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 Stimulation of defective Gunn-rat liver uridine diphosphate glucuronyltransferase activity in vitro by alkyl ketones

1979 ◽  
Vol 177 (3) ◽  
pp. 993-995 ◽  
Author(s):  
E N Lalani ◽  
B Burchell
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Wistar Rat ◽  
Uridine Diphosphate ◽  
Gunn Rat ◽  
Alkyl Ketone ◽  
Genetic Deficiency ◽  
Liver Homogenates ◽  
Stimulation Of

Addition of alkyl ketone (10mM) to Gunn-rat liver homogenates increased UDP-glucuronyltransferase activity towards 2-aminophenol by 10–20 fold, up to enhanced values of enzyme activity observed with similarly treated Wistar-rat liver homogenates. Alkyl ketones also activate the defective enzyme purified from Gunn-rat liver. This genetic deficiency of UDP-glucuronyltransferase activity is no longer apparent when assayed in the presence of alkyl ketones.

Purification and stabilization of galactosyl transferase from serum and lysolecithin extracted microsomes

1980 ◽  
Vol 58 (10) ◽  
pp. 878-884 ◽  
Author(s):  
Ian H. Fraser ◽  
Patricia Wadden ◽  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
High Speed ◽  
Specific Activity ◽  
Active Form ◽  
Liver Microsomes ◽  
Affinity Column ◽  
Chromatographic Technique ◽  
Rat Liver Microsomes ◽  
Galactosyl Transferase ◽  
Membrane Enzyme

Rat liver microsomes treated with increasing concentrations of lysolecithin released, after a brief lag, progressively increasing amounts of UDPgalactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. A second extraction of the microsomes with lysolecithin (8 mM) resulted in a total release of about 45% enzyme. The specific activity of the enzyme in the second extract was 12 times higher than that of the first extract. The galactosyl-transferase present in the extract was purified 417-fold by an affinity column chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. During purification, the column and elution buffers required 0.1% lysolecithin to keep the enzyme in active form. For purposes of comparison the soluble serum galactosyltransferase was also purified by identical techniques, which also required 0.1% lysolecithin in column and elution buffers to prevent the loss of enzyme activity. The pure serum and membrane galactosyltransferase contained no sialyltransferase and ran as a double band on polyacrylamide gels (molecular weight 63 000–64 000). The pure enzyme had an absolute requirement for Mn2+, not replaceable by Cu2+, Mg2+, Zn2+, and Co2+. The enzymes were active over a wide pH range, with optimum pH of 6.5. The apparent Km's for UDPgalactose for the serum and membrane enzymes were 12.05 and 11.8 μM, respectively. The specific activities of these two purified enzymes were also remarkably similar, 3.99 × 106 for serum and 3.84 × 106 for membrane enzyme. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose (DSG-fetuin). The enzyme activity with DSG-fetuin acceptor was inhibited to a lesser extent by α-lactalbumin.Effect of various additives on the stability of purified membrane and serum galactosyltransferase was studied at 0–5 °C and at −20 °C up to 60 days. At both temperatures, albumin was found to be the best stabilizer. Ammonium sulfate was a good stabilizer for the serum but not for the membrane enzyme. Glycerol showed some stabilizing effect for both enzymes. EDTA, p-methylbenzenesulfonyl fluoride, N-acetylglucosamine-Mn2+, and water did not offer any stabilization of the pure enzyme.

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.

scholarly journals Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase

1984 ◽  
Vol 223 (2) ◽  
pp. 461-465 ◽  
Author(s):  
B Burchell ◽  
N Blanckaert
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Total Bilirubin ◽  
Glucuronic Acid ◽  
Liver Microsomes ◽  
Wistar Rat ◽  
Zero Order ◽  
Rat Liver Microsomes ◽  
Order Kinetic ◽  
Hepatic Microsomes

Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.

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.

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.

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