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:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.
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PMID:Enzyme and phospholipid asymmetry in liver microsomal membranes. 19 Feb 41

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from 3-methylcholanthrene (MC)-treated and untreated rats. Treatments with PenCB and MC resulted in about 8- and 7-fold increases of cytosolic DT-diaphorase activity, respectively. Purification of the enzyme preparations from untreated, and PenCB- and MC-treated rats were conducted by using DE-52, DEAE-Sephadex A-50, hydroxylapatite, and Bio-Gel P-150 column chromatographies. Both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all of the final preparations from the three origins were homogeneous and had the same molecular weight of 59,000, consisting of two subunits with molecular weights of 30,000. Further studies on amino acid composition, Km value, optimum pH, and catalytic activities for various substrates also indicated that both PenCB- and MC-inducible DT-diaphorases were identical with that from the untreated rats. All three DT-diaphorases contained about 2 mol of FAD per mol of enzyme. Partial digestion of the enzymes by trypsin and subsequent analysis by HPLC revealed that the three preparations were indistinguishable. The identity among the three purified DT-diaphorases was finally confirmed by Ouchterlony immunodiffusion employing anti-serum raised against each enzyme preparation.
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PMID:Comparison of DT-diaphorases purified from the liver cytosol of untreated, and 3,4,5,3',4'-pentachlorobiphenyl- and 3-methylcholanthrene-treated rats. 312 17

NAD(P)H:menadione oxidoreductase induction by polycyclic hydrocarbons is known to be governed by the aromatic hydrocarbon-responsive (Ah) locus. This cytosolic enzyme was isolated from 3-methylcholanthrene-treated rat liver by a rapid two-step procedure with the use of affinity gel purification and fast-protein liquid chromatography. Polyclonal antiserum to menadione reductase was raised in rabbits. On Western (immuno) blot analysis, large increases in this hepatic menadione reductase protein (NMOR1) of 3-methylcholanthrene-treated C57BL/6N but not DBA/2N mice confirmed that induction of this enzyme by 3-methyl-cholanthrene is regulated by the Ah receptor. A cDNA expression library was constructed in lambda gt11 and screened with antiserum. Positive cDNA clones were plaque purified and further characterized by showing enhanced hybridization to 3-methylcholanthrene-induced poly(A+) RNA from rats; the longest cDNA clone (1,501 base pairs) has an open reading frame (bases 75-899) and a nucleotide sequence consistent with a new gene family. On Northern blot analysis, a single 3-methylcholanthrene-inducible rat liver mRNA (approximately 1.6 kilobases) hybridizes to the cDNA probe. On Southern blot analysis a total of 14-16 kilobases of rat genomic DNA fragments hybridize to the cDNA probe, indicating one or a small number of menadione reductase genes in this family. The amino acid sequence (274 residues) and Mr of 30,946 compare well with the size of the rat enzyme (32 kDa) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of two internal fragments of the trypsin-digested purified NMOR1 protein is in complete agreement with that deduced from the cDNA nucleotide sequence. This study represents the first cloning and sequencing of a cDNA encoding a Phase II drug-metabolizing enzyme under control of the Ah locus.
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PMID:NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation. 353 15

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
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PMID:A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase. 751 54

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

Both regular physical exercise and low levels of H(2)O(2) administration result in increased resistance to oxidative stress. We measured the accumulation of reactive carbonyl derivatives and the activities of proteasome complex and DT-diaphorase in cardiac muscle of trained and untrained rats after chronic i.p. administration of 1 ml t-butyl H(2)O(2) (1 mmol/kg for 3 weeks every second day). Twenty-four rats were randomly assigned to a control group administered with saline, control administered with H(2)O(2), and exercised administered either saline or H(2)O(2). The activity of DT-diaphorase significantly increased in H(2)O(2) administered and exercised groups, indicating that an increase in H(2)O(2) levels stimulate the activity of this enzyme. The cardiac muscle of H(2)O(2) administered nonexercised animals accumulated significantly more carbonyl than control group (P < 0.05). The exercise and H(2)O(2) administration resulted in less oxidatively modified protein than found in nonexercised groups (P < 0.05). The peptide-like activity of proteasome complex was induced by the treatment of H(2)O(2) and exercise and exercise potentiate the effect of H(2)O(2). On the other hand, the chymotrypsin-like and trypsin-like activities were stimulated only by physical training and H(2)O(2) administration. The data suggest that chronic administration of H(2)O(2) after exercise training decreases the accumulation of carbonyl groups below the steady-state level and induces the activity of proteasome and DT-diaphorase. Hence, the stimulating effect of physical exercise on free radical generation is an important phenomenon of the exercise-induced adaptation process since it increases resistance to oxidative stress. Regular exercise training is a valuable physiological means of preconditioning the myocardium to prolonged oxidative stress.
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PMID:Exercise preconditioning against hydrogen peroxide-induced oxidative damage in proteins of rat myocardium. 1077 9

The biochemical mechanisms by which regular exercise significantly benefits health and well being, including improved cognitive function, are not well understood. Four-week-old (young) and 14-month-old (middle aged) Wistar rats were randomly assigned to young control and young exercised, middle-aged control and middle-aged exercised groups. Exercise groups were exposed to a swimming regime of 1 h a day, 5 days a week for 9 weeks. The passive avoidance test showed that middle-aged exercised rats had significantly (P<0.05) better short- (24 h) and long-term (72 h) memory than aged-matched control rats. Conditioned pole-jumping avoidance learning was improved markedly in both age groups by exercise. Brain thiobarbituric acid-reactive substances and 8-hydroxy-2'deoxyguanosine content in the DNA did not change significantly, while the protein carbonyl levels decreased significantly (P<0.05) in both exercised groups. This decrease was accompanied by an increase in the chymotrypsin-like activity of proteasome complex in the exercised groups, whereas trypsin-like activity did not differ significantly between all groups. The DT-diaphorase activity increased significantly (P<0.05) in the brain of young exercised animals. These data show that swimming training improves some cognitive functions in rats, with parallel attenuation of the accumulation of oxidatively damaged proteins.
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PMID:Regular exercise improves cognitive function and decreases oxidative damage in rat brain. 1091 84

The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained unaltered. The diaphorase activity of mutant 104 and the terminal-NR activity of mutant 305 of C. reinhardii, which are the sole activities related to NR present in these mutants, responded to tryptic treatment to the same extent as the corresponding activities of the wild enzyme complex. Trypsin disassembled the 220-kd NR native complex by destroying the aggregation capability of the diaphorase subunits without affecting their activity nor molecular size (45 kd). A 67-kd thermostable protein, containing molybdenum co-factor, was also released from trypsin-treated NR. This protein lacked diaphorase and NR activities but was able to reconstitute the overall-NR complex by complementation with untreated diaphorase subunit of mutant 104. Our results support a tetrameric structure for the C. reinhardii NR complex, containing two kinds of subunits.
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PMID:Heteromultimeric structure of the nitrate reductase complex of Chlamydomonas reinhardii. 1645 30

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l-lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.
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PMID:In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor. 1666 Oct 24


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