-
Nature Biotechnology Oct 2001An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps:...
An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.
Topics: Affinity Labels; Amino Acid Sequence; Cell Differentiation; HL-60 Cells; Humans; Mass Spectrometry; Microsomes; Molecular Sequence Data; Proteins; Software; Tetradecanoylphorbol Acetate
PubMed: 11581660
DOI: 10.1038/nbt1001-946 -
The Journal of Clinical Investigation Mar 1988Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized...
Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The Km value of guinea pig lung LTC4 synthase for LTA4 was 3 microM and the Vmax was 108 nmol/3 min per microgram; the Km values for LTA3 and LTA5 were similar, and the Vmax values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective Ki values of 1.5, 3.3, and 2.8 microM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 microM LTA4-me with 5 mM GSH were 2.1 microM and 0.3 microM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 greater than 40 microM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.
Topics: Animals; Glutathione Transferase; Guinea Pigs; Kinetics; Lung; Microsomes; Subcellular Fractions; Substrate Specificity
PubMed: 3343345
DOI: 10.1172/JCI113396 -
Proceedings of the National Academy of... Dec 1975About 300 carcinogens and non-carcinogens of a wide variety of chemical types have been tested for mutagenicity in the simple Salmonella/microsome test. The test uses...
About 300 carcinogens and non-carcinogens of a wide variety of chemical types have been tested for mutagenicity in the simple Salmonella/microsome test. The test uses bacteria as sensitive indicators for DNA damage, and mammalian liver extracts for metabolic conversion of carcinogens to their active mutagenic forms. Quantitative mutagenicity data from linear dose-response curves are presented: potency varies over a 10(6)-fold range. There is a high correlation between carcinogenicity and mutagenicity: 90% (156/174) of carcinogens are mutagenic in the test and despite the severe limitations inherent in defining non-carcinogenicity, few "non-carcinogens" show any degree of mutagenicity. The results also demonstrate the great utility, and define the limitations, of the test in detecting environmental carcinogens.
Topics: Carcinogens; Drug Evaluation, Preclinical; Microsomes; Mutagens; Salmonella
PubMed: 1061098
DOI: 10.1073/pnas.72.12.5135 -
British Journal of Industrial Medicine May 1979The effect of tricresylphosphate (TCP) was studied in vitro and in vivo on the rat liver and brain enzymes acetylcholinesterase (ACC), butyrylcholinesterase (CHE),...
The effect of tricresylphosphate (TCP) was studied in vitro and in vivo on the rat liver and brain enzymes acetylcholinesterase (ACC), butyrylcholinesterase (CHE), arylesterase (ARE), aliesterase (ALI), and the microsomal nicotinamide-adenine dinucleotide phosphate oxidase (NADPH2-oxidase) system. The results show that, in the male rat, TCP given intraperitoneally induces an increase in liver microsomal ARE AND NADPH2-oxidase and a decrease in ALI and cholinesterase; no activation of ARE and NADPH2-oxidase is observed in female rats.
Topics: Animals; Brain; Cresols; Drug Synergism; Enzyme Induction; Esterases; Female; Hexanes; In Vitro Techniques; Male; Microsomes; Microsomes, Liver; Rats; Tritolyl Phosphates
PubMed: 465377
DOI: 10.1136/oem.36.2.153 -
FEBS Letters Jan 1986A factor which markedly activates Ca2+-dependent thiol protease (calpain) is associated with Triton X-100-insoluble materials, presumably structural elements such as...
A factor which markedly activates Ca2+-dependent thiol protease (calpain) is associated with Triton X-100-insoluble materials, presumably structural elements such as cytoskeletons, of bovine brain microsomal fraction. This factor is extracted with 0.6 M KC1, and purified partially by sucrose density gradient centrifugation and hydroxyapatite column chromatography. The factor appears to be a heat-stable protein with an approximate Mr of 15 000. With casein as substrate this factor activates both calpain I and calpain II several-fold up to more than 10- fold without alteration of their affinity to Ca2+. Calmodulin is unable to substitute for this factor. A similar factor is associated with human platelet insoluble materials.
Topics: Animals; Brain Chemistry; Calpain; Caseins; Cattle; Centrifugation, Density Gradient; Enzyme Activation; Hydrolysis; Microsomes; Proteins; Solubility
PubMed: 3000821
DOI: 10.1016/0014-5793(86)80060-6 -
Journal of Pharmacy & Pharmaceutical... 2012The antipsychotic drug haloperidol can be metabolised to pyridinium metabolites haloperidol pyridinium (HP+) and reduced haloperidol pyridinium (RHP+). These pyridinium...
