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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.
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PMID:Differences in redox and kinetic properties between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase. 272 58

The alkalophile NADH dehydrogenase (NADH: 2,6-dichlorophenolindophenol oxidoreductase) [EC 1.6.99.3] consists of two identical subunits of 65 kDa, and each subunit contains the catalytic and liposome-binding regions. On treatment with trypsin, the polypeptide exhibiting the liposome-binding property in one of the subunits was digested to form an enzymatically active hetero-dimer (40 and 65 kDa), and then the polypeptide in the other subunit was digested to form an active homo-dimer (40 and 40 kDa). The hetero-dimer bound to liposomes, but the homo-dimer did not. Kinetic analysis showed that removal of one or two of the polypeptides in the enzyme slightly affects its kinetic parameters. For all the enzyme species, NAD inhibited competitively with respect to NADH and non-competitively with respect to 2,6-dichlorophenolindophenol. The partially determined amino acid sequence of this alkalophile enzyme suggested that (i) a long random-coiled peptide (58 amino acid residues) or a portion of the peptide is located between the polypeptides with liposome-binding and catalytic properties, (ii) the polypeptide exhibiting liposome-binding property is in the amino terminal region of the enzyme, (iii) the amino acid sequences around the subtilisin and trypsin cleavage sites of the peptide are hydrophilic and on the surface of the protein molecule and therefore are susceptible to digestion, and (iv) the FAD-binding site is located near the amino terminal region of the catalytic region.
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PMID:Tryptic digestion of NADH dehydrogenase from alkalophilic Bacillus. 276 20

Rat liver deflavoxanthine dehydrogenase has been prepared by incubating native enzyme with calcium chloride. On reconstitution with FAD, about 85% of the original activity is recovered, all which is the O2-dependent type. In contrast, when dithiothreitol-treated deflavoenzyme is incubated with FAD, the recovery of activity is almost the same as above, but most of the recovered activity is of the NAD-dependent type. Deflavoenzyme with or without previous treatment with dithiothreitol was also reconstituted with two artificial FAD analogues, 8-mercapto-FAD and 6-OH-FAD. The difference spectra between the reconstituted enzymes and the initial deflavoenzyme indicate that, in each case, the FAD analogue is bound in its neutral form in dithiothreitol-treated enzyme, whereas it is bound in the anionic form in enzyme without previous dithiothreitol treatment. Furthermore, the protonated forms can be converted into the anionic forms on storage with a concomitant change of activity from the NAD-dependent to the O2-dependent type. This clearly indicates different environments around FAD in the two types of enzyme protein, which are shown to be interconvertible through oxidation-reduction of enzyme cysteinyl residues.
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PMID:Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study. 277 72

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.
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PMID:5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity. 284 89

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.
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PMID:Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme. 285 Feb 66

Age-related changes affecting mitochondrial adenine nucleotide metabolism may underlie age-related decreases in hepatic metabolic activities. Oxidative activity coupled with phosphorylation, the apparent Km and Vmax of the adenine nucleotide translocase (AdNT), the adenine nucleotide pool size and membrane lipid composition were determined for hepatic mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. The age-related decreases in state 3 respiration supported by NAD-linked substrates were 2-4-fold greater than that of an FAD-linked substrate. The 32% (P less than 0.05) decrease in the AdNT Vmax calculated for the aged rats was accompanied by a 17% decrease in the AdNT Km. The exchangeable pool of adenine nucleotides in mitochondria from aged rats was 72% (P less than 0.05) that in the young rats. While the age-related increase in the cholesterol/phospholipid-Pi ratio and changes in the phospholipid head group pattern were not significant, the overall change in the fatty acid pattern effected a 20% (P less than 0.05) decrease in the n-6/n-3 fatty acid ratio. These data suggest that the reduced Vmax of the AdNT is a consequence of a diminished pool of exchangeable adenine nucleotides. The lower AdNT velocity may reflect the effect of changes in the lipid environment of the membrane in which it is embedded. The major shifts in these parameters occurred during the second year of life.
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PMID:Age-related changes in respiration coupled to phosphorylation. I. Hepatic mitochondria. 285 81

Trout liver mitochondria were incubated in the presence of micromolar concentration of potassium dichromate (Cr(VI)) under several experimental conditions. Cr(VI) strongly inhibited both state 3 and state 4 of respiration supplemented by NAD-linked substrates; it also slightly affected the respiration of FAD-linked substrates. Evidence is provided that the respiratory inhibition induced by dichromate is partially coupled to the Cr(VI) reduction mechanism occurring in mitochondria.
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PMID:Effects of hexavalent chromium on trout mitochondria. 290 88

Adrenodoxin reductase is an NADP dependent flavoenzyme which functions as the reductase of mitochondrial P 450 systems. We sequenced two adrenodoxin reductase cDNAs isolated from a bovine adrenal cortex cDNA library. The deduced amino acid sequence shows no similarity to the sequence of the microsomal P 450 systems or other known protein sequences. Nonetheless, by sequence analysis and c comparisons with known sequences of dinucleotide-binding folds of two NADP-binding flavoenzymes, two regions of adrenodoxin reductase sequence were identified as the FAD- and NADP-binding sites. These analyses revealed a consensus sequence for the NADP-binding dinucleotide fold (GXGXXAXXXAXXXXXXG, in one-letter amino acid code) that differs from FAD and NAD-binding dinucleotide-fold sequences. In the data base of protein sequences, the NADP-binding-site sequence appears solely in NADP-dependent enzymes, the binding sites of which were not known to date. Thus, this sequence may be used for identification of a certain type of NADP-binding site of enzymes that show no significant sequence similarity.
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PMID:cDNA sequence of adrenodoxin reductase. Identification of NADP-binding sites in oxidoreductases. 292 77

Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.
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PMID:The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide. 292 14

The effect of hyperthermia (1 hr, 41 degrees C) on the functional properties of Ehrlich ascites tumor mitochondria was investigated. Mitochondria isolated from Ehrlich ascites tumor after exposure of whole cells to 41 degrees C for 1 hr still phosphorylate and maintain a normal acceptor control ratio (ACR). The temperature decreases state 4 and ADP-and FCCP-stimulated respiration on various substrates entering at three energy-conserving sites of the respiratory chain. The inhibition of oxygen consumption by NAD- and FAD-linked substrates was 40% for state 4 and 70% for ADP- or FCCP-stimulated respiration. State 4 and FCCP-stimulated respiration of mitochondria on TMPD + ascorbate was affected 38% and 45%, respectively. ATPase activity was unaffected by hyperthermia, indicating that under these experimental conditions, the inhibition of ADP-stimulated respiration does not depend on an effect on either Fo F1-ATPase or adenine translocase, the activity of which is required for ATP entry prior to ATPase activity. Because of the inability to detect a specific site of action of temperature, it is conceivable that hyperthermia might inhibit substrate oxidation by altering some components of the inner mitochondrial membrane, which regulates the kinetic properties of the membrane-associated enzymes.
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PMID:Effect of hyperthermia on electron transport in Ehrlich ascites tumor mitochondria. 295 47


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