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: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.
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PMID:The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes. 641 23

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.
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PMID:Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase. 722 57

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.
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PMID:Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420. 740 43

Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of approximately 1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of approximately 89- and approximately 79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.
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PMID:Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. 751 50

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.
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PMID:Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. 866 Mar 10

We have recently demonstrated that neurotrophins induce reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in cultured spinal cord neurons. One prominent neuron population of the spinal cord expressing NADPH-diaphorase activity in vivo are preganglionic sympathetic neurons, including those innervating the adrenal medulla. These neurons receive trophic support from their target. We have shown previously that chromaffin cells contain as yet unidentified neurotrophic molecules, which may include releasable factors relevant for the survival and differentiation of developing preganglionic sympathetic neurons. We have studied the influence of proteins derived from bovine chromaffin cells and released by nicotine on NADPH-diaphorase expression in spinal cord cultures established from 16-day-old rat embryos. At this embryonic age, NADPH-diaphorase activity becomes apparent in the spinal cord and predominantly expressed in sympathetic nuclei. Similar to brain-derived neurotrophic factor and neurotrophin-4, a heat- and trypsin-sensitive component from chromaffin cells contained in granule preparations up-regulated the number of NADPH-diaphorase-positive neurons in spinal cord cultures. Combined application of this activity and neurotrophin-4 resulted in an additive effect, indicating that the effect of the chromaffin cell-derived active component is not mediated by one of the trk B ligands. This was confirmed by co-treatment studies with the trk-signalling pathway inhibitor K252b, which did not inhibit the effect of the chromaffin cell-derived protein(s). Further studies revealed that NADPH-diaphorase reactivity is inducible in spinal cord neurons at any time point throughout the entire culture period of six days, suggesting de novo induction of the enzyme rather than a survival-promoting effect of the activity from chromaffin cells. Culture supernatants from nicotine-stimulated bovine chromaffin cells induced NADPH-diaphorase-positive neurons at the same magnitude as the material obtained from chromaffin granule preparations. Our data suggest that chromaffin cell-derived proteins are capable of up-regulating NADPH-diaphorase activity or to induce de novo this transmitter phenotype in neuron populations of the spinal cord, which may include preganglionic sympathetic neurons.
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PMID:A chromaffin cell-derived protein induces the NADPH-diaphorase phenotype in cultured rat spinal cord neurons. 868 18

Treatment of red cell ghosts with ozone inhibited both AChE (marking the outside of the membrane) and G3PDH (marking the inside of the membrane). There was no change in tryptophan fluorescence of the ghosts after the ozone treatment. Band 3 protein was isolated from the ozone-treated ghosts. The protein was digested with trypsin to obtain water soluble peptides from the cytoplasmic N-terminal tail and the interhelical loops. Fluorescent peptides included GWVIHPLGLR from the outer loop between helices 7 and 8, and peptide WMEAAR from the N-terminal cytoplasmic tail. Neither one of these peptides was oxidized by ozone. This was true whether or not the ghosts were sealed. We conclude that the position of these tryptophans either in the membrane structure, or because of binding to other proteins in the cytoplasmic tail, protects them from oxidation by ozone. Treatment of horse heart cytochrome c with ozone did not change the absorbance spectrum in the heme region or the tryptophan absorbing region. HPLC of the ozone-treated cytochrome c showed that cytochrome c was being modified, indicated by a change in the elution time. Treatment of cytochrome c with ozone did not change the activity in the NADH-cytochrome c reductase assay. Digestion of the ozone-treated cytochrome c with trypsin gave peptides which demonstrated normal fluorescence. (Cytochrome c has abnormally low fluorescence, which is not changed by ozone exposure.) The peptides were separated by HPLC. The fluorescence of the tryptophan-containing peptide (GITWK) was not decreased by treatment of the cytochrome c by ozone. Amino acid analysis of the ozone-treated cytochrome c indicated that methionine was oxidized. We conclude that tryptophan in cytochrome c is protected from oxidation by ozone because of the interaction with the porphyrin ring. Bovine serum albumin and human serum albumin were treated with ozone. There was a monotonic decrease in tryptophan fluorescence in both cases. Digestion of BSA with trypsin produced two fluorescent peptides. The peptide FWGK was identified by coelution with the authentic peptide. The putative peptide AWSVAR was not the same as the chemically synthesized peptide. The peptide sequences FWGK and "AWSVAR" were both oxidized in ozone-treated bovine serum albumin, with no detectable discrimination. Tryptic digestion of the ozone-treated human serum albumin produced a single fluorescent peptide, which was oxidized by ozone. The putative peptide AWAVAR in the tryptic digest of HSA was distinct from chemically synthesized peptide. The oxidation of tryptophans in proteins by ozone is markedly influenced by position in tertiary structure, position in membrane structure, and by chemical interactions within the protein.
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PMID:Reaction of ozone with protein tryptophans: band III, serum albumin, and cytochrome C. 902 65

