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 (Mr = 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 an 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.
Highlights
From theLaboratory of Deuelopmental Pharmacology, National Institute of Child Health and Human Deuelopment, National Institutes of Health, Bethesda, Maryland20892
This latency, which can be removed by detertranscription-translationreaction, thecDNA directed gents, is thought to result from the lipid bilayer acting as a the synthesis of a 52,000-dalton polypeptide with im- permeability barrier between the enzyme and its substrates munologic epitopes characteristic noaftive microsomal ( l l ), especially the common cosubstrate, UDP-glucuronic
This invitro synthe- acid, which is negatively charged at physiological pH. This sized polypeptide was cleaved and glycosylated when observation implies that thecatalytic site is either buried deep dogpancreaticmicrosomes were includedinthe in within the membrane or is located on the luminal side of the vitro reaction.Thecodingregion ofthe cDNA was microsomal vesicle
Summary
Expressionof UDPGT,-Z-The plasmid pUDPGTr-2Fwas digested indicated that t h e PstI insert of pUDPGT,-2 [15] did not with BamHI, and the resultant fragments separated on agarose gels contain a translation initiation codon oar poly(A) additional [20]. The XhoI insert of pUDPGT,2F, containing the UDPGT,-2 sequence, was isolated and digested with PstI andEcoRI. Nucleotide Sequence of pUDPGTr-2F cDNA-The cDNA was 1961 base pairs in leanngdtchontained a putative poly(A) addition signal 24 nucleotides upstream of the terminating assayed for activity towards 4-methylumbelliferone as described [29]. The initiator codon, as defined by Kozak
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