of the activity of GTP-cyclohydrolase, the enzyme of the first step of flavinogenesis in yeasts]. 73 22

The reaction mechanism of adenosine 5'-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s-1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH-) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25-35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2-. the activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.
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PMID:A study on the reaction mechanism of adenosine 5'-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein. 83 49

AMP-deaminases were isolated and partially purified from subfractions of soluble mitochondrial proteins of rat liver under normal conditions and in ethanol intoxication. Repeated freezing and thawing of the mitochondrial fractions from liver of rats, which were treated with ethanol (1 ml of 32% solution daily for 7 days, intraperitoneally), liberated into the subfraction of soluble mitochondrial proteins significantly less AMP-deaminases, as compared with the control animals. The enzyme preparations obtained from intoxicated and intact animals were quite similarly inactivated by controlled heating, deaminated at similar rates AMP, ADP, FAD and some other nitrogenous compounds (but did not deaminate adenosine and some structural analogues of AMP). However, an inhibitory effect of the structural analogues of AMP and of nucleosides was significantly higher towards the AMP-deaminase from healthy rats as compared with the corresponding enzyme preparations obtained from the ethanol-treated animals. The increase in velocity of enzymatic AMP deamination in the subfraction of soluble mitochondrial proteins apparently does not represent a suitable target for possible therapeutic approaches to control the phenomenon, observed in the experimental ethanol intoxication, of stimulation of the deaminating activity in total mitochondrial fraction of rat liver.
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PMID:[Adenylate deaminase of the liver mitochondria in normal state and in alcoholic intoxication]. 103 94

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
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PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 120 21


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