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. 1988 Nov;85(22):8381–8385. doi: 10.1073/pnas.85.22.8381

Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells.

D Harding 1, S Fournel-Gigleux 1, M R Jackson 1, B Burchell 1
PMCID: PMC282461  PMID: 3141926

Abstract

A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDP-glucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector lambda gt11. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of approximately 55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of approximately 3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arias I. M., Gartner L. M., Cohen M., Ezzer J. B., Levi A. J. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am J Med. 1969 Sep;47(3):395–409. doi: 10.1016/0002-9343(69)90224-1. [DOI] [PubMed] [Google Scholar]
  2. Bansal S. K., Gessner T. A unified method for the assay of uridine diphosphoglucuronyltransferase activities toward various aglycones using uridine diphospho[U-14C]glucuronic acid. Anal Biochem. 1980 Dec;109(2):321–329. doi: 10.1016/0003-2697(80)90655-7. [DOI] [PubMed] [Google Scholar]
  3. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  4. Bock K. W., Bock-Hennig B. S. Differential induction of human liver UDP-glucuronosyltransferase activities by phenobarbital-type inducers. Biochem Pharmacol. 1987 Dec 1;36(23):4137–4143. doi: 10.1016/0006-2952(87)90572-7. [DOI] [PubMed] [Google Scholar]
  5. Bock K. W., Brunner G., Hoensch H., Huber E., Josting D. Determination of microsomal UDP-glucuronyltransferase in needle-biopsy specimens of human liver. Eur J Clin Pharmacol. 1978 Dec 18;14(5):367–373. doi: 10.1007/BF00611908. [DOI] [PubMed] [Google Scholar]
  6. Burchell B., Coughtrie M. W., Jackson M. R., Shepherd S. R., Harding D., Hume R. Genetic deficiency of bilirubin glucuronidation in rats and humans. Mol Aspects Med. 1987;9(5):429–455. doi: 10.1016/0098-2997(87)90007-0. [DOI] [PubMed] [Google Scholar]
  7. Coughtrie M. W., Burchell B., Shepherd I. M., Bend J. R. Defective induction of phenol glucuronidation by 3-methylcholanthrene in Gunn rats is due to the absence of a specific UDP-glucuronosyltransferase isoenzyme. Mol Pharmacol. 1987 Jun;31(6):585–591. [PubMed] [Google Scholar]
  8. Dragacci S., Hamar-Hansen C., Fournel-Gigleux S., Lafaurie C., Magdalou J., Siest G. Comparative study of clofibric acid and bilirubin glucuronidation in human liver microsomes. Biochem Pharmacol. 1987 Nov 15;36(22):3923–3927. doi: 10.1016/0006-2952(87)90459-x. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  10. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  12. Irshaid Y. M., Tephly T. R. Isolation and purification of two human liver UDP-glucuronosyltransferases. Mol Pharmacol. 1987 Jan;31(1):27–34. [PubMed] [Google Scholar]
  13. Iyanagi T., Haniu M., Sogawa K., Fujii-Kuriyama Y., Watanabe S., Shively J. E., Anan K. F. Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase. J Biol Chem. 1986 Nov 25;261(33):15607–15614. [PubMed] [Google Scholar]
  14. Jackson M. R., Craft J. A., Burchell B. Nucleotide and deduced amino acid sequence of human liver microsomal epoxide hydrolase. Nucleic Acids Res. 1987 Sep 11;15(17):7188–7188. doi: 10.1093/nar/15.17.7188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson M. R., Kennedy S. M., Lown G., Burchell B. Induction of UDP-glucuronyl transferase mRNA in embryonic chick livers by phenobarbital. Biochem Pharmacol. 1986 Apr 1;35(7):1191–1198. doi: 10.1016/0006-2952(86)90159-0. [DOI] [PubMed] [Google Scholar]
  16. Jackson M. R., McCarthy L. R., Harding D., Wilson S., Coughtrie M. W., Burchell B. Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA. Biochem J. 1987 Mar 1;242(2):581–588. doi: 10.1042/bj2420581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kwok S. C., Ledley F. D., DiLella A. G., Robson K. J., Woo S. L. Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase. Biochemistry. 1985 Jan 29;24(3):556–561. doi: 10.1021/bi00324a002. [DOI] [PubMed] [Google Scholar]
  19. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Leakey J. E., Hume R., Burchell B. Development of multiple activities of UDP-glucuronyltransferase in human liver. Biochem J. 1987 May 1;243(3):859–861. doi: 10.1042/bj2430859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mackenzie P. I. Rat liver UDP-glucuronosyltransferase. cDNA sequence and expression of a form glucuronidating 3-hydroxyandrogens. J Biol Chem. 1986 Oct 25;261(30):14112–14117. [PubMed] [Google Scholar]
  23. Mahu J. L., Preaux A. M., Mavier P., Berthelot P. Characterization of microsomal bilirubin and p-nitrophenol uridine diphosphate glucuronosyltransferase activities in human liver: a comparison with rat liver. Enzyme. 1981;26(2):93–102. doi: 10.1159/000459155. [DOI] [PubMed] [Google Scholar]
  24. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miners J. O., Lillywhite K. J., Matthews A. P., Jones M. E., Birkett D. J. Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes. Biochem Pharmacol. 1988 Feb 15;37(4):665–671. doi: 10.1016/0006-2952(88)90140-2. [DOI] [PubMed] [Google Scholar]
  26. Mishina M., Kurosaki T., Tobimatsu T., Morimoto Y., Noda M., Yamamoto T., Terao M., Lindstrom J., Takahashi T., Kuno M. Expression of functional acetylcholine receptor from cloned cDNAs. Nature. 1984 Feb 16;307(5952):604–608. doi: 10.1038/307604a0. [DOI] [PubMed] [Google Scholar]
  27. Oesch F. Purification and specificity of a human microsomal epoxide hydratase. Biochem J. 1974 Apr;139(1):77–88. doi: 10.1042/bj1390077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Otani G., Abou-El-Makarem M. M., Bock K. W. UDP-glucuronyltransferase in perfused rat liver and in microsomes - III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin. Biochem Pharmacol. 1976 Jun 1;25(11):1293–1297. doi: 10.1016/0006-2952(76)90092-7. [DOI] [PubMed] [Google Scholar]
  29. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Parquet M., Pessah M., Sacquet E., Salvat C., Raizman A., Infante R. Glucuronidation of bile acids in human liver, intestine and kidney. An in vitro study on hyodeoxycholic acid. FEBS Lett. 1985 Sep 23;189(2):183–187. doi: 10.1016/0014-5793(85)81020-6. [DOI] [PubMed] [Google Scholar]
  31. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Skinner M. K., Griswold M. D. Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem J. 1983 Jan 1;209(1):281–284. doi: 10.1042/bj2090281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wishart G. J., Campbell M. T. Demonstration of two functionally heterogenous groups within the activities of UDP-glucuronosyltransferase towards a series of 4-alkyl-substituted phenols. Biochem J. 1979 Feb 15;178(2):443–447. doi: 10.1042/bj1780443. [DOI] [PMC free article] [PubMed] [Google Scholar]

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