scholarly journals The composition of rat liver microsomes. The structural proteins of rat liver microsomes

1969 ◽  
Vol 114 (1) ◽  
pp. 41-48 ◽  
Author(s):  
K. A. Ward ◽  
J K Pollak
Keyword(s):  
Rat Liver ◽  
Ribosomal Proteins ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Microsomes ◽  
Electrophoretic Pattern ◽  
Structural Proteins ◽  
Rat Liver Microsomes ◽  
Structural Protein ◽  
Electrophoretic Patterns

1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum.

scholarly journals Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

1987 ◽  
Vol 245 (3) ◽  
pp. 811-819 ◽  
Author(s):  
H Yoshida ◽  
N Tondokoro ◽  
Y Asano ◽  
K Mizusawa ◽  
R Yamagishi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Concanavalin A ◽  
Protein Fraction ◽  
Proteolytic Enzymes ◽  
Binding Capacity ◽  
Liver Microsomes ◽  
Binding Activity ◽  
Rat Liver Microsomes ◽  
Ribosome Binding

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.

Solubilization of receptors for pancreatic polypeptide from rat liver membranes

1995 ◽  
Vol 268 (2) ◽  
pp. G215-G223
Author(s):  
T. D. Nguyen ◽  
M. S. Wolfe ◽  
G. G. Heintz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Pancreatic Polypeptide ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Receptor Complexes ◽  
High Affinity

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

scholarly journals Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.

1980 ◽  
Vol 85 (3) ◽  
pp. 516-526 ◽  
Author(s):  
J Meldolesi ◽  
G Corte ◽  
G Pietrini ◽  
N Borgese
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Water Soluble ◽  
Rat Liver Microsomes ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.

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.

scholarly journals Oxidation of docosahexaenoic acid by rat liver microsomes.

1984 ◽  
Vol 259 (9) ◽  
pp. 5776-5783 ◽  
Author(s):  
M VanRollins ◽  
R C Baker ◽  
H W Sprecher ◽  
R C Murphy
Keyword(s):  
Docosahexaenoic Acid ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes
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