Nucleotide and deduced amino acid sequence of rat liver 17 beta-hydroxysteroid UDP-glucuronosyltransferase.
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Amino Acid Sequence↗
Biomedical subjects
B Burchell
Publications and source records attributed to B Burchell.
Development of multiple activities of UDP-glucuronyltransferase in human liver.
UDP-glucuronyltransferase activities towards eight substrates were assayed in samples of foetal, term and adult human liver. Activities towards bilirubin, androsterone, testosterone, 1-naphthol, 4-nitrophenol and 2-aminophenol were present in foetal and term liver samples at less than 14% of adult values, whereas activity towards 5-hydroxytryptamine was present in foetal and term liver at 109 and 121% of adult values respectively. Thus a 'foetal' form of UDP-glucuronyltransferase may exist in human liver that is more restricted in substrate specificity than are those of the rat or rhesus monkey.
Glucuronosyltransferase↗
The molecular basis of the inherited deficiency of androsterone UDP-glucuronyltransferase in Wistar rats.
A major UDP-glucuronyltransferase isoenzyme in rat liver (51 kDa), corresponding to androsterone glucuronidating activity, has been identified by immunoblot analysis. This isoenzyme is absent from Wistar rats exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity phenotype. Northern blot analysis of total RNA from normal and androsterone glucuronidation deficient Wistar rats demonstrated that the mRNA encoding this protein was not synthesised. Differences in restriction fragment length observed on Southern blotting of genomic DNA from LA Wistar rats indicate that this inherited deficiency is the result of a deletion in the androsterone UDP-glucuronyltransferase gene.
Androsterone↗
Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.
A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).
Animals↗
Purification and properties of rat kidney UDP-glucuronosyltransferase.
Rat kidney microsomes catalysed the glucuronidation of 1-naphthol, 4-nitrophenol, bilirubin and beta-estradiol. Unlike rat hepatic microsomes, UDP-glucuronosyltransferase activity towards morphine and testosterone was not detectable. Treatment of rats with beta-naphthoflavone resulted in a 3-fold induction of renal UDPGT activity towards 1-naphthol, 4-nitrophenol and phenol, and a 2-fold induction of bilirubin and beta-estradiol glucuronidation. No induction of renal UDPGT was observed after phenobarbital treatment, but renal bilirubin UDPGT activity was specifically induced after treatment of rats with clofibrate. UDPGT activity was purified from rat kidney by a combination of ion-exchange chromatography, gel filtration and affinity chromatography on UDP-hexanolamine Sepharose. One major protein-staining polypeptide was observed on silver-stained SDS-polyacrylamide gels, of molecular weight 55,000 Da, and a minor band of 54,000 Da was also present. Indeed, immunoblot analysis of purified renal UDPGTs with anti-rat liver UDPGT antibodies revealed two immuno-reactive polypeptides of molecular weight 55,000 and 54,000 Da. The highly purified preparations catalysed the glucuronidation of 1-naphthol and bilirubin. Glucuronidation of bilirubin by purified renal UDPGT preparations required the presence of phospholipid, the activity being further enhanced by incubation with rat lung microsomes. The data presented indicate that two UDPGT isoenzymes have been copurified.
Animals↗
Genetic deficiency of bilirubin glucuronidation in rats and humans.
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Animals↗
The molecular biology of UDP-glucuronyltransferases.
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Animals↗
Defective induction of phenol glucuronidation by 3-methylcholanthrene in Gunn rats is due to the absence of a specific UDP-glucuronosyltransferase isoenzyme.
Antiserum directed against purified rat kidney UDP-glucuronosyltransferase (UDPGT) was raised in goats. IgG prepared from this antiserum exhibited specificity for only two UDPGT isoenzymes (bilirubin and phenol) on immunoblot analysis of Wistar rat liver microsomes. Use of this antibody preparation to probe Western blots of liver microsomes prepared from Gunn rats confirmed that the defective phenol glucuronidation was due to the absence of a 53-kDa, 3-methylcholanthrene-inducible UDPGT isoenzyme. Results obtained from enzyme activity measurements and immunoblot analysis of microsomes isolated from xenobiotic-treated Wistar and Gunn rat liver are consistent with the 3-methylcholanthrene/UDPGT induction deficiency in the Gunn rat being due to the absence of this phenol UDPGT isoenzyme. The contribution of other UDPGT isoenzymes to the greatly reduced glucuronidation of planar phenols observed in the Gunn rat is discussed.
Animals↗
A general assay for UDPglucuronosyltransferase activity using polar amino-cyano stationary phase HPLC and UDP[U-14C]glucuronic acid.
The assay of UDPglucuronosyltransferase activity toward various substrates using UDP[U-14C]glucuronic acid is described. HPLC on a polar amino-cyano bonded phase column was used to separate radioactive glucuronides from unmetabolized UDP[U-14C]glucuronic acid and other labeled reaction products. Radioactivity was measured using flow-through scintillation counting. All the glucuronides analyzed, with one exception, chromatographed with the same retention time (9.0-9.6 min) under the conditions described. Glucuronide conjugates were identified by comparison with retention times of commercial glucuronide standards, using radioactive aglycones, or hydrolysis with beta-glucuronidase. The method provides a unified, sensitive (100-200 pmol of glucuronide product) and reproducible assay for a wide variety of UDPglucuronosyltransferase substrates, and could be extended to include many others.
