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.

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.

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 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.

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.

scholarly journals A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

1962 ◽  
Vol 15 (3) ◽  
pp. 563-578 ◽  
Author(s):  
Lars Ernster ◽  
Lois C. Jones
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Physiological Role ◽  
Sodium Deoxycholate ◽  
Inorganic Pyrophosphatase ◽  
Rat Liver Microsomes ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
No Activation

Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment. Different approaches toward the function of the liver microsomal nucleoside tri- and diphosphatases are reported, and the possible physiological role of the two enzymes is discussed.

scholarly journals STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES

1960 ◽  
Vol 8 (3) ◽  
pp. 665-673 ◽  
Author(s):  
Jay S. Roth
Keyword(s):  
Enzyme Activity ◽  
Biological Activity ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rnase Activity ◽  
Rat Liver Microsomes ◽  
Rnase Inhibitor ◽  
Normal Constituent ◽  
Liver Ribosomes

Attempts have been made to prepare rat liver microsomes and ribosomes free of RNase activity. Washing of microsomes with a large number of reagents, as well as preparation of microsomes by homogenizing the liver in the presence of a variety of reagents chosen to remove or inhibit RNase activity, failed to abolish completely the enzyme activity. However, when rat liver was homogenized in the presence of optimal concentrations of ATP the microsomes subsequently obtained showed no RNase activity. The composition of such microsomes was compared to controls prepared without the use of ATP. Preparation of microsomes with the use of ATP apparently repressed but did not remove the RNase activity for, when such microsomes were treated with 1 per cent deoxycholate to obtain ribosomes, the latter exhibited normal RNase activity. A possible explanation for these results based on several experiments is given. The incorporation of C14 of L-leucine-C14 into control and ATP-treated microsomes was measured. Repression of RNase activity by use of ATP or with RNase inhibitor, significantly reduced the incorporation. As a result of these and other experiments it is tentatively concluded that an alkaline RNase is a normal constituent of rat liver ribosomes and plays a role in the biological activity of these particles.

Sign in / Sign up

Export Citation Format

Share Document