PURIFICATION OF A PHENOBARBITAL-INDUCIBLE MORPHINE UDP-GLUCURONOSYLTRANSFERASE ISOFORM, ABSENT FROM GUNN RAT-LIVER

A morphine UDP-glucuronyltransferase (morphine UGT(PB)) was purified from liver microsomes of Sprague-Dawley rats treated with phenobarbital. UDP-glucuronyl-transferases in the liver microsomes were solubilized with Emulgen 911 and separated by omega-(beta-carboxypropionylamino)octyl Sepharose 4B column chromatography, which has been developed in our laboratory. Morphine UDP-glucuronyltransferases were eluted into two fractions, Peak I and Peak II, which have different substrate specificities. Morphine UGT(PB) was purified by two times of Chromatofocusing from Peak II which was more specific to morphine. The purified morphine UGT(PB) gave an apparent pI of 8.0 on chromatofocusing and displayed a subunit molecular weight of 55 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the glucuronidation of 3-hydroxyl group of morphine and small extent of 4-hydroxybiphenyl, but not of androsterone, bilirubin, chloramphenicol, codeine, 4-methylumbelliferone, 4-nitrophenol, testosterone, and 6-hydroxyl group of morphine. The N-terminal amino acid sequences of morphine UGT(PB) were identical to those of UGT1*01P which is deficient to homozygous Gunn rat. Peak II was absent from the fraction of omega-(beta-carboxypropionylamino)octyl Sepharose 4B column chromatography of Liver microsomes of Gunn rats treated with phenobarbital, whereas morphine UGT in Peak I was PB-inducible in Gunn rats. Present results suggest that an isoform of morphine UDP-glucuronyltransferase belongs to the UGT1 family and is phenobarbital-inducible. (C) 1994 Academic Press, Inc.