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The Journal of Biophysical and... Jun 1960Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point...
Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.
Topics: Animals; Lipids; Liver; Membranes; Microsomes; Microsomes, Liver; Proteins; RNA; Rats
PubMed: 14424705
DOI: 10.1083/jcb.7.3.547 -
Journal of Lipid Research Mar 1968The role of 7alpha,12alpha-dihydroxycholest-4-en-3-one as an intermediate in the formation of 5alpha-bile acids from cholesterol was investigated with liver preparations...
The role of 7alpha,12alpha-dihydroxycholest-4-en-3-one as an intermediate in the formation of 5alpha-bile acids from cholesterol was investigated with liver preparations of Iguana iguana in vitro. The microsomal fraction of iguana liver catalyzed the transformation of 7alpha,12alpha-dihydroxycholest-4-en-3-one to 5alpha-cholestane-3alpha,7alpha,12alpha-triol in good yield. 7alpha,12alpha-dihydroxy-5alpha-cholestan-3-one served as an intermediate. Under the conditions employed, formation of the corresponding 5beta-isomers could not be detected. High speed supernatant solution and mitochondrial fraction of iguana liver did not reduce 7alpha,12alpha-dihydroxycholest-4-en-3-one to a measurable extent. The microsomal enzyme system required NADPH as hydrogen donor and was inactive in the presence of NADH. It is suggested that 7alpha,12alpha-dihydroxycholest-4-en-3-one may serve as a common intermediate in the formation of 5alpha- and 5beta-bile acids from cholesterol.
Topics: Animals; Bile Acids and Salts; Centrifugation; Cholestanes; Chromatography; Chromatography, Gas; Chromatography, Thin Layer; Liver; Lizards; Microsomes; Oxidation-Reduction; Tritium
PubMed: 5640503
DOI: No ID Found -
Biochemical Pharmacology Oct 1967
Topics: Alkanes; Aminopyrine; Aniline Compounds; Animals; Carbon Tetrachloride; Chloroform; Cytochromes; Liver; Male; Microsomes; Rats; Spectrophotometry
PubMed: 6065969
DOI: 10.1016/0006-2952(67)90317-6 -
Gastroenterology Jan 1993Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of...
BACKGROUND
Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined.
METHODS
(1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed.
RESULTS
(1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4.
CONCLUSIONS
It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.
Topics: Autoantibodies; Cell Membrane; Cytochrome P-450 Enzyme System; Epitopes; Fluorescent Antibody Technique; Hepatectomy; Humans; Immunoblotting; Immunoenzyme Techniques; Kidney; Liver; Microsomes; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Oxygenases; Perfusion
PubMed: 7678237
DOI: 10.1016/0016-5085(93)90853-5 -
Drug Metabolism and Disposition: the... Jun 1995There have been considerable interlaboratory variations in the reported levels of rat brain microsomal cytochrome P450 and associated monooxygenase activities. To...
There have been considerable interlaboratory variations in the reported levels of rat brain microsomal cytochrome P450 and associated monooxygenase activities. To ascertain if the variability could be accountable, at least in part, to different methodologies used for microsome preparation, cytochrome P450 monooxygenase components and activities were directly compared herein using brain microsome prepared by various methods. Rat brain microsome isolated using a calcium aggregation method in the presence of dithiothreitol and glycerol contained approximately 100 pmol of cytochrome P450/mg protein. Considerably lower cytochrome P450 levels (e.g. 20-40 pmol/mg protein) were found in brain microsome prepared in a more conventional manner using Tris or phosphate buffers without glycerol and dithiothreitol. The NADPH cytochrome c reductase activity was consistently approximately 23-25 nmol of cytochrome c reduced/min/mg protein, whatever the method of preparation of the brain microsome. Cytochrome P450-associated monooxygenase activities, namely morphine N-demethylase and ethoxycoumarin O-deethylase, were dependent on the amount of protein in the incubation medium, the length of incubation, and the ratio of the concentration of the substrate to the amount of protein in the incubation mixture. The specific activity of morphine N-demethylase was constant over a range of protein concentration, if the ratio of the concentration of the substrate to the protein was kept constant.
