scholarly journals Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

1969 ◽  
Vol 115 (5) ◽  
pp. 1063-1069 ◽  
Author(s):  
T. Scott-Burden ◽  
A. O. Hawtrey
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Lithium Chloride ◽  
Ribosomal Rna ◽  
Gradient Analysis ◽  
Extraction Procedure ◽  
Liver Microsomes ◽  
Sucrose Density Gradient ◽  
Rat Liver Microsomes ◽  
Phenol Extraction

1. Treatment of washed rat liver microsomes in a medium containing 0·12m-sucrose, 12·5mm-potassium chloride, 2·5mm-magnesium chloride and 25mm-tris–hydrochloric acid buffer, pH7·6, with 2m-lithium chloride at 5° for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting 14C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH–2,6-dichlorophenol-indophenol reductase, NADH–neo-tetrazolium reductase, NADH–cytochrome c reductase and ribonuclease activities. 5. 3H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of 3H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

A Factor Capable of Dissociating Rat Liver Ribosomes

1971 ◽  
Vol 49 (12) ◽  
pp. 1301-1306 ◽  
Author(s):  
G. Ross Lawford ◽  
Jutta Kaiser ◽  
W. C. Hey
Keyword(s):  
Ionic Strength ◽  
Ammonium Sulfate ◽  
Density Gradient ◽  
Rat Liver ◽  
Messenger Rna ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Small Subunit ◽  
Temperature Dependent ◽  
Liver Ribosomes

A factor capable of dissociating rat liver monomeric ribosomes into 60 S and 40 S subunits has been partially purified and characterized.The factor was prepared by extracting a fraction of rat liver enriched in its content of native subunits with 0.05 M triethanolamine–HCl, 1.0 M KCl, 0.01 M MgSO4, and 2 mM dithiothreitol. The activity of the preparation was assayed by testing its ability to dissociate monomeric ribosomes into subunits which were detected by sucrose density gradient analysis. The ribosomes used as substrate were prepared by dissociating polysomes in the presence of puromycin, 0.5 M KCl, and 3 mM MgSO4 and subsequently reassociating the subunits into monomers by lowering the ionic strength. The factor acts only on ribosomes freed of both messenger RNA and nascent protein by associating with the small subunit. The activity was time and temperature dependent, reaching a plateau after 30 min at 30 °C.The factor has been partially purified by ammonium sulfate fractionation between 35% and 65% saturation and by treatment at 40 °C for 15 min to precipitate ribosome-aggregating substances.

scholarly journals ULTRASTRUCTURAL AND BIOCHEMICAL STUDIES ON THE PRECURSOR RIBOSOMAL PARTICLES ISOLATED FROM RAT LIVER NUCLEOLI

1974 ◽  
Vol 63 (2) ◽  
pp. 629-640 ◽  
Author(s):  
S. Matsuura ◽  
T. Morimoto ◽  
Y. Tashiro ◽  
T. Higashinakagawa ◽  
M. Muramatsu
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Rat Liver ◽  
Ribosomal Rna ◽  
Sucrose Density Gradient ◽  
Spherical Particles ◽  
Maturation Process ◽  
Biochemical Studies

Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80–100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80–100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.

Metabolic Analysis of Triptolide Microspheres in Human, Dog, Rabbit and Rat Liver Microsomes with UPLC-MS/MS Method

2020 ◽  
Vol 17 ◽  
Author(s):  
LiJuan Wang ◽  
Yan Liu ◽  
Rui Li ◽  
DongXian He
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Internal Standard ◽  
Gradient Elution ◽  
Rat Liver Microsomes ◽  
Good Precision ◽  
Kinetics Parameters ◽  
Measure Concentration ◽  
Standard Curve ◽  
Variation Of Parameters

Objectives: Triptolide (TPL) has been shown to have a good clinical effect on rheumatoid arthritis (RA). We designed TPL microspheres (TPL-MS) and investigated its metabolic behavior in human, dog, rabbit and rat liver microsomes (HLM, DLM, RLM and SDRLM) with UPLC-MS/MS method. Methods: First, a UPLC-MS/MS method was established to measure concentration of TPL in samples. The sample was separated on a C18 column (2.1×100 mm, 1.8μm) and eluted with a gradient elution. The precursor ion/product ion were m/z 378.1/361.0 for TPL and 260.0/116.2 for the internal standard. Then T1/2, Vmax and CLint were calculated from the above data. Finally, the metabolites of TPL-MS were identified by high-resolution UPLC-MS/MS. The sample was separated on a C18 column (2.1×100 mm, 2.2 μm) and eluted with isocratic elution. Mass spectrometric detection was carried out on a thermo Q-exactive mass spectrometer with HESI. The scanning range of precursor ions was from m/z 50 to m/z 750. Result and Discussion: Through several indicators including standard curve, precision, accuracy, stability, matrix effect and recovery rate, the enzymatic kinetics parameters including T1/2, Vmax and CLint were completed. Several metabolites of TPL-MS were identified. Conclusion: UPLC-MS/MS method is an accurate and sensitive method for determination of TPL in liver microsome samples with good precision, accuracy and stability. The variation of parameters indicated that the microspheres can delay the elimination of TPL in liver microsomes. The metabolism of TPL-MS varied among species, but no new metabolites appeared.

scholarly journals The metabolism of gelsevirine in human, pig, goat and rat liver microsomes

2021 ◽  
Author(s):  
Hua‐Hai Zhang ◽  
Wen‐Jia Yang ◽  
Ya‐Jun Huang ◽  
Wen‐Jing Li ◽  
Shuo‐Xin Zhang ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes

scholarly journals Detoxification of the tricyclic antidepressant opipramol and its analog – IS-noh by UGT enzymes before and after activation by phase I enzymes in rat liver microsomes

2021 ◽  
Author(s):  
Anna Mieszkowska ◽  
Koleta Hemine ◽  
Anna Skwierawska ◽  
Ewa Augustin ◽  
Zofia Mazerska
Keyword(s):  
Phase I ◽  
Rat Liver ◽  
Phase Ii ◽  
Oxidation Product ◽  
Liver Microsomes ◽  
Correct Selection ◽  
Rat Liver Microsomes ◽  
Recombinant Enzymes ◽  
Data Set ◽  
Phase I Enzymes

AbstractThe present studies were carried out to evaluate the simultaneous one-pot metabolism of opipramol (IS-opi) and analog (IS-noh) by phase I and phase II enzymes present in rat liver microsomes (RLM) as an alternative to separate testing with recombinant enzymes. This approach allows for more time-saving and cost-effective screening of the metabolism of newly discovered drugs. We also considered that the lack of results for phase II, including UGT, often creates problems in correct selection of valuable compounds. Moreover, microsomes data set is richer in the contest and provides medical scientist to determine also the susceptibility of drugs to undergo phase I and then phase II. In the present work, we have shown that IS-noh was metabolized in vitro by phase I enzymes to the oxidation product, which was next transformed with UGTs to glucuronide. The results showed also that the previously known oxidation product of opipramol was changed to previously no reported glucuronidation product by UDP-glucuronosyltransferases. In addition, unlike IS-noh, opipramol did not prove to be the substrate for UGTs. Therefore, tricyclic antidepressants depending on the structure can trigger a different response after contact with UGT enzymes. Some will metabolize directly with UGTs, others only after activation by phase I enzymes.

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