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M W Coughtrie

Publications and source records attributed to M W Coughtrie.

87 records · Page 5Linked to original sources
PubMed1990

Inhibition of UDP-glucuronosyltransferase activity by possible transition-state analogues in rat-liver microsomes.

A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4-nitrophenol glucuronidation was more effectively inhibited than that of 1-naphthol, bilirubin or testosterone. 2-(1-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were the most effective inhibitors. Their inhibitory effect was competitive towards both UDP-glucuronic acid and 4-nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP-glucuronosyl transferase have different active sites.

Animals↗
PubMed1990

The role of xenobiotic glucuronidating enzymes in drug resistance of tumour tissues and cells.

Xenobiotic and endobiotic glucuronidation is regulated by many cellular features such as (a) access of substrates to a family of microsomal enzymes, the UDP-glucuronosyltransferases (UDPGTs) and (b) mechanisms of transport and excretion of glucuronides from the cell. We have isolated molecular biological probes identifying human UDPGTs to facilitate the examination of mechanisms regulating the functional expression of UDPGTs during natural development, in genetic diseases and in cancer cells. UDPGTs are encoded by a multigene family. Seven rat and four human UDPGT cDNAs have been cloned and expressed in cell culture to determine their substrate specificity and ability to glucuronidate xenobiotics. Analysis of the amino acid sequences of cloned UDPGTs has provided information about their method of synthesis and topological orientation with the lumen of the endoplasmic reticulum (ER). The location of hepatic UDPGT raises interesting questions about the role of possible transporters essential for functional glucuronidation. Measurement of the UDPGT activities and UDPGT mRNA in carcinoma tissue and in hepatic nodules from rats suggest that increases in the level of a phenol-UDPGT may contribute to drug resistance in cancer. Human UDPGTs stably expressed in cell lines have been used to study the glucuronidation of carcinogens and anticancer drugs. Human phenol UDPGT will catalyse the glucuronidation of carcinogens. The high levels of expression of human phenol UDPGT in tissue culture cells could also lead to an increased resistance to xenobiotic toxicity caused by mitoxantrone, mitomycin C, and adriamycin. This molecular cell biology approach is being used to further assess the glucuronidation and effective toxicity of anticancer drugs.

Animals↗
PubMed1989

The enantioselective glucuronidation of morphine in rats and humans. Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme.

The formation of morphine glucuronides is enantio- and regioselective in rats and humans. In rat liver microsomes, natural (-)-morphine formed only the 3-O-glucuronide, whereas the unnatural (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions, with the 6-O-glucuronide being the principal product. In human liver microsomes, both the 3-OH-and 6-OH positions were glucuronidated with each of the enantiomers, with the 3-O-glucuronide being the major product with (-)-morphine, and the 6-OH position preferred with the (+)-enantiomer. By using a series of biochemical and biological situations such as induction by xenobiotics, ontogeny, selective inhibition and genetic deficiencies, which are considered to be diagnostic of UDP-glucuronosyltransferase heterogeneity, we determined that two UDP-glucuronosyltransferase isoenzymes were responsible for the glucuronidation of morphine in rat liver. One isoenzyme (the so-called "morphine UDP-glucuronosyltransferase") was responsible for the glucoronidation at the (-)-3-OH and (+)-6-OH positions of morphine, whereas the other formed only the (+)-morphine-3-glucuronide. Evidence from enzyme induction and the genetically deficient deficient Gunn rat suggested that bilirubin UDPGT may be responsible for the (+)-morphine-3-UDP-glucuronosyltransferase activity. In human kidney, glucuronidation of both (-)- and (+)-enantiomers at the 6-OH position was deficient, whereas the activity at the 3-OH positions was still present, which indicated the involvement of two UDP-glucuronosyltransferases in the glucuronidation of morphine in man, as well as rats.

Age Factors↗
PubMed1988

The inadequacy of perinatal glucuronidation: immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human liver microsomes.

Two anti-rat UDP-glucuronosyltransferase (UDPGT) antibody preparations, exhibiting different specificity of recognition of UDPGT isoenzymes on immunoblot analysis, were used to investigate the molecular basis of the perinatal inadequacy of glucuronidation in rats and humans. Immunoblot analysis of microsomes from developing rat liver demonstrated that the deficiency in bilirubin and testosterone glucuronidation in the fetus was due to the absence of the UDPGT isoenzyme proteins responsible for these conjugations. In contrast, phenol UDPGT enzyme activity and protein was detectable in significant amounts in fetal rat liver (greater than 30% of adult levels). In human liver, only one major immunoreactive polypeptide was observed in fetal microsomes. The remaining UDPGTs present in adult human liver developed postnatally, in parallel with the appearance of enzyme activities. Therefore, there was a correlation between the development of enzyme activity and enzyme protein. The possible consequences of developmental inadequacy of conjugation reactions for the fetus is discussed.

Adult↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1986

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↗