PURPOSE
The antipsychotic drug haloperidol can be metabolised to pyridinium metabolites haloperidol pyridinium (HP+) and reduced haloperidol pyridinium (RHP+). These pyridinium metabolites were proposed to contribute to the extrapyramidal side effects of haloperidol, because they are structural analogues of N-methyl-4-phenylpyridinium (MPP+), a well-known neurotoxin. RHP+ can be oxidized to HP+ by CYP1A1. In the current study, the oxidation of RHP+ to HP+ was investigated using human placenta microsomal preparations which contain relatively high levels of CYP1A1.
METHODS
Cytochrome P450 isoenzymes responsible for the metabolism of RHP+ were characterized in vitro using human placenta microsomal preparations from smokers and non-smokers.
RESULTS
A comparison of the metabolic activities between smokers and non-smokers suggests that smokers had higher activities for the oxidation of RHP+. A selective antibody against CYP1A1 was a partial inhibitor of RHP+ oxidase in placenta from smokers but had no effect in placenta from non-smokers. Furafylline and ketokonazole were shown to be stronger inhibitors of the oxidation of RHP+ to HP+ in liver than in placenta. This seems to indicate important contributions of CYP1A1 and CYP3A7 as compared to CYP1A2 and CYP3A4, respectively, because furafylline and ketokonazole are stronger inhibitors of CYP1A2 and CYP3A4 than CYP1A1 and CYP3A7, respectively. Interestingly, α-naphathoflavone enhanced the metabolic activity in liver microsomes due to its activator effect on CYP3A4. On the other hand, α-naphathoflavone partially inhibited the activity in placenta microsomes, indicating a role played by CYP1A1 or CYP1A2 in the oxidation of RHP+ in placenta.
CONCLUSIONS
These data indicate that CYP1A1 plays an important role in the oxidation of RHP+ to HP+ in placenta from smokers. CYP3A7 and CYP3A4 could also play important roles in the metabolism of RHP+ in placenta microsomes. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Topics: Antipsychotic Agents; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP3A; Female; Haloperidol; Humans; Microsomes; Microsomes, Liver; Oxidation-Reduction; Placenta; Pregnancy; Pyridinium Compounds; Smoking
PubMed: 23106957
DOI: 10.18433/j31w20 -
Bioorganic & Medicinal Chemistry Letters Dec 2015Adamantyl ureas are good soluble epoxide hydrolase (sEH) inhibitors; however they have limited solubility and rapid metabolism, thus limiting their usefulness in some...
Adamantyl ureas are good soluble epoxide hydrolase (sEH) inhibitors; however they have limited solubility and rapid metabolism, thus limiting their usefulness in some therapeutic indications. Herein, we test the hypothesis that nodal substitution on the adamantane will help solubilize and stabilize the compounds. A series of compounds containing adamantane derivatives and isoxazole functional groups were developed. Overall, the presence of methyl on the nodal positions of adamantane yields higher water solubility than previously reported urea-based sEH inhibitors while maintaining high inhibition potency. However, it did not improve microsomal stability.
Topics: Adamantane; Drug Stability; Enzyme Inhibitors; Epoxide Hydrolases; Humans; Inhibitory Concentration 50; Isoxazoles; Microsomes; Solubility; Structure-Activity Relationship; Urea
PubMed: 26520661
DOI: 10.1016/j.bmcl.2015.10.066 -
Genetics and Molecular Research : GMR Jun 2004Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects. This activation involves cytochrome P450s...
Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects. This activation involves cytochrome P450s (CYP), which generates unstable metabolites that react with the DNA of cells in the immediate vicinity of metabolite formation. Although NDEA is carcinogenic, it has been considered a weak mutagen in classic genotoxicity assays. We used optimized Salmonella/mammalian microsome genotoxicity assays to assess the mutagenicity and toxicity of low concentrations of NDEA. Using a fixed concentration of NDEA (36.5 mg/ml), we varied the length of preincubation in the presence of different concentrations of an S9 metabolic activation mixture. Salmonella typhimurium strains TA97 and TA102 were resistant to NDEA-induced mutagenesis, even after a preincubation of up to 120 min and the use of different concentrations of the S9 mix. Strain TA98 was susceptible to mutagenesis by NDEA in the absence of the S9 mix and after preincubation with NDEA for 90 min. When bacteria of this strain were preincubated with NDEA for 60 min, mutagenesis was detected at an S9 mix concentration >9.55 mg/ml. NDEA also induced mutagenesis in strain TA100 after preincubation for 90 or 120 min, and this effect was dependent on the S9 concentration. E. coli strain BH990 also showed a concentration-dependent response, with only 60% of the cells surviving after a 120-min preincubation with NDEA in the presence of 19.1 mg S9 mix/ml.
Topics: Alkylating Agents; Biotransformation; Diethylnitrosamine; Dose-Response Relationship, Drug; Escherichia coli; Microsomes; Mutagenicity Tests; Salmonella typhimurium; Time Factors
PubMed: 15266397
DOI: No ID Found -
Environmental Health Perspectives Apr 1981Studies were carried out to determine the actions of and interactions between ascorbate, NADPH, Fe2+, and Fe3+ on lipid peroxidation in adrenal and testicular...
