UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
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The second-order rate constants obtained in solution for the reduction of horse cytochrome c (cytc; net charge +7) by either Clostridium beijerinckii flavodoxin semiquinone (Fld; net charge -16) or reduced spinach ferredoxin (Fd; net charge -15) decrease monotonically with increasing ionic strength, as expected for reactions between oppositely charged species. Although the rate constant for the Fld reaction is almost two orders of magnitude larger at low ionic strength than that for Fd, the values extrapolated to infinite ionic strength are closely similar, indicating comparable reactivities when electrostatic effects are eliminated. Furthermore, Fld has a much larger value for the electrostatic interaction energy, and thus a larger apparent active site charge, than does Fd, accounting for the rate constant disparity at low ionic strength. Electrostatically binding cytc at low ionic strength to a negatively charged lipid bilayer vesicle (membranes containing mixtures of egg phosphatidylcholine (PC) and cardiolipin (CL)) results in a marked decrease of the observed electron transfer rate constant (k(obs)) for reduction of the cytochrome by both Fld and Fd. The magnitude of this decrease is proportional to the mole percent of CL present in the membrane (10- to 20-fold change over 5-60 mol%). With Fld, k(obs) decreases monotonically with increasing ionic strength at a fixed CL concentration. With Fd an increase in k(obs) occurs as the ionic strength is increased, which maximizes at intermediate ionic strength at a value larger than that obtained in the absence of lipid vesicles. When Fld is electrostatically bound to a positively charged vesicle composed of 40 mol% dioctadecyldimethylammonium ion (DODAC) and 60 mol% PC, again k(obs) for electron transfer to cytc is decreased over that obtained in solution, and the magnitude is diminished monotonically by increasing ionic strength. In contrast, k(obs) for electron transfer from Fd to cytc is unaffected by the presence of the positively charged membrane. The implications of these results for the role of membrane surface charge in modulating protein-protein interactions is discussed.
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PMID:Comparison of electron transfer kinetics between redox proteins free in solution and electrostatically complexed to a lipid bilayer membrane. 817 24

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to readily promote formation of cross-links between spinach ferredoxin-NADP+ reductase and bacterial flavodoxins. The covalent complex between ferredoxin-NADP+ reductase and the Desulfovibrio vulgaris flavodoxin had a stoichiometry of 1 mol of flavodoxin per mole of the reductase, as assessed by denaturing electrophoresis, gel filtration and spectral analysis. The reductase moiety of the cross-linked complex gained the capacity to catalyze at a high rate the electron transfer from NADPH to cytochrome c without addition of free flavodoxin in the assay. The pH optimum for this activity was shifted to the alkaline region with respect to that for the noncovalent complex. FMN, the prosthetic group of flavodoxin, is required for electron transfer from the reductase FAD to cytochrome c. Structural studies carried out on the cross-linked complex allowed the identification of the peptide regions of the proteins involved in the interaction. The CNBr peptide 61-155 of the reductase was found cross-linked to the uncleaved flavodoxin, while the cross-linked region in flavodoxin appeared to be within the tryptic peptide 37-86. Treatment of flavodoxin with the carbodiimide in the presence of glycine ethyl ester brought about the modification of a few carboxyl groups and prevented its interaction with the reductase. It can be concluded that the bacterial flavodoxin binds to the reductase in a way similar to that of the physiological substrate ferredoxin (G. Zanetti, D. Morelli, S. Ronchi, A. Negri, A. Aliverti, and B. Curti, 1988, Biochemistry 27, 3753-3759). The cross-linked complex here described represents an useful model for studying electron transfer between the two flavoproteins.
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PMID:A functional heterologous electron-transfer protein complex: Desulfovibrio vulgaris flavodoxin covalently linked to spinach ferredoxin-NADP+ reductase. 820 13

A manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium catalyzed the reduction of cytochrome c in a reaction mixture containing H2O2, Mn(II)-tartrate, and p-hydroquinone. Electron spin resonance studies have shown that the hydroquinone-dependent reductive activity of MnP is due to the benzosemiquinone formed upon the one-electron oxidation of p-hydroquinone by Mn(III)-tartrate, which is formed upon the oxidation of Mn(II) by MnP. The reductive activity increased linearly with an increase in the concentration of p-hydroquinone. The reductive activity was also observed using other hydroquinones such as methylhydroquinone, 2,5-dimethylhydroquinone, and trimethylhydroquinone. The apparent Km values for Mn(II) and H2O2 for the hydroquinone-dependent reductive activity were similar to those for oxidative reactions of MnP. A stoichiometry study showed that about 1.5 mol of cytochrome c was reduced per mole of H2O2 consumed. The stoichiometry decreased with an increase in the concentration of H2O2. The optimal pH for the reductive activity was 5.0, approximately the physiological pH of the fungus. The reduction of cytochrome c was also observed using a quinone and cellobiose:quinone oxidoreductase isolated from the extracellular medium of the fungus.
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PMID:Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium. 821 23

Mitochondrial NADPH-linked aquacobalamin reductase was purified and characterized to clarify its enzymatic properties. The enzyme was purified about 360-fold over rat liver mitochondrial membranes in a yield of 7.5%. The purified enzyme was homogenous in SDS-PAGE. The molecular mass (M(r)) of the enzyme was calculated to be 65 kDa by SDS-PAGE and by Toyopearl HW55 gel filtration, indicating that the enzyme is a monomeric polypeptide with M(r) of 65 kDa. The enzyme was a flavoprotein containing 1 mol of FAD and FMN per mole of the enzyme. The enzyme was specific for NADPH as electron donor and had the ability to reduce cytochrome c (15.4 mumol.min-1 x mg protein-1), potassium ferricyanide (4.9 mumol.min-1 x mg protein-1) and 2,6-dichlorophenolindophenol (16.8 mumol.min-1.mg protein-1) as well as aquacobalamin (6.4 mumol.min-1 x mg protein-1). Although the enzyme immunoreacted with an antibody against NADPH-cytochrome P-450 reductase, which had the activity of the NADPH-linked aquacobalamin reductase in rat liver microsomes, the mitochondrial enzyme and the microsomal enzyme had different enzymological properties.
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PMID:Mitochondrial NADPH-linked aquacobalamin reductase is distinct from the NADPH-linked enzyme from microsomal membranes in rat liver. 822 2

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.
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PMID:Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean. 846 4

The basic chromosomal protein histone H1 binds avidly to liposomes containing acidic phospholipids and with characteristics somewhat resembling the lipid association of cytochrome c (cyt c) [Koiv et al. (1995) Biochemistry 34, 8018-8027]. Membrane association of histone H1 strongly attenuates the lipid lateral diffusion in large unilamellar vesicles containing phosphatidylglycerol (PG) as revealed by the decrease in the excimer to monomer ratio Ie/Im of the pyrene fatty acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphogly cer ol (PPDPG) fluorescence. Similarly, an increase in fluorescence anisotropy of the membrane-incorporated probe, diphenylhexatriene (DPH), due to histone H1 indicates that the membrane becomes more rigid. Increasing the mole fraction of PG (XPG) increases in a linear manner the concentration [H1]s required for the maximal decrease in Ie/Im or increase in fluorescence anisotropy, thus allowing to estimate the binding site for H1 to be constituted by approximately 20 PG molecules. Domain formation is also supported by differential scanning calorimetry measurements. Subsequently, we studied the detachment of cyt c from PG-containing liposomes by H1 by measuring its efficiency in decreasing resonance energy transfer between PPDPG and the heme of cyt c. The A-site interaction of 1 microM cyt c with 25 microM PG/PC (XPG = 0.20) liposomes is fully inhibited by low (0.1 microM) histone concentrations. Upon XPG being increased, the concentration [H1]D required for complete detachment of cyt c increases. Irrespective of the [cyt c] present (varying between 0.1 and 10 microM), the C-site-mediated binding of cyt c to neat PG liposomes (XPG = 1.0) is fully prevented at [H1] = 0.6 microM. These measurements indicate that the affinity of histone H1 to liposomes exceeds that of cyt c. The above effects of H1 were subsequently compared with those of different basic membrane-associating peptides. Notably, the effects of HI were remarkably well-reproduced by polylysine (K19). The high affinity of H1 to acidic phospholipids suggests that this feature might also contribute to its physiological function.
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PMID:Dissociation of cytochrome c from liposomes by histone H1. Comparison with basic peptides. 860 3

