scholarly journals Recovery of dolichyl diphosphate oligosaccharide in methanolic aqueous phase prepared from rat liver microsomal fractions

1986 ◽  
Vol 236 (3) ◽  
pp. 913-916
Author(s):  
M Sarkar ◽  
S Mookerjea
Keyword(s):  
Aqueous Phase ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Rat Liver Microsomes ◽  
Endogenous Protein ◽  
Phase Map ◽  
Chloroform Methanol ◽  
Cellulose Column

The synthesis of dolichyl diphosphate oligosaccharide was studied by incubating rat liver microsomes (microsomal fractions) with GDP-[14C]mannose, UDP-glucose, UDP-N-acetylglucosamine and [3H]dolichol phosphate. The labelled products obtained by the first step of extraction of the microsomes in methanolic aqueous phase (MAP fraction in chloroform/methanol/water; 3:2:1, by vol.) and in CMW fraction (chloroform/methanol/water; 10:10:3, by vol.) obtained by extraction of the interphase after the first step of extraction were analysed on a DEAE-cellulose column. With the progress of incubation, the radioactivity in unchanged GDP-mannose decreased, whereas the labelled dol-P-P-oligo in the MAP fraction increased about 5-6-fold. The lipid oligosaccharide in this fraction accounted for about 50-60% of the GDP-mannose used, whereas the recovery of the labelled lipid oligosaccharide in the CMW fraction was about 10%. The lipid oligosaccharide from both reactions after mild acid hydrolysis were analysed by gel filtration on Bio-Gel P-4. The oligosaccharide from the MAP fraction gave a peak of higher Mr distinctly separate from the lower-Mr peak obtained from the CMW fraction. Microsomes incubated with labelled lipid oligosaccharide from the MAP fraction showed incorporation of the label into endogenous protein.

scholarly journals Inhibition of phospholipid methylation by a cytosolic factor

1984 ◽  
Vol 218 (2) ◽  
pp. 637-639 ◽  
Author(s):  
V Alvarez Chiva ◽  
J M Mato
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Rat Liver Microsomes ◽  
Stable Factor ◽  
Heat Stable ◽  
Ph Dependent ◽  
Chloroform Methanol ◽  
Cytosolic Factor ◽  
Rat Liver Cytosol

Rat liver cytosol contains a heat-stable factor which inhibits phospholipid methylation by rat liver microsomes. The effect of this factor on lipid methylation was dose- and pH-dependent. This factor has an Mr of approx. 3200 as estimated by gel filtration. It could not be extracted by chloroform/methanol (2:1, v/v), and its action was inhibited by incubation with subtilisin.

Isolation of rat liver microsomes by gel filtration

1973 ◽  
Vol 54 (2) ◽  
pp. 597-603 ◽  
Author(s):  
O. Tangen ◽  
J. Jonsson ◽  
S. Orrenius
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Rat Liver Microsomes

scholarly journals Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA

1993 ◽  
Vol 295 (1) ◽  
pp. 81-86 ◽  
Author(s):  
J J Mukherjee ◽  
F T Jay ◽  
P C Choy
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Neutral Lipids ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Rat Liver Microsomes ◽  
Terminal Sequence ◽  
Physiological Control ◽  
Apparent Homogeneity ◽  
Hydrolysis Of

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

Aldehyde Dehydrogenases in Rat Liver

1972 ◽  
Vol 50 (7) ◽  
pp. 741-748 ◽  
Author(s):  
G. T. Shum ◽  
A. H. Blair
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Aldehyde Dehydrogenases ◽  
Low Concentrations ◽  
Enzyme I ◽  
Cellulose Column ◽  
Aldehyde Dehydrogenase Activity

Two enzymes (I and II) with NAD+-dependent aldehyde dehydrogenase activity have been separated and partially purified from the supernatant fraction of rat liver. Resolution was effected by DEAE-cellulose column chromatography. In addition to the differences in charge properties, these two proteins differ in substrate specificity, that of enzyme II being comparatively restricted. Enzyme I has a relatively sharp optimum in activity at pH 8 whereas enzyme II exhibits an optimal range between pH 8 and 9.5. Both enzymes are strongly inhibited by low concentrations of p-chloromercuribenzene sulfonic acid and this inhibition can be reversed by dithiothreitol. Both enzymes are also inhibited by arsenite; inhibition of enzyme I is enhanced by mercaptoethanol but inhibition of enzyme II is not so affected. Molecular weight estimation by gel filtration indicates each protein has a molecular weight of approximately 180 000.

scholarly journals Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties

2003 ◽  
Vol 376 (1) ◽  
pp. 261-268 ◽  
Author(s):  
Lourdes RODRIGO ◽  
Fernando GIL ◽  
Antonio F. HERNANDEZ ◽  
Olga LOPEZ ◽  
Antonio PLA
Keyword(s):  
Affinity Chromatography ◽  
Rat Liver ◽  
Specific Activity ◽  
Liver Microsomes ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Rabbit Serum ◽  
Rat Liver Microsomes ◽  
Apparent Homogeneity

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435–33442] and liver [Ozols (1999) Biochem. J. 338, 265–275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE–Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A–Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, Km and heat and pH stability.

scholarly journals Dolichyl monophosphate and its sugar derivatives in plants

1977 ◽  
Vol 161 (1) ◽  
pp. 93-101 ◽  
Author(s):  
C T Brett ◽  
L F Leloir
Keyword(s):  
Enzyme Preparation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sodium Deoxycholate ◽  
Soya Bean ◽  
Pea Seedlings ◽  
Lipid Moiety ◽  
Sugar Derivatives ◽  
Chloroform Methanol ◽  
Cellulose Column

A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.

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
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