Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.
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PMID:Solubilization and partial purification of heme oxygenase from rat liver. 1 77

Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.
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PMID:The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli. 35 Aug 72

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.
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PMID:Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes. 42 35

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.
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PMID:Transformation of mercuric chloride and methylmercury by the rumen microflora. 53 20

Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant.
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PMID:Mutagenesis of the N- and C-terminal cysteine pairs of Tn501 mercuric ion reductase: consequences for bacterial detoxification of mercurials. 254 Aug 17

Heme oxygenase has been purified to electrophoretic homogeneity from detergent solubilized adult human liver microsomes. Treatment of microsomes with Triton X-100, sodium cholate and subsequent batchwise DEAE-cellulose, 2', 5' ADP-sepharose 4B, Sepharose CLB and hydroxylapatite column resulted in 17% yield of the purified heme oxygenase. The reconsituted system of heme oxygenase, composed of heme oxygenase, NADPH cytochrome c (P450) reductase and biliverdin reductase was equiactive with 1 mM NADPH and 4 nM NADH and showed complete dependence on added heme for catalytic activity. The Km values for NADPH and NADH were .046 and .526 mM, respectively. While NADPH concentration was held constant, the Km value for heme was 1.01 microM with a specific activity of 583 unit/mg protein. The activity of the reconstituted heme oxygenase system was not affected by preincubation with heavy metals despite their inhibitory effect of NADPH cytochrome c (P450) reductase and biliverdin reductase. However, the metalloporphyrins of these heavy metals were found to be strong inhibitors of the reconsituted system with Ki values of 0.015, 0.6, 2.3 and 5 microM for Sn-, Co-, Zn- and Mg- protoporphyrins, respectively. Similarly, the sulfhydryl inactivating reagents, HgCl2, iodoacetamide and p-chloromercurylbenzoate, inhibited the reconstituted heme oxygenase activity. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from rat and human liver as well as HepG2 cells were identified on dot and Western blots by their reaction with the anti-heme oxygenase similar to the purified enzyme protein. Anti-heme oxygenase precipitated quantitatively, the entire heme oxygenase of rat liver microsomes obtained from animals maintained on standard diet. The human bone marrow microsomal heme oxygenase activity was also quantitatively precipitated by this antibody. Antibody inhibition of rat and human heme xoygenase demonstrated a degree of conservation of both enzyme proteins between the species. As judged by Western blotting, the anti-heme oxygenase recognized only a single protein in spleen, liver, kidney, brain, heart, bone marrow, integtine and corneal epithelium. The human heme oxygenase cDNA was isolated by screening a cDNA library in the Okayama-Berg vector with a rat liver cDNA and was subjected to nucleotide sequence analysis. The deducted human heme oxygenase is also composed of 288 amino acids with a molecular mass of 32,800 Da.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Expression of heme oxygenase in hemopoiesis. 314 8

Mercuric reductase contains FAD and a redox-active disulfide which is reduced to a thiol/thiolate pair in two-electron reduced enzyme (EH2) (Fox, B. and Walsh, C.T. (1982) J. Biol. Chem. 257, 2498-2503). A charge transfer interaction between the thiolate and oxidized FAD gives EH2 a characteristic absorption spectrum, very similar to that found with other flavoprotein disulfide oxidoreductases. We have examined the reaction of EH2 with HgCl2 (+/- mercaptoethanol) in stopped-flow kinetic and static titration experiments. In the absence of mercaptoethanol, reaction of EH2 with HgCl2 yields a final spectrum which is indistinguishable from that of oxidized enzyme. The nature of the final species was examined by titration of enzyme thiols with 5,5'-dithiobis-2,2'-nitrobenzoic acid under denaturing conditions in the presence of NaI to displace any Hg(II) bound to enzyme thiols. These studies demonstrate that EH2 tightly complexes Hg(II) with its active site thiols, but is incapable of reducing Hg(II) to Hg0. For the latter reaction to occur, additional reducing equivalents are required. In catalysis, the enzyme must first be reduced to EH2 after which it cycles between EH2 and EH2 X NADPH forms. This is in contrast to other flavoprotein disulfide oxidoreductases which cycle between Eox and EH2 forms in catalysis (Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 13, pp. 89-173, Academic Press, New York). With mercuric reductase, exogenous thiols are required for catalytic reduction of Hg(II) to Hg0. We have shown that this is due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of EH2.
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PMID:Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction. 352 63