Studies were carried out to determine the actions of and interactions between ascorbate, NADPH, Fe2+, and Fe3+ on lipid peroxidation in adrenal and testicular microsomes. Ascorbate-induced malonaldehyde production was maximal in adrenal and testicular microsomes at an ascorbate concentration of 1 X 10(-4)M. Fe2+, at levels between 10(-6) and 10(-3)M, produced concentration-dependent increases in lipid peroxidation in adrenal and testicular microsomes; Fe2+ had a far greater effect than Fe3+ in both tissues. In liver microsomes, by contrast, Fe2+ and Fe3+ had quantitatively similar effects on lipid peroxidation. NADPH alone had no effect on malonaldehyde production in adrenal or testicular microsomes. However, in the presence of low Fe2+ concentrations (10(-6)M), NADPH stimulated adrenal malonaldehyde production. The stimulation of lipid peroxidation by NADPH plus low Fe2+ was not demonstrable in testicular microsomes nor in adrenal microsomes which had been heat-treated to inactivate microsomal enzymes. Testicular malonaldehyde production was stimulated by NADPH if Fe3+ (5 X 10(-5) to 1 X 10(-3)M) was added to the incubation medium; the stimulation was not demonstrable in heat-treated microsomes. Fe3+ plus NADPH had little effect on adrenal lipid peroxidation. In the presence of high Fe2+ levels (10(-3)M), NADPH produced a concentration-dependent inhibition of adrenal lipid peroxidation; the inhibition was fully demonstrable in heat-treated microsomes. NADPH similarly inhibited ascorbate-induced lipid peroxidation in adrenal microsomes. In testicular microsomes, NADPH did not inhibit ascorbate or Fe2+-induced lipid peroxidation. The results indicate that various endogenous substances may be important in the control of adrenal and testicular lipid peroxidation and that the nature of the regulation differs from tissue to tissue.
Topics: Adrenal Glands; Animals; Ascorbic Acid; Guinea Pigs; Iron; Kinetics; Lipid Peroxides; Male; Malonates; Malondialdehyde; Microsomes; Testis
PubMed: 7238441
DOI: 10.1289/ehp.8138105 -
European Journal of Biochemistry May 1996Two distinct long-chain-acyl-CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland. One was localized in the...
Two distinct long-chain-acyl-CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland. One was localized in the microsomes and the other in the mitochondria. The relative V(max) values of the microsomal enzyme were 8.1, 1.7 and 1 and the apparent Km values were 66.7, 12.0 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values of the mitochondrial enzyme were 2.7, 3.5 and 1 and the apparent Km values were 33.3, 29.9 and 30.0 microM for palmitic, linoleic and arachidonic acids, respectively. The relative V(max) values for the liver microsomal enzyme were 2.0, 2.5 and 1, while those of the liver mitochondrial enzyme were 4.1, 3.9 and 1 with palmitic, linoleic and arachidonic acids, respectively. There were no difference between the microsomal and the mitochondrial enzymes in the liver, regarding apparent Km values; these were 38.4, 29.9 and 22.0 microM for palmitic, linoleic and arachidonic acids, respectively. Thus, the substrate specificity and catalytic rate of the mitochondrial enzyme in Harderian gland for palmitic, linoleic and arachidonic acids were similar to the liver enzyme, but not to the microsomal enzyme in Harderian gland. On the other hand, the antiserum raised against the rat liver enzyme immune-titrated and immuno-blotted the enzymes from Harderian gland microsomes and liver, but not so the enzyme from Harderian gland mitochondria. Thus, the microsomal enzyme in Harderian gland had a common immunogenic epitope(s) with the liver enzyme, but the mitochondrial enzyme did not. The Harderian gland mitochondrial enzyme was a distinct protein from liver enzymes. The catalytic and immunogenic characteristics suggest that the enzyme proteins in the Harderian gland are unique, that is, different from that in the liver. The large V(max) value of the Harderian gland microsomal enzyme for palmitic acid suggests that it contributes to the synthesis of a large amount of the secretory lipid and the high Km value to maintenance of cellular lipid in this organ. The evidence that long-chain-acyl-CoA synthetase in the mitochondria is distinct from that in the microsomes was first found in guinea pig Harderian gland.
Topics: Animals; Blotting, Western; Chromatography, Gel; Coenzyme A Ligases; Guinea Pigs; Harderian Gland; Kinetics; Lipid Metabolism; Liver; Microsomes; Mitochondria; Rats; Repressor Proteins; Saccharomyces cerevisiae Proteins; Tissue Distribution; Titrimetry
PubMed: 8665926
DOI: 10.1111/j.1432-1033.1996.0104q.x