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BMC Pharmacology Oct 2004Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an...
BACKGROUND
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.
RESULTS
Exposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 +/- 0.005 mM and 1.25 +/- 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.
CONCLUSION
These results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.
Topics: Animals; Carbon Monoxide; Dose-Response Relationship, Drug; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Male; Microsomes; Nitric Oxide; Nitrites; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds
PubMed: 15498099
DOI: 10.1186/1471-2210-4-26 -
Applied and Environmental Microbiology Oct 1981Seventeen commonly used dyes and 16 of their metabolites or derivatives were tested in the Salmonella-mammalian microsome mutagenicity test. Mutagens active with and... (Comparative Study)
Comparative Study
Seventeen commonly used dyes and 16 of their metabolites or derivatives were tested in the Salmonella-mammalian microsome mutagenicity test. Mutagens active with and without added Aroclor-induced rat liver microsome preparations (S9) were 3-aminopyrene, lithol red, methylene blue (USP), methyl yellow, neutral red, and phenol red. Those mutagenic only with S9 activation were 4-aminopyrazolone, 2,4-dimethylaniline, N,N-dimethyl-p-phenylenediamine, methyl red, and 4-phenyl-azo-1-naphthylamine. Orange II was mutagenic only without added S9. Nonmutagenic azo dyes were allura red, amaranth, ponceau R, ponceau SX, sunset yellow, and tartrazine. Miscellaneous dyes not mutagenic were methyl green, methyl violet 2B, and nigrosin. Metabolites of the azo dyes that were not mutagenic were 1-amino-2-naphthol hydrochloride, aniline, anthranilic acid, cresidine salt, pyrazolone T,R-amino salt (1-amino-2-naphthol-3,6-disulfonic disodium salt), R-salt, Schaeffer's salt (2-naphthol-6-sulfonic acid, sodium salt), sodium naphthionate, sulfanilamide, and sulfanilic acid. 4-Amino-1-naphthalenesulfonic acid sodium salt was also not mutagenic. Fusobacterium sp. 2 could reductively cleave methyl yellow to N,N-dimethyl-p-phenylenediamine which was then activated to a mutagen.
Topics: Coloring Agents; Microsomes, Liver; Mutagenicity Tests; Mutagens; Salmonella; Salmonella typhimurium
PubMed: 7039509
DOI: 10.1128/aem.42.4.641-648.1981 -
Future Medicinal Chemistry Jul 2021This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to...
This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE biosynthesis while maintaining prostacyclin synthesis. We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE biosynthesis alone without affecting COX-2 coupled to PGI synthase (PGIS) for PGI biosynthesis was obtained. Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.
Topics: Benzene Derivatives; Cyclooxygenase 1; Cytochrome P-450 Enzyme System; Drug Evaluation, Preclinical; HEK293 Cells; Humans; Intramolecular Oxidoreductases; Microsomes; Protein Engineering
PubMed: 34080888
DOI: 10.4155/fmc-2021-0056 -
The Journal of Biological Chemistry Apr 1984Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by...
Electron spin resonance spin-trapping techniques were used to investigate the in vitro and in vivo formation of free radicals during 3-methylindole (3MI) metabolism by goat lung. Utilizing the spin trap phenyl-t-butylnitrone, a nitrogen-centered free radical was detected 3 min after the addition of 3MI to an in vitro incubation system containing goat lung microsomes in the presence of NADPH and O2. The spectrum of the spin adduct was identical to that observed when 3MI was irradiated with ultraviolet light. A carbon-centered radical was also observed which increased in concentration with increasing incubation time. Microsomal incubations containing ferrous sulfate in the absence of 3MI to initiate lipid peroxidation produced the same carbon-centered free radical as obtained by spin-trapping. Malondialdehyde, and end product of lipid peroxidation, was also found to increase in concentration with increasing incubation time of 3MI. The concept that 3MI causes lipid peroxidation in the lung was supported by the in vivo study in which a carbon-centered radical was spin-trapped by phenyl-t-butylnitrone in lungs of intact goats infused with 3MI. This carbon-centered radical had hyperfine splitting constants identical to those carbon-centered free radicals trapped in in vitro incubations of 3MI. These data demonstrate that microsomal metabolism of 3MI produces a nitrogen-centered radical from 3MI which initiates lipid peroxidation in vitro and in vivo causing the formation of carbon-centered radicals from microsomal membranes.
