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 Purification and characterization of paraoxon hydrolase from rat liver

1997 ◽  
Vol 321 (3) ◽  
pp. 595-601 ◽  
Author(s):  
Lourdes RODRIGO ◽  
Fernando GIL ◽  
Antonio F. HERNANDEZ ◽  
Anabel MARINA ◽  
Jesus VAZQUEZ ◽  
...  
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Rabbit Serum ◽  
Heat Inactivation ◽  
Ph Profile ◽  
Apparent Homogeneity ◽  
Nitrophenyl Phosphate ◽  
Cibacron Blue 3Ga ◽  
Serum Paraoxonase

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.

scholarly journals Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

1982 ◽  
Vol 201 (3) ◽  
pp. 653-656 ◽  
Author(s):  
B Burchell
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Specific Activity ◽  
Liver Microsomes ◽  
Wistar Rat ◽  
Transferase Activity ◽  
Rat Liver Microsomes ◽  
Phosphatidylcholine Liposomes ◽  
Gunn Rat ◽  
Rigid Environment

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.

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 Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes

1992 ◽  
Vol 287 (3) ◽  
pp. 725-731 ◽  
Author(s):  
E Axén ◽  
T Bergman ◽  
K Wikvall
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Vitamin D3 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Pig Liver ◽  
Specific Content ◽  
Apparent Homogeneity ◽  
Structural And Functional Properties ◽  
Cytochrome P 450

A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.

CHANGES IN OESTROGEN METABOLISM INDUCED IN RAT LIVER MICROSOMES BY SUBCUTANEOUS PELLETS OF OESTRONE AND TESTOSTERONE

1967 ◽  
Vol 39 (1) ◽  
pp. 99-104 ◽  
Author(s):  
P. H. JELLINCK ◽  
JANETTE WOO
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Microsomes ◽  
Hepatic Metabolism ◽  
Water Soluble ◽  
High Yield ◽  
Male Rat ◽  
Rat Liver Microsomes ◽  
Time Intervals ◽  
Rate Of Absorption

SUMMARY Oestrone administered in the form of subcutaneous pellets produced marked changes in the metabolism of [14C]oestradiol by male rat liver microsomes. The high yield of both 2-hydroxyoestradiol and water-soluble metabolites was decreased to the level normally observed in females and this effect was induced by relatively small amounts of oestrogen within a few days after implantation. The action of testosterone on the hepatic metabolism of oestrogens was also investigated together with the effect of removing the hormone pellets at different time intervals. In addition, the rate of absorption of the steroids was determined by direct weighing and, in the case of oestrone, controlled by using radioactive pellets of known specific activity.

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.

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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