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)

A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.
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PMID:The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1. 634 42

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.
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PMID:Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase. 669 18

Carbon monoxide dehydrogenase (CO dehydrogenase) has been purified from the homoacetate-fermenting bacterium, Clostridium thermoaceticum. By use of 63Ni, it has been determined that the dehydrogenase is a metallo nickel enzyme. Nickel was rapidly taken up by the organism and most of the ingested metal was found to be incorporated into CO dehydrogenase. As estimated by gel filtration, the native enzyme has a molecular weight of 410,000. Ferredoxin and a membrane-bound b-type cytochrome, both obtained from C. thermoaceticum, are rapidly reduced by the enzyme in the presence of carbon monoxide and both are considered to be native electron carriers. FMN and Desulfovibrio vulgaris cytochrome c3 were also reduced by the enzyme, while spinach ferredoxin, FAD, NAD, and NADP were not. CO dehydrogenase activity was not appreciably affected by propyl iodide, methyl iodide, carbon tetrachloride, or metal chelators, but was reversibly inhibited by KCN. A method for the in situ assay of CO dehydrogenase in polyacrylamide gels is presented.
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PMID:Purification of carbon monoxide dehydrogenase, a nickel enzyme from Clostridium thermocaceticum. 689 49

Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.
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PMID:Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles. 702 Jul 57

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57

The enzyme responsible for the respiratory burst in human neutrophils is a membrane-bound oxidase that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We describe further properties of the solubilized oxidase. The rates of O2- production and NADPH consumption are consistent with the stoichiometry: 2 O2 + NADPH leads to 2 O2- + NADP The enzyme is highly specific for oxygen, failing to reduce several artificial electron acceptors including ferricyanide. FAD, an essential cofactor, binds tightly to the enzyme, as indicated by a Km of 61 nM. A requirement for a free --SH group is suggested by a 2-fold increase in activity in the presence of low concentrations of dithiothreitol; the higher dithiothreitol concentrations lead to inhibition. The solubilized enzyme is highly unstable, losing one-half its activity after 1 h at room temperature. Loss of activity is accelerated 2- to 3-fold by salts and EDTA. The substrate analog 2',5'-ADP is similar to other salts in its effect on the inactivation rate. ATP, on the other hand, causes loss of activity in seconds, raising the possibility that ATP is a physiological regulator of the catalytic activity of the enzyme.
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PMID:The O2--producing enzyme of human neutrophils. Further properties. 746 39

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

Fatty acid subterminal (omega-1 approximately omega-3) hydroxylase of the fungus Fusarium oxysporum was solubilized from the microsomal fraction and partially purified. The hydroxylase activity was recovered into a single active fraction, and its spectral nature showed the presence of cytochrome P-450 (P-450). Fatty acid hydroxylase activity was markedly restored upon addition of FAD, FMN, and/or hemin to the eluted fraction. The fraction also exhibited other properties characteristic of both a hemeprotein and a flavin-containing reductase. These results are highly indicative that the fungal hydroxylase is a fused protein containing both P-450 and its reductase domains. In this aspect the fungal enzyme resembles bacterial P-450BM3, although it is membrane-bound unlike the bacterial counterpart.
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PMID:Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase. 803 65

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 possible flavoprotein related to the electron transport between NADPH and cytochrome b558. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome 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. It is the purpose of this review to focus on recent data concerning electron transfer mechanisms of the activated neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease.
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PMID:Mechanisms for the activation/electron transfer of neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease. 803 32

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F420H2: quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-beta-D-maltoside the complex was purified 32-fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDS/PAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non-heme iron and 7 mol acid-labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmol/mg membrane protein). The purified F420H2: quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone (apparent Km 190 microM) with reduced coenzyme F420 (apparent Km 50 microM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65 degrees C (temperature optimum). 2-Methyl-1,4-naphthoquinone (menadione), 2-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-1,4- benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2: quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.
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PMID:F420H2: quinone oxidoreductase from Archaeoglobus fulgidus. Characterization of a membrane-bound multisubunit complex containing FAD and iron-sulfur clusters. 805 20


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