Topics: Animals; Cysteine; Cytochrome P-450 Enzyme System; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Goats; Indoles; Kinetics; Lung; Male; Microsomes; Mixed Function Oxygenases; Phenobarbital; Skatole; Ultraviolet Rays
PubMed: 6323473
DOI: No ID Found -
FEBS Letters May 1994N-Oligosaccharyltransferase catalyzes the N-glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved... (Comparative Study)
Comparative Study
N-Oligosaccharyltransferase catalyzes the N-glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved in all eukaryotes. An enzymatically active complex was isolated from microsomal membranes from Saccharomyces cerevisiae, which is composed of four proteins: Wbp1p and Swp1p (previously found to be encoded by two essential genes necessary for N-glycosylation in vivo and in vitro) and two additional proteins with a molecular mass of 60/62 kDa and 34 kDa. The 60/62 component represents differentially glycosylated forms of a protein that has sequence homology to ribophorin I. Wbp1p and Swp1p reveal homology to mammalian OST 48 and ribophorin II, respectively. Ribophorin I and II and OST 48 were recently shown to be constituents of the mammalian transferase from dog pancreas. The data reveal a high conservation of the organization of this enzyme activity.
Topics: Amino Acid Sequence; Animals; Asparagine; Dogs; Glycosylation; Hexosyltransferases; Intracellular Membranes; Membrane Proteins; Microsomes; Molecular Sequence Data; Pancreas; Saccharomyces cerevisiae; Sequence Homology; Transferases
PubMed: 8181570
DOI: 10.1016/0014-5793(94)00356-4 -
FEBS Letters Nov 1991We have determined the effect of prolonged ethanol treatment on several enzyme activities related to lipid metabolism in chick-brain and liver microsomes. Ethanol... (Comparative Study)
Comparative Study
We have determined the effect of prolonged ethanol treatment on several enzyme activities related to lipid metabolism in chick-brain and liver microsomes. Ethanol increased microsome cholesterol levels in both organs. The treatment caused a marked increase in the hepatic HMG-CoA reductase and ACAT activities while in the brain a clear decrease was found in these enzyme activities. At the same time the activity of reacylation of phospholipids, was clearly modified in both brain and liver. Thus, while in the liver the turnover of acyl moieties of phosphatidylethanolamine, sphingomyelin and phosphatidylinositol was enhanced by ethanol consumption, in the brain only the reacylation of phosphatidylserine increased to any significant extent. These results indicate that ethanol exerts a differential action in brain and liver, namely cholesterol synthesis and esterification decreased in brain and increased in chick liver. Ethanol also induces faster phospholipid metabolism in both brain and liver microsomes.
Topics: Animals; Brain; Chickens; Drug Administration Schedule; Ethanol; Hydroxymethylglutaryl CoA Reductases; Lipid Metabolism; Male; Microsomes; Microsomes, Liver; Sterol O-Acyltransferase
PubMed: 1959666
DOI: 10.1016/0014-5793(91)81190-j -
The Biochemical Journal Jan 19691. A study was made of the hydroxylation of trans-stilbene in rabbits, guinea pigs and mice, as well as by rabbit liver microsomes. 2. In the rabbit in vivo,...