Spin-trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) was used to demonstrate that 3-nitrotyrosine (nitrotyrosine) promotes the formation of substantial amounts of reactive oxygen species (O2.- and *OH), when incubated with NAD(H)-cytochrome c reductase and a corresponding electron donor. Spin adduct formation is strongly inhibited by the presence of superoxide dismutase (SOD); spin adduct formation requires aerobic conditions. Nitration of leucine enkephalin, a tyrosine-containing pentapeptide, results in a similar generation of O2*- and *OH species. Both nitrotyrosine and nitrated leucine enkephalin stimulate acetylated ferricytochrome c reduction in the presence of NAD(H)-cytochrome c reductase with typical Michaelis-Menten kinetics and Km's of 104 +/- 14 and 0.78 +/- 0.11 microM, respectively. No stimulation of acetylated ferricytochrome c reduction is observed in the presence of SOD. Catalase and the metal chelators DTPA and deferoxamine mesylate do not influence observed stimulation of acetylated ferricytochrome c reduction by nitrotyrosine. Nitration of two tyrosines (of four) within the sequence of the 6.5-kDa globular protein bovine pancreas trypsin inhibitor (BPTI) fails to stimulate O2*- generation implying steric restrictions for BPTI-reductase interactions. However, nitrated BPTI subjected to trypsin digestion stimulated reduction of acetylated ferricytochrome c. These results suggest that, as with other nitroaromatic compounds, nitrotyrosine may be enzymatically reduced to the corresponding nitro anion radical (ArNO2*-) which is then oxidized by molecular oxygen to yield O2*- and regenerate ArNO2. Thus, once formed in vivo, nitrotyrosine may act to promote oxidative stress by means of repetitive redox cycling.
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PMID:Enzymatic reduction of 3-nitrotyrosine generates superoxide. 958 80

The NADH oxidase of Amphibacillus xylanus shows high NADH-peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of a 22-kDa disulfide-containing protein component (Y. Niimura, L. B. Poole, and V. Massey, J. Biol.Chem. 270, 25645-25650, 1995). It was found that the membrane-bound NADH dehydrogenase of an alkaliphilic Bacillus (YN-1) involved in the respiratory chain also exhibits reductase activity for hydrogen peroxide and cumene hydroperoxide in the presence of the 22-kDa component from Amphibacillus xylanus. Vmax values for these substrates were as high as those of the NADH oxidase of A. xylanus. Although the 38-kDa protein produced by trypsin treatment of NADH dehydrogenase retains NADH dehydrogenase activity, it exhibited no peroxide reductase activity in the presence of the 22-kDa component from A. xylanus. The NADH dehydrogenase of YN-1 might not only catalyze electron flow from NADH to the respiratory chain, but also function for scavenging peroxide.
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PMID:Peroxide reductase activity of NADH dehydrogenase of an alkaliphilic Bacillus in the presence of a 22-kDa protein component from Amphibacillus xylanus. 964 49

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.
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PMID:Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase. 1279 75


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