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)

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.
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PMID:Kinetic properties of guinea pig liver microsomal NADPH-cytochrome P-450 reductase. 632 Oct 97

The nucleotide sequence of a 3614 base-pair segment of DNA containing the sdhA gene, encoding the flavoprotein subunit of succinate dehydrogenase of Escherichia coli, and two genes sdhC and sdhD, encoding small hydrophobic subunits, has been determined. Together with the iron-sulphur protein gene (sdhB) these genes form an operon (sdhCDAB) situated between the citrate synthase gene (gltA) and the 2-oxoglutarate dehydrogenase complex genes (sucAB): gltA-sdhCDAB-sucAB. Transcription of the gltA and sdhCDAB gene appears to diverge from a single intergenic region that contains two pairs of potential promoter sequences and two putative CRP (cyclic AMP receptor protein)-binding sites. The sdhA structural gene comprises 1761 base-pairs (587 codons, excluding the initiation codon, AUG) and it encodes a polypeptide of Mr 64268 that is strikingly homologous with the flavoprotein subunit of fumarate reductase (frdA gene product). The FAD-binding region, including the histidine residue at the FAD-attachment site, has been identified by its homology with other flavoproteins and with the flavopeptide of the bovine heart mitochondrial succinate dehydrogenase. Potential active-site cysteine and histidine residues have also been indicated by the comparisons. The sdhC (384 base-pairs) and sdhD (342 base-pairs) structural genes encode two strongly hydrophobic proteins of Mr 14167 and 12792 respectively. These proteins resemble in size and composition, but not sequence, the membrane anchor proteins of fumarate reductase (the frdC and frdD gene products).
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PMID:Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli. 638 59

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

An L-amino acid oxidase (L-amino-acid oxygen oxidoreductase (deaminating), EC 1.4.3.2) from the blue-green alga Anacystis nidulans has been purified to homogeneity with an overall yield of about 10%. Purification included ammonium sulfate fractionation and CM-Sephadex, DEAE-Sephadex, and hydroxyapatite chromatography. The purified enzyme has an absorption spectrum which is characteristic of a flavoprotein, and contains 1 mol FAD per mol enzyme. The native enzyme has a molecular weight of 98 000 as determined by gel exclusion chromatography. Electrophoresis in SDS-polyacrylamide gels gives a single protein band corresponding to a molecular weight of 49 000, which suggests that the native enzyme is composed of 2 subunits of equal molecular weight. As previously demonstrated, the enzyme catalyzes the oxidative deamination of the basic amino acids: L-arginine, L-lysine, L-ornithine and L-histidine. In the presence of catalase and of any of these amino acids, 0.5 mol O2 is consumed, and 1 mol ammonia is formed for each mol amino acid oxidized. HCN is formed from L-histidine when the L-amino acid oxidase is supplemented with peroxidase. In addition to the unusual substrate specificity of this L-amino acid ozidase, it also has an unusual set of inhibitors including o-phenanthroline as well as divalent cations of which Cu2+, Zn2+, and Cd2+ are the most effective ones, but Mg2+ and Ca2+ also inhibit. This inhibition can be reversed by chelating agents such as EDTA. ATP and ADP, but not AMP, can also overcome the inhibition caused by Mg2+, for example. The inhibitory effect of cations can be demonstrated in vivo.
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PMID:Some properties of a basic L-amino-acid oxidase from Anacystis nidulans. 676 43

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.
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PMID:Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide. 678 Mar 52