The effects of metal ions on the activities of biliverdin reductase in the rat kidney and liver were examined; the pH optimum and the cofactor requirement for the enzyme activity in the kidney were also studied. The reduction of biliverdin IX alpha by biliverdin reductase in the rat kidney cytosol fraction could be supported by NADPH and NADH. The activity was optimal around pH 8.7 when NADPH was the cofactor. The activity with NADH was undetectable at this pH. NADH-dependent biliverdin reductase was optimal at pH 7.0, where the NADPH-dependent activity was negligible. Biliverdin reductase activity was not inducible in the kidney or liver in response to treatment of rats with metal ions--Co2+, Ni2+, Pb2+, Sn2+, Zn2+, Cd2+, and Cu2+ or sodium selenite. Rather, both NADPH- and NADH-dependent activities in the kidney were decreased markedly in a time- and dose-related manner following the administration of HgCl2 (10-30 mumoles/kg, 24 hr). The pretreatment of rats (30 min) with sodium selenite (5 mumoles/kg) effectively blocked the Hg2+ (20 mumoles/kg, 24 hr) inhibition of the kidney cytosol biliverdin reductase activity. Similarly, in vitro Hg2+ was an effective inhibitor of the kidney biliverdin reductase. In addition, highly purified biliverdin reductase also was extremely sensitive to Hg2+ and the thiol reagent, 5,5'-dithiobis-(2-nitrobenzoic acid). The inhibition of purified reductase by 5,5'-dithiobis-(2-nitrobenzoic acid), but not by Hg2+, could be reversed by dithiothreitol.
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PMID:Biliverdin reductase: characterization in the rat kidney and the inhibition of activity by mercuric chloride. 622 39

The plasmid-determined mer operon, which provides resistance to inorganic mercury compounds, was subject to a 2.5-fold decrease in expression when glucose was administered at the same time as the inducer HgCl2. This glucose-mediated transient repression of the operon was overcome by the addition of cyclic AMP. Permanent catabolite repression of the operon was observed in the 1.6- to 1.9-fold decrease in expression in mutants lacking either adenyl cyclase (cya) or the catabolite activator protein (crp). The effect of the cya mutation on mer expression could be overcome by the addition of cyclic AMP at the time of induction, In addition to these effects on the whole cells of a wild-type strains, we examined the effect of catabolite repression on the expression of the mercuric ion [Hg(II)] reductase enzyme, assayable in cell extracts, and on the Hg(II) uptake system, assayable in a mutant strain which lacked reductase activity. There was a two- to threefold effect of repression on the Hg(II) reductase enzyme assayable in vitro after induction under catabolite repressing conditions (either with glucose or in the crp and cya mutants). We did not find a similar repressing effect on the induction of the Hg(II) uptake system, which is also determined by the mer operon.
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PMID:Effect of catabolite repression on the mer operon. 627 43

Transpositional mutagenesis of the mer operon of the IncFII plasmid, R100, has revealed a second, trans-acting positive regulatory function. Mutants in this function do not synthesize any of the three small mer operon peptides and have no inducible Hg(II) uptake activity. This second regulatory function is part of complementation group B and so depends upon the activity of the previously described trans-acting positive regulatory function merR. All mutants in this new function map in the amino-terminal 20 kDal of the Hg(II) reductase, suggesting either that this enzyme is also a regulatory protein or that there is a distinct protein whose reading frame is superimposed on that of the Hg(II) reductase. While we have only seen the five previously described mer operon peptides of 69, 66, 15.1, 14 and 12 (13) kDal encoded in minicells by single-copy plasmids, we have observed two new HgCl2-inducible polypeptides of approx. 20 kDal in minicells carrying a multicopy derivative of the mer operon of R100. Sequence data for the Hg(II) reductase region of the related mer operon of the transposon, Tn501 [Brown, N.L., Ford, S.J., Pridmore, R.D. and Fritzinger, D.C., Biochemistry 22 (1983) 4089-4095], shows a second reading frame very rich in cysteine and arginine which overlaps the amino-terminal 20 kDal of the Hg(II) reductase structural gene. We believe that this reading frame is the structural gene for this new regulatory function and propose the name merC (for control).
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PMID:A second positive regulatory function in the mer (mercury resistance) operon. 631 37


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