Animals↗
Induction of UDP-glucuronyl transferase mRNA in embryonic chick livers by phenobarbital.
Administration of phenobarbital to chick embryos increased hepatic microsomal UDP-glucuroyltransferase activity some 25-fold. The large phenobarbital-induced increase of UDP-glucuronyltransferase activity was correlated to an equivalent increase of immunochemically measurable UDP-glucuronyltransferase protein. Poly(A+) RNA isolated from the livers of chick embryos treated with either phenobarbital or saline was translated in vitro. Immunochemical analysis of the translation products indicated that phenobarbital induced a 30-fold increase in UDP-GT mRNA. Fractionation of hepatic poly(A+) RNA from phenobarbital-treated chick embryos by sucrose density gradient centrifugation indicated that the size of the UDP-GT mRNA was 21S. These data show that phenobarbital induction of chick embryo liver UDP-glucuronyltransferase activity correlates with a similar large increase in the level of translatable mRNA for this enzyme.
Animals↗
The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family.
Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT.
Amino Acid Sequence↗
Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride.
Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.
Aluminum↗
Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.
Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity.
Animals↗
Congenital jaundice in rats due to the absence of hepatic bilirubin UDP-glucuronyltransferase enzyme protein.
Three major UDP-glucuronyltransferase isoenzymes (50-54 kDa) have been identified by immunoblot analysis. Bilirubin UDP-glucuronyltransferase (54 kDa) was specially induced by treatment of the rats with clofibrate. This isoenzyme was not detectable in liver microsomal extracts from congenitally jaundiced Gunn rats and was not induced by treatment of these animals with clofibrate. Phenol UDP-glucuronyltransferase, the only isoenzyme determined to be present in foetal Wistar rat liver microsomes was not detected by enzyme assay or immunoblot analysis of foetal Gunn rat liver microsomal extracts. These results provide the first indication that bilirubin UDP-glucuronyltransferase and possible phenol UDP-glucuronyltransferase proteins are not present in the congenitally jaundiced Gunn rat.
Animals↗
Genotoxicity studies of rodents exposed to coal dust and diesel emission particulates.
Genotoxicity studies with mice and/or rats have been conducted to evaluate the potential mutagenic hazard associated with exposures of coal miners to diesel emission particulates (DEP) and/or coal dusts (CD). Rats and mice were exposed to filtered air, DEP, and/or CD for periods ranging from 3 months to 2 years. Levels of respirable particulates were maintained at 2 mg/m3 in all exposed groups. DEP and/or CD were collected in the inhalation chambers in which animals were exposed. Urine samples were collected for 5 consecutive days from rats exposed to DEP and/or CD for 3, 6, and 24 months. The particulate samples extracted with dichloromethane and the urine samples concentrated with XAD-2 columns were analyzed for mutagenic activity by the Ames Salmonella/microsome assay system. Peripheral blood lymphocytes from rats exposed for 3 months were analyzed for sister chromatid exchanges (SCE). The femur bone marrow cells from rats exposed for 24 months and mice exposed for 6 months were analyzed for micronuclei in both polychromatic and normochromatic erythrocytes. The results indicate that the solvent extract of DEP was mutagenic, while no mutagenic activity was found for the CD extract. Combination of CD and DEP did not show any synergistic effect. No mutagenic activity was found for urine samples from rats exposed to DEP and/or CD for up to 2 years. A slight increase in the micronucleated polychromatic erythrocytes over the control level was found in mice exposed to DEP and DEP plus CD for 6 months but the increase was not statistically significant. No increase in micronuclei was detected in rats exposed for 24 months. The frequencies of SCE in the peripheral lymphocytes of the 3-month-exposed rats were similar for control and DEP plus CD-exposed groups.
Animals↗
Cloning of cDNAs coding for rat hepatic microsomal UDP-glucuronyltransferases.
Radioiodinated, affinity-purified, anti-UDP-glucuronyltransferase (UDPGT) antibodies have been used to isolate cDNAs coding for UDPGT(s) from a rat liver cDNA library cloned in the expression vector bacteriophage lambda gt11. The sizes of ten cloned cDNAs range from 0.3-2.1 kb. The identity of the cDNAs was confirmed by the hybrid-select translation and immunochemical analyses. Restriction mapping indicates that two classes of cDNA coding for different UDPGT mRNAs have been cloned.
Animals↗
Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase.
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.
Animals↗
Specific inactivation of the phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system by diethyl pyrocarbonate.
We have examined the interactions of the histidine-specific reagent diethyl pyrocarbonate (DEPC) with the components of the rat hepatic glucose-6-phosphatase system (EC 3.1.3.9). DEPC is the first known reagent that satisfies the criteria of an active-site-specific label for the phosphohydrolase component. (a) It inactivates through formation of a stable covalent bond. (b) It is effective at reasonably low concentrations (2-4 mM) under relatively mild conditions (e.g. 30 degrees C at neutral pH). (c) Inactivation is substantially blocked by glucose 6-phosphate, Pi and NaF, compounds which are known to interact quite specifically with the phosphohydrolase. (d) Under conditions where glucose 6-phosphate and NaF protect the enzyme, no protection is provided against DEPC-mediated inactivation of two other functional components of the membrane, the glucose 6-phosphate translocase and UDP-glucuronyltransferase. DEPC also shows potential for use at 0 degree C as a label for UDP-glucuronyltransferase.
Animals↗