The total -SH content of purified NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) from rabbit liver microsomes accessible to an excess equivalent of PCMB was 7.0 +/- 0.3 mol thiol groups/mol protein. The modification of four -SH groups at low concentration of PCMB stimulated the activity of the enzyme. On the other hand, further blocking of -SH groups (6-7 mol -SH groups/Mol protein) with an excess amount of PCMB completely inhibited cytochrome c (or DCPI) reductase activity. The fluorescence quenching of the flavin was rapidly removed by binding of PCMB to a fifth and sixth -SH group during a gradual titration. Kinetic and fluorimetric analyses confirmed the suggestion that these two -SH groups essential for catalytic function were partly protected by NADP+ or 2'-AMP against the reaction with PCMB. Excess PCMB begins to compete with the ligand preincubated with the enzyme. The spectral perturbation on the addition of approx. 6-7 equiv. PCMB/mol enzyme is accompanied by a slight blue shift of the absorbance maximum at 380 nm, with the appearance of a pronounced shoulder at 475 nm. In contrast to the native enzyme, 3-electron-reduced semiquinone form of PCMB-treated enzyme showed the same absorption spectrum as 1-electron-reduced semiquinone which has an absorption maximum at 585 nm with a broad shoulder around 635 nm. An inhibitory effect may be attributable to the fact that NADPH is less accessible to the FAD binding site as well as the pyridine nucleotide binding site, since the rate of FAD reduction becomes extremely slow after complete modification.
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PMID:Location of functional -SH groups in NADPH-cytochrome P-450 reductase from rabbit liver microsomes. 679 74

Six sulfhydryl group were determined after complete denaturation of NADPH-cytochrome P-450 reductase; of these, about 5.2 in both the native holoenzyme and FMN-depleted enzyme are accessible to p-hydroxychloromercuribenzoate (pCMB), which may be differentiated as follows: four --SH groups are modified by low concentration of the reagent but are not essentially involved in the catalytic function; additional block of one --SH group at high concentrations of pCMB completely inhibited the reductase activity. The fluorescence quenching of the FAD in the FMN-depleted enzyme was removed after the fifth --SH group was reacted slowly with pCMB. Kinetic and fluorometric analysis indicated that this finally modified --SH group was assumed to be essential for the activity and significantly protected by either 1 mM NADP+ or 2'-AMP against attack by mercurial compounds. A strong negative ellipticity at around 450 nm is clearly decreased upon binding of pCMB to an essential --SH group, while the CD spectra in the near and far UV region show only minor differences during the modification of --SH groups. Removal of the FMN prosthetic group from the native holoprotein results in 1.25-fold greater tryptophan fluorescence with a slight red shift of the emission maximum from 332 to 336 nm, and FMN reconstitution reduces the protein fluorescence quantum yield to approximately that of the holoprotein. Oxidation of tryptophan indol rings of the FMN-depleted enzyme is associated with a loss of FMN binding ability to the protein which causes the inactivation of cytochrome c reductase activity, but ferricyanide reductase activity is not strongly affected by tryptophan modification.
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PMID:Studies on FAD- and FMN-binding domains in NADPH-cytochrome P-450 reductase from rabbit liver microsomes. 681 23

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.
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PMID:Purification and characterization of NADH dehydrogenase from Bacillus subtilis. 681 92

Isolation and partial purification of AMP-deaminase from subfraction of soluble proteins of the mitochondrial fraction from rat liver is described. The enzyme preparations obtained deaminated AMP at the highest rate from pH 6.4 to 6.6. At the optimal pH value and in presence of optimal AMP concentrations the AMP-deaminase preparation was not activated by ATP or K+ and was inhibited by inorganic phosphate. Relationship was noted between both the content of protein in the enzyme preparations and length of the interval from composing the samples to monitoring the enzymatic activity and the following parameters of the AMP-deaminase: (a) shape of curves describing the rate of AMP deamination as a function of the nucleotide concentration, (b) reversible decrease in the AMP-deaminating activity after dialysis, (c) properties to deaminate, besides AMP, also some other nucleotides (ADP, NAD, FAD), (d) dynamics of inactivation of the enzyme preparations by controlled heating. The properties of the partially purified AMP-deaminase from the subfraction of rat liver soluble mitochondrial proteins were not identical with those described previously for other AMP-deaminases.
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PMID:[Partial purification and properties of the adenylate deaminase from subfractions of soluble mitochondrial proteins of rat liver]. 728 62

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].
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PMID:Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase. 743 74


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