Cytosol-mediated reduction of resorufin: a method for measuring quinone oxidoreductase.
AuthorsNims RW, Prough RA, Lubet RA
JournalArch Biochem Biophys
PubMed ID6422857
The reduction of resorufin (7-hydroxyphenoxazone) fluorescence was catalyzed by enzymes present in the hepatic cytosol of rats and hamsters. This reaction was mediated by either NADH or NADPH, was completely inhibited by 10 microM dicumarol, and was not affected by anaerobic conditions (purging the reaction cuvette with nitrogen). The enzyme-mediated ... More
Enzymatic analysis of cDNA-expressed human CYP1A1, CYP1A2, and CYP1B1 with 7-ethoxyresorufin as substrate.
AuthorsChang TK, Waxman DJ,
JournalMethods Mol Biol
PubMed ID16719376
'Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYPB1. ... More
Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta.
'The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in ... More
Stochastic inhibitor release and binding from single-enzyme molecules.
AuthorsGorris HH, Rissin DM, Walt DR,
JournalProc Natl Acad Sci U S A
PubMed ID17965235
'Inhibition kinetics of single-beta-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of beta-galactosidase could be clearly distinguished, and the large array size ... More
Resorufin inhibits the in vitro metabolism and mutagenesis of benzo(a)pyrene.
AuthorsJablonski JE, Sullivan PD
JournalBiochem Biophys Res Commun
PubMed ID2423085
'7-Hydroxyphenoxazin-3-one, commonly known as resorufin, strongly inhibits benzo(a)pyrene-induced mutation in the Ames bacterial reversion assay. The antimutagenic mechanism is due in part to redox cycling of resorufin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen. The diversion of electrons from cytochrome P-450 enzymes results in a ... More
Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase.
AuthorsDutton DR, Reed GA, Parkinson A
JournalArch Biochem Biophys
PubMed ID2464338
'The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The ... More
Characterization of the oxidase activity in mammalian catalase.
'Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell ... More
Glucose localization in maize ovaries when kernel number decreases at low water potential and sucrose is fed to the stems.
AuthorsMcLaughlin JE, Boyer JS
JournalAnn Bot (Lond)
PubMed ID15159218
'BACKGROUND AND AIMS: Around the time of anthesis, young ovary development in maize (Zea mays) is vulnerable to 2 or 3 d of water deficits that inhibit photosynthesis. Abortion can result, and fewer kernels are produced. A breakdown of stored ovary starch is associated with the abortion and was investigated ... More
An enzymatic fluorimetric assay for glucose-6-phosphate: application in an in vitro Warburg-like effect.
AuthorsZhu A, Romero R, Petty HR,
JournalAnal Biochem
PubMed ID19454216
'Recently, there has been a resurgence of interest in the regulatory role of cell metabolism in tumor biology and immunology. To assess changes in metabolite levels in cell populations and tissues, especially from small clinical samples, highly sensitive assays are required. Based on the reaction of glucose-6-phosphate (G6P) and the ... More
Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins).
AuthorsBurke MD, Mayer RT
JournalChem Biol Interact
PubMed ID6883573
'The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed ... More
Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers.
'Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation ... More
Continuous fluorometric assay of phenol sulfotransferase.
AuthorsBeckmann JD
JournalAnal Biochem
PubMed ID1785696
'Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3''-phosphoadenosine 5''-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the ... More
Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins.
'The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed ''PB1c'' and ''PB2c''. These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from ... More
A resorufin-based fluorescent assay for quantifying NADH.
AuthorsBatchelor RH, Zhou M
JournalAnal Biochem
PubMed ID12018954
Metabolism of alkoxyphenoxazones by channel catfish liver microsomes: effects of phenobarbital, Aroclor 1254 and 3-methylcholanthrene.
AuthorsAnkley GT, Reinert RE, Mayer RT, Burke MD, Agosin M
JournalBiochem Pharmacol
PubMed ID3109440
Efficient analysis of cytochrome P4501A catalytic activity, porphyrins, and total proteins in chicken embryo hepatocyte cultures with a fluorescence plate reader.
AuthorsKennedy SW, Jones SP, Bastien LJ
JournalAnal Biochem
PubMed ID7793639
An efficient method for measuring polychlorinated biphenyl-mediated induction of cytochrome P4501A, using the ethoxyresorufin-O-deethylase (EROD) assay, and total porphyrins in chicken embryo hepatocyte cultures is described. Hepatocytes were cultured in 48-well plates, assays were carried out within the wells, and concentrations of the product of the EROD reaction (resorufin), porphyrins, ... More
Automation of the resazurin reduction test using fluorometry of microtitration trays.
AuthorsAli-Vehmas T, Louhi M, Sandholm M
JournalZentralbl Veterinarmed B
PubMed ID1927150
Microtitration plate technique and -fluorometry was applied to automate the resazurin reduction test for monitoring bacterial numbers in broth cultures and milk. The effect of resazurin and resorufin concentration, bacterial species, growth medium, pH, and redox potential on the fluorescence response was studied. The timing of the appearance of maximum ... More
Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.
AuthorsDreier J, Breitmaier EB, Gocke E, Apfel CM, Page MG
JournalMutat Res
PubMed ID11719102
Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly ... More
Cytochrome P-450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes.
AuthorsBarnett CR, Abbott RA, Bailey CJ, Flatt PR, Ioannides C
JournalBiochem Pharmacol
PubMed ID1575780
The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence ... More
Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies.