Topics: Animals; Brain; Cytochrome P-450 Enzyme System; Kinetics; Male; Microsomes; Oxidoreductases, N-Demethylating; Oxygenases; Rats; Rats, Wistar
PubMed: 7587947
DOI: No ID Found -
The Journal of Cell Biology Jan 1963The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated...
The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated at 37 degrees C and centrifuged thereafter under the conditions of their initial isolation. The trypsin-treated microsomes and the untreated controls were fixed in unbuffered osmium tetroxide and embedded first in gelose and then in methacrylate. In the trypsin-treated microsomes, there was a removal of the ribosomes from the rough vesicles. Parallel chemical determinations showed that the total nitrogen and total phosphorus of the pellet were lowered. Particles, densely stained with phosphotungstic acid (PTA) and homogeneous in appearance, were found within microsome smooth vesicles in a fluffy layer which collects on the top of the microsome pellet. The morphology of these PTA-stained particles remained unchanged after incubation with trypsin.
Topics: Animals; Liver; Microsomes; Microsomes, Liver; Rats; Ribosomes; Trypsin
PubMed: 13931790
DOI: 10.1083/jcb.16.1.81 -
Mutation Research Dec 1975
Topics: Animals; Bacteria; Methods; Microsomes; Mutagens; Mutation
PubMed: 1219484
DOI: 10.1016/0165-1161(75)90047-3 -
Biochemical Pharmacology Oct 1967
Topics: Animals; Benactyzine; Liver; Male; Meprobamate; Microsomes; Rats
PubMed: 6065972
DOI: 10.1016/0006-2952(67)90323-1 -
Drug Metabolism and Disposition: the... 1981Tamoxifen is a nonsteroidal antiestrogen which is used as an adjuvant form of chemotherapy for breast carcinomas containing estrogen receptors. Tamoxifen citrate (2... (Comparative Study)
Comparative Study
Tamoxifen is a nonsteroidal antiestrogen which is used as an adjuvant form of chemotherapy for breast carcinomas containing estrogen receptors. Tamoxifen citrate (2 mg/rat/day) administration to male rats significantly decreased hepatic microsomal aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase activities and cytochrome P-450 content. These effects may be exerted through an antiandrogenic activity of tamoxifen, because plasma testosterone concentrations were also decreased. In male rats, tamoxifen treatment also depressed lung and intestinal microsomal aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase activities. Tamoxifen citrate treatment of female rats had no effect on hepatic, pulmonary, or intestinal microsomal aryl hydrocarbon hydroxylase or 7-ethoxycoumarin O-de-ethylase activities or hepatic cytochrome P-450 content. The results support the contention that estrogens at physiologic levels do not exert a significant regulatory effect on xenobiotic metabolism. Furthermore, androgens are known to influence drug metabolism, and the results indicate that tamoxifen has some antiandrogenic activity.
Topics: 7-Alkoxycoumarin O-Dealkylase; Animals; Cytochrome P-450 Enzyme System; Female; Intestines; Kinetics; Lung; Male; Microsomes; Microsomes, Liver; Mixed Function Oxygenases; Organ Specificity; Oxidoreductases; Oxygenases; Rats; Sex Factors; Tamoxifen; Testosterone
PubMed: 6114831
DOI: No ID Found -
Drug Metabolism and Disposition: the... 1976Cytochrome P-450-metabolic intermediate complexes were formed from N-hydroxyamphetamine, benzphetamine, norbenzphetamine, and d- and l-amphetamine in lung microsomal...
Cytochrome P-450-metabolic intermediate complexes were formed from N-hydroxyamphetamine, benzphetamine, norbenzphetamine, and d- and l-amphetamine in lung microsomal fractions and from N-hydroxyamphetamine in small intestinal mucosa microsomal fractions. Complexes were not formed in either tissue from SKF 525-A or propoxyphene. The rates of metabolic intermediate complex formation, per mole of cytochrome P-450, were higher in lung than in liver or small intestine. In contrast to that in liver, metabolic intermediate complex formation in the extrahepatic tissues was not dramatically affected by phenobarbital or 3-methylcholanthrene treatment.
Topics: Amphetamines; Animals; Cytochrome P-450 Enzyme System; Hemeproteins; In Vitro Techniques; Intestines; Lung; Male; Methylcholanthrene; Microsomes; Microsomes, Liver; Phenobarbital; Rabbits
PubMed: 11977
DOI: No ID Found