Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.
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PMID:Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase. 130 86

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids. The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system. Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da. The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components. FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration. The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents. The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues. Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide. Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group. The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed. The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.
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PMID:P450BM-3: reduction by NADPH and sodium dithionite. 156 20

The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans. The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system minus ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 mumol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N2O. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO/NOH) by thiol.
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PMID:Oxidation of dithiothreitol during turnover of nitric oxide reductase: evidence for generation of nitroxyl with the enzyme from Paracoccus denitrificans. 156 12

Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column. The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH. These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values. The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method. The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit. The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000). The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)). Each subunit therefore contained only one heme group. The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A. The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen. It seemed not to be inactivated by aerobic solutions. These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J. (1990) Biochem. J. 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa. denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G. (1989) J. Bacteriol. 171, 3288-3297). There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa. denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C. (1989) J. Biol. Chem. 264, 7556-7563).
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PMID:Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics. 164 15

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the Km and an 80% decrease in kcat/Km (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109-130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.
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PMID:Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450. 192 97

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.
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PMID:Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae. 212 25

The Azotobacter vinelandii mutant strain UW45 contains a mutation in the nifB gene and produces an inactive dinitrogenase protein that can be activated by the addition of purified iron-molybdenum cofactor (FeMoco). This FeMoco-deficient dinitrogenase (Apo I) has now been purified 96-fold to greater than 95% purity and is FeMoco-activatable to 2200 nmol of C2H2 reduced/(min.mg of protein). The Apo I complex was found to contain two molecules of a 20-kDa protein, in addition to the alpha 2 beta 2 tetramer found for isolated holodinitrogenase (Holo I). The Apo I complex contained 15 +/- 2 mol of Fe per mole, but no Mo. While the presence of dinitrogenase reductase caused a 2-fold stimulation in the activation of the purified Apo I complex by FeMoco, this enhancement resulted from the stabilization of Apo I by dinitrogenase reductase to the denaturing effects of N-methylformamide. When the activation was performed in the absence of N-methylformamide, there was no enhancement by dinitrogenase reductase alone or by dinitrogenase reductase-Mg-ATP complex. The Apo I complex is more sensitive to O2 than Holo I, with a half-life in air of 6 min; however, the addition of dithiothreitol to Apo I during the exposure to air (or after exposure) resulted in a half-life very similar to that seen for Holo I. This suggests that sulfhydryl(s) is (are) important for the FeMoco-activation reaction.
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PMID:Apodinitrogenase: purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase. 216 95

1. Sheep NADPH-ferredoxin reductase (E.C. 1.18.1.2) was purified from the adrenocortical mitochondria. The reductase was typical flavoenzyme and crystallized in ammonium sulfate solution. 2. The properties of the reductase were investigated physicochemically and immunochemically. The minimum molecular weight of the reductase was 52,000 and the reductase has one FAD per mole as a coenzyme. 3. The sheep NADPH-ferredoxin reductase showed a precipitate line against antibody to bovine NADPH-ferredoxin reductase. 4. The compositions and sequences of amino acid residues of this reductase and porcine, bovine, and human enzymes were compared. In spite of differences of mammalian species, the sequence of amino acid residues in the amino-terminal regions were highly homologous. 5. It is suggested that the amino-terminal region may be essential for the function of the NADPH-ferredoxin reductase.
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PMID:Crystallization and comparative characterization of reduced nicotinamide adenine dinucleotide phosphate-ferredoxin reductase from sheep adrenocortical mitochondria. 236 76


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