AuthorsGorris HH, Walt DR,
JournalJ Am Chem Soc
PubMed ID19338338
Many individual horseradish peroxidase (HRP) molecules were isolated and observed simultaneously by fluorescence microscopy in an array of 50 000 femtoliter chambers chemically etched into the surface of a glass optical fiber bundle. The substrate turnovers of hundreds of individual HRP molecules were readily analyzed, and the large number of ... More
Multi-level analysis of organic anion transporters 1, 3, and 6 reveals major differences in structural determinants of antiviral discrimination.
AuthorsTruong DM, Kaler G, Khandelwal A, Swaan PW, Nigam SK,
JournalJ Biol Chem
PubMed ID18174163
Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues. Organic anion transporters (OATs) are believed to mediate the cellular uptake, and hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various OAT ... More
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions.
The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in ... More
Induction of cytochrome P-448 activity as exemplified by the O-deethylation of ethoxyresorufin. Effects of dose, sex, tissue and animal species.
AuthorsIwasaki K, Lum PY, Ioannides C, Parke DV
JournalBiochem Pharmacol
PubMed ID3778512
The effects of tissue, sex, animal species and dose on the induction of cytochrome P-448 activity by various inducing agents were investigated using O-ethoxyresorufin as a model substrate. The liver was by far more effective in catalysing the O-deethylation of ethoxyresorufin (EROD) than the lung and kidney. The extent of ... More
Alkoxyresorufin O-dealkylases: association with the murine Ah locus.
AuthorsLum PY, Burke MD, Mayer RT, Ioannides C
JournalCancer Lett
PubMed ID3768851
The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
Resazurin reduction assay for ram sperm metabolic activity measured by spectrophotometry.
AuthorsWang S, Holyoak GR, Panter KE, Liu Y, Evans RC, Bunch TD
JournalProc Soc Exp Biol Med
PubMed ID9452144
Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a redox dye that can be reduced by metabolically active spermatozoa to resorufin and manifested as visual color change from blue (resazurin) to pink (resorufin). This study combined the resazurin reduction test with spectrophotometric methods and investigated the correlation between metabolic activity and fertilization potential of ... More
Induction by xenobiotics of phase I and phase II enzyme activities in the human keratinocyte cell line NCTC 2544.
AuthorsGelardi A, Morini F, Dusatti F, Penco S, Ferro M
JournalToxicol In Vitro
PubMed ID11698172
This study analyses the expression and induction of several drug-metabolising enzyme activities involved in either phase I or phase II biotransformations in NCTC 2544 human keratinocytes. The phase I activities 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depenthylase (PROD) were easily detectable in basal conditions. During incubations lasting up ... More
Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.
AuthorsKobayashi Y, Fang X, Szklarz GD, Halpert JR
JournalBiochemistry
PubMed ID9578551
A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order ... More
Glycine N-methyltransferase is an example of functional diversity. Role as a polycyclic aromatic hydrocarbon-binding receptor.
AuthorsBhat R, Bresnick E
JournalJ Biol Chem
PubMed ID9261130
The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 269, 5750-5756). The role of GNMT as ... More
Physiological ecology of Methanobrevibacter cuticularis sp. nov. and Methanobrevibacter curvatus sp. nov., isolated from the hindgut of the termite Reticulitermes flavipes.
AuthorsLeadbetter JR, Breznak JA
JournalAppl Environ Microbiol
PubMed ID8837417
Two morphologically distinct, H2- and CO2-utilizing methanogens were isolated from gut homogenates of the subterranean termite, Reticulitermes-flavipes (Kollar) (Rhinotermitidae). Strain RFM-1 was a short straight rod (0.4 by 1.2 micron), whereas strain RFM-2 was a slightly curved rod (0.34 by 1.6 microns) that possessed polar fibers. Their morphology, gram-positive staining ... More
Reverse micelles as a water-property-control system to investigate the hydration/activity relationship of alpha-chymotrypsin.
AuthorsDorovska-Taran V, Veeger C, Visser AJ
JournalEur J Biochem
PubMed ID8281919
alpha-Chymotrypsin, solubilized in hydrated reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in n-octane, was used as a model system for studying the involvement of different water structures (strongly bound water, disordered water, water clusters and bulk water) in the development of the catalytically active conformation of the enzyme. Results presented in ... More
Paraquat increases cyanide-insensitive respiration in murine lung epithelial cells by activating an NAD(P)H:paraquat oxidoreductase: identification of the enzyme as thioredoxin reductase.
AuthorsGray JP, Heck DE, Mishin V, Smith PJ, Hong JY, Thiruchelvam M, Cory-Slechta DA, Laskin DL, Laskin JD
JournalJ Biol Chem
PubMed ID17229725
Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity ... More
Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities.
AuthorsPasanen M, Haaparanta T, Sundin M, Sivonen P, Vakakangas K, Raunio H, Hines R, Gustafsson JA, Pelkonen O
JournalToxicology
PubMed ID1693794
The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase ... More
Defective one- or two-electron reduction of the anticancer anthracycline epirubicin in human heart. Relative importance of vesicular sequestration and impaired efficiency of electron addition.
AuthorsSalvatorelli E, Guarnieri S, Menna P, Liberi G, Calafiore AM, Mariggiò MA, Mordente A, Gianni L, Minotti G
JournalJ Biol Chem
PubMed ID16423826
One-electron quinone reduction and two-electron carbonyl reduction convert the anticancer anthracycline doxorubicin to reactive oxygen species (ROS) or a secondary alcohol metabolite that contributes to inducing a severe form of cardiotoxicity. The closely related analogue epirubicin induces less cardiotoxicity, but the determinants of its different behavior have not been elucidated. ... More