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 soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.
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PMID:Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation. 117 Aug 94

Serum glutathione reductase (NADPH-GSSG oxidoreductase, EC. 1.6.4.2 (GR)) has been examined in cystic fibrosis subjects (CF), obligate CF heterozygotes, and control subjects. Serum protein concentration was similar in the three groups. Regardless of the units used to express activity (milligrams of protein or milliters of serum) or whether or not samples were dialyzed against water or phosphate buffer, mean serum GR in CF was greater than in control subjects (P less than or equal to 0.002) in all series over several years. Under the above assay conditions no difference in serum GR between control subjects and carriers was detected. Calculated and assayed values of combined control and CF sera agreed as did expected and observed 50% activity in 1:2 sera dilutions in CF, control subjects, and carriers. Addition of FAD to incubation media did not effect enzyme activity in the three groups. Differences between CF and control subjects persisted after dialysis in membranes permitting passage of molecules of approximately 12,000 mol wt or less. These findings would tend to exclude the effect of extraneous serum factors in explaining the diffferences between CF and control subjects. The percentage of initial GR activity after four days storage (0-4 degrees) was significantly greater in CF than in control subjects (P less than 0.025). The effect of heparin on serum GR was recorded as the percentage of activity after incubation with heparin vs. activity in the standard assay for individual subjects. The effect of incubation with 5 mug/ml heparin on serum GR activity was greater in control subjects than in carriers (P less than 0.0005) and CF (P less than 0.0005). Mean serum GR activity in CF and carriers was unaffected by heparin, whereas mean activity in control subjects was decreased. In no control was the percentage of initial activity with heparin greater than the mean of CF and carrier groups. Only 3 of 20 CF and 4 of 20 carrier individuals had percentages lower than the control mean. The CF and carrier distributions were clearly different from the control distribution. Serum GR was determined in seven non-CF individuals with chronic obstructive pulmonary disease (COPD). Activity in the COPD was different from CF and no different from control subjects. In none of these controls or COPD was serum GR as great as the CF mean. Serum GR in no CF was as low as the mean of control subjects or COPD. It is concluded that serum GR activity is greater in CF than in control subjects, carriers, and non-CF COPD subjects; that the difference in activity is not attributable to an extraneous serum factor, that the activity difference is not secondary to chronic respiratory disease; that in comparison with control subjects, GR from CF serum behaves differently after storage; and that serum GR from CF and carriers behaves differently from control GR in the presence of heparin.
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PMID:Serum glutathione reductase and cystic fibrosis. 119 5

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

Exposure of primary cultures of neonatal rat cortical astrocytes to bacterial lipopolysaccharide (LPS) results in the appearance of nitric oxide synthase (NOS) activity. The induction of NOS, which is blocked by actinomycin D, is directly related to the duration of exposure and dose of LPS, and a 2-hr pulse can induce enzyme activity. Cytosol from LPS-treated astrocyte cultures, but not from control cultures, produces a Ca(2+)-independent conversion of L-arginine to L-citrulline that can be completely blocked by the specific NOS inhibitor NG-monomethyl-L-arginine. The induced NOS activity exhibits an apparent Km of 16.5 microM for L-arginine and is dependent on NADPH, FAD, and tetrahydrobiopterin. LPS also induces NOS in C6 glioma cells and microglial cultures but not in cultured cortical neurons. The expression of NOS in astrocytes and microglial cells has been confirmed by immunocytochemical staining using an antibody to the inducible NOS of mouse macrophages and by histochemical staining for NADPH diaphorase activity. We conclude that glial cells of the central nervous system can express an inducible form of NOS similar to the inducible NOS of macrophages. Inducible NOS in glia may, by generating nitric oxide, contribute to the neuronal damage associated with cerebral ischemia and/or demyelinating diseases.
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PMID:Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures. 127 98

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

Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.
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PMID:Effect of KCl on the interactions between NADPH:cytochrome P-450 reductase and either cytochrome c, cytochrome b5 or cytochrome P-450 in octyl glucoside micelles. 131 30

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.
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PMID:Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies. 131 54

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.
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PMID:Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. 132 Mar 78

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.
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PMID:Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. 132 65

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P450. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.
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PMID:Dechlorination of pentachlorophenol by membrane bound enzymes of Rhodococcus chlorophenolicus PCP-I. 136 74


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