The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.
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PMID:The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein. 930 29

Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large unilamellar liposomes composed of egg phosphatidylcholine (eggPC) with varying content of the acidic phospholipid phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate of binding of cyt c to membranes was attenuated upon increasing the mole fraction of the acidic phospholipid (XPG). For example, at 50 microM phospholipid and 5 microM cyt c, when XPG was increased from 0.20 to 0.40 the half-time for the single-exponential decay in fluorescence increased from 4.7 to 8.6 ms. A similar observation was made for the membrane binding of another cationic protein, histone H1. We suggest that the formation of cooperative hydrogen-bonded networks by deprotonated and protonated PG in the vesicle surface retards the binding of cyt c to the liposome surface. However, once formed, the complex of cyt c with acidic phospholipids is stabilized by increasing XPG. Accordingly, significantly prolonged half-times of dissociation of cyt c from liposomes by NaCl, ATP, and different cationic proteins are measured upon increasing XPG. Differences between the latter cationic membrane binding ligands most likely reflect the varying relative contributions of hydrophobicity and Coulombic forces to their attachment to liposomes. Our data on the release and binding of cyt c to liposomes as a function of XPG and in the presence of ATP also provide the first direct experimental evidence for multiple lipid binding sites in cyt c.
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PMID:Binding and dissociation of cytochrome c to and from membranes containing acidic phospholipids. 947 68

We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
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PMID:Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans. 973 72

A large number of pharmaceutically active compounds have a high affinity to acidic phospholipids; good examples are the cationic compounds lidocaine, propranolol, and gentamycin. These drugs influenced the lipid dynamics of liposomes composed of phosphatidylcholine and the acidic phosphatidylglycerol, as judged by the excimer/monomer emission intensity ratio for a pyrene-labeled phospholipid analog, as well as by polarization of DPH fluorescence. When the mole fraction X of PG (XPG) was 0.20, lidocaine increased membrane fluidity. The opposite was true for propranolol, which caused the formation of pyrene lipid-enriched microdomains. Gentamycin had no apparent effect. At XPG = 1.00, all these drugs rigidified membrane. Subsequently, we investigated the detachment of a cationic peripheral membrane protein, cytochrome c (cyt c), by these compounds from liposomes. This was accomplished by monitoring resonance energy transfer from a pyrene-labeled phospholipid to the heme of cyt c. The efficiency of the above compounds to dissociate cyt c varied considerably. In brief, significantly lower concentrations of gentamycin than propranolol or lidocaine were required for half-maximal dissociation of cyt c from liposomes, although the final extent of protein detachment by gentamycin was less complete. ATP augmented the dissociation of cyt c from membranes by lidocaine and propranolol. Stopped-flow measurements also revealed that the half-times differed for the release of cyt c from the membranes. Our results are likely to reflect differences in the contributions of the electrostatic interactions and hydrophobicity to the drug/lipid interaction and comply with two different acidic phospholipid binding sites in cyt c.
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PMID:Detachment of cytochrome c by cationic drugs from membranes containing acidic phospholipids: comparison of lidocaine, propranolol, and gentamycin. 976 16

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