Rat liver UDP-glucuronosyltransferase. Sequence and expression of a cDNA encoding a phenobarbital-inducible form

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Abstract

The cDNA encoding a phenobarbital-inducible form of rat liver UDP-glucuronosyltransferase has been isolated, sequenced, and expressed to yield a catalytically active enzyme. The cDNA was found to be 1,961 nucleotides in length and to have an open reading frame of 1,590 nucleotides flanked by 25 and 346 base pairs of 5' and 3' untranslated regions, respectively. The open reading frame encodes a protein of 529 residues (M(r) = 60,484) and contains amino-terminal and carboxy-terminal segments characteristic of a signal peptide and a transmembrane-anchoring region, respectively. Two potential asparagine-linked glycosylation sites are located between these segments. In a in vitro transcription-translation reaction, the cDNA directed the synthesis of a 52,000-dalton polypeptide with immunologic epitopes characteristic of native microsomal UDP-glucuronosyltransferase. This in vitro synthesized polypeptide was cleaved and glycosylated when dog pancreatic microsomes were included in the in vitro reaction. The coding region of the cDNA was inserted into a SV40 recombinant vector, and this construct transfected into permissive monkey kidney cells. Seventy hours after transfection, the glucuronidation of 4-methylumbelliferone was detected in lysates of cells containing the cDNA in the correct orientation with respect to SV40 transcription signals. This experimental approach should allow definitive characterization of the structure, function, and heterogeneity of this major family of drug-metabolizing enzymes.

Original languageEnglish
Pages (from-to)6119-6125
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number13
Publication statusPublished - 5 May 1986
Externally publishedYes

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