Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin.
A chicken anti-rat polyclonal antibody to a purified rat liver UDP-glucuronosyltransferase (UGT) with catalytic activity toward opioid substrates was used to screen a liver cDNA library prepared from phenobarbital-treated Wistar rats. A number of positive clones were obtained, and one of these clones, pM1, was further characterized. Clone pM1 was found to be a full length cDNA coding for a member of the rat UGT1 gene family. Specifically, pM1 represents the full length homologue of the Gunn rat liver pseudo-gene product UGT1.1P and, therefore, has been designated UGT1.1r. The cDNA insert has an open reading frame of 1605 base pairs, which codes for a protein of 535 amino acids and is flanked by 2 and 632 base pairs of 5' and 3' noncoding sequence, respectively. The deduced amino acid sequence of pM1 contains amino acid sequences identical to the amino-terminal and internal peptides of the purified rat liver opioid UGT and to sequences reported for a rat liver bilirubin UGT [FEBS Lett. 299:183-186 (1992)]. Stable expression of UGT1.1r in human embryonic kidney 293 cells showed that a protein with a subunit molecular mass (56 kDa) identical to that of the purified protein was produced. Expressed UGT1.1r protein catalyzed the glucuronidation of buprenorphine and bilirubin at high rates. Other opioids, such as nalorphine and morphine, were also substrates for the expressed UGT1.1r protein. These results show that bilirubin and opioids can be conjugated by the same rat liver UGT.
Coffman
B L
BL
Department of Pharmacology, University of Iowa, Iowa City 52242, USA.
Green
M D
MD
King
C D
CD
Tephly
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005965:Glucuronates / Q000378:metabolism; D020723:Glucuronic Acid; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009294:Narcotics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005965:Glucuronates; D009294:Narcotics; D020723:Glucuronic Acid; D014453:Glucuronosyltransferase; D001663:Bilirubin