1. A study was made of the hydroxylation of trans-stilbene in rabbits, guinea pigs and mice, as well as by rabbit liver microsomes. 2. In the rabbit in vivo, trans-stilbene is converted into 4-hydroxy-,4,4'-dihydroxy-,3-hydroxy-4-methoxy-and 4-hydroxy-3-methoxy-stilbene, and hydroxylation plays a more significant role in the metabolism of trans-stilbene than has previously been reported. 3. Investigation of the hydroxylation of 4-hydroxystilbene in the rabbit in vivo demonstrated its ready conversion into 4,4'-dihydroxystilbene and established its intermediacy in the formation of this compound and the methylated analogues of 3,4-dihydroxystilbene. 4. Hydroxylation of trans-stilbene in the guinea pig was found to follow a pattern similar, both qualitatively and quantitatively, to that in the rabbit. 5. Studies in the mouse revealed only limited yields of 4,4'-dihydroxystilbene. 6. Studies of the hydroxylation of trans-stilbene and 4-hydroxystilbene by rabbit liver microsomes located two of the reactions that occur with these compounds in vivo. 7. Work with a solubilized liver-microsomal preparation provided evidence that ;stilbene hydroxylase' activity is not completely lost on solubilization, thus allowing for future microsomal enzyme-isolation studies.
Topics: Animals; Chromatography, Paper; Chromatography, Thin Layer; Guinea Pigs; In Vitro Techniques; Liver; Mice; Microsomes; Mixed Function Oxygenases; Rabbits; Stilbenes
PubMed: 5775688
DOI: 10.1042/bj1110035 -
Drug Metabolism and Disposition: the... Oct 20164-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and...
4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.
Topics: Animals; Cattle; Dogs; Female; Glucuronides; Humans; Kidney; Liver; Lung; Macaca fascicularis; Male; Mice, Inbred C57BL; Microsomes; Oxidation-Reduction; Rabbits; Rats; Rats, Sprague-Dawley; Species Specificity; Terpenes
PubMed: 27468999
DOI: 10.1124/dmd.116.070003 -
Drug Metabolism and Disposition: the... Jul 2019Nonspecific drug partitioning into microsomal membranes must be considered for in vitro-in vivo correlations. This work evaluated the effect of including lipid...
Nonspecific drug partitioning into microsomal membranes must be considered for in vitro-in vivo correlations. This work evaluated the effect of including lipid partitioning in the analysis of complex TDI kinetics with numerical methods. The covariance between lipid partitioning and multiple inhibitor binding was evaluated. Simulations were performed to test the impact of lipid partitioning on the interpretation of TDI kinetics, and experimental TDI datasets for paroxetine (PAR) and itraconazole (ITZ) were modeled. For most kinetic schemes, modeling lipid partitioning results in statistically better fits. For MM-IL simulations (K = 0.1 M, k = 0.1 minute), concurrent modeling of lipid partitioning for an f range (0.01, 0.1, and 0.5) resulted in better fits compared with post hoc correction (AICc: -526 vs. -496, -579 vs. -499, and -636 vs. -579, respectively). Similar results were obtained with EII-IL. Lipid partitioning may be misinterpreted as double binding, leading to incorrect parameter estimates. For the MM-IL datasets, when f = 0.02, MM-IL, and EII model fits were indistinguishable (AICc = 3). For less partitioned datasets (f = 0.1 or 0.5), the inclusion of partitioning resulted in better models. The inclusion of lipid partitioning can lead to markedly different estimates of K and k A reasonable alternate experimental design is nondilution TDI assays, with post hoc f incorporation. The best fit models for PAR (MIC-M-IL) and ITZ (MIC-EII-M-IL and MIC-EII-M-Seq-IL) were consistent with their reported mechanism and kinetics. Overall, experimental f values should be concurrently incorporated into TDI models with complex kinetics, when dilution protocols are used.
Topics: Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Itraconazole; Lipid Metabolism; Microsomes; Models, Biological; Paroxetine
PubMed: 31043439
DOI: 10.1124/dmd.118.085969 -
The Biochemical Journal Nov 19651. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the...
1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.
Topics: Animals; In Vitro Techniques; Liver; Microscopy, Electron; Microsomes; Phenylalanine; Polynucleotides; Protein Biosynthesis; Rats; Ribosomes; Subcellular Fractions; Uracil Nucleotides
PubMed: 5880015
DOI: 10.1042/bj0970422