PURIFICATION OF A PHENOBARBITAL-INDUCIBLE UDP-GLUCURONOSYLTRANSFERASEISOFORM FROM DOG LIVER WHICH CATALYZES MORPHINE AND TESTOSTERONE GLUCURONIDATION

A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle d og treated with phenobarbital, Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The mic rosomes were solubilized with Emulgen 911 and applied on a column of h emisuccinate derivative of Sepharose 4B column which has been develope d in our laboratory, An isoform of UGT in the eluate was purified furt her by chromatofocusing and UDP-hexanolamine-affinity chromatography, A purified enzyme, UGT(DOG-PB), was homogeneous on sodium dodecyl sulf ate polyacrylamide gel electrophoresis and two-dimensional electrophor esis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybi phenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but no t toward chloramphenicol and the 6-hydroxyl group of morphine. The sub strate specificity of UGT(DOG-PB) is similar to that of stably express ed UGT2B1 which is considered a phenobarbital-inducible morphine UGT i n the rat except that UGT(DOG-PB) is capable of glucuronidating 4-nitr ophenol but not chloramphenicol. The NH2-terminus until the 30th resid ue of UGT(DOG-PB) is highly homologous to UGT2B subfamily, and the NH2 -terminal 15 residues of UGT(DOG-PB), are completely identical to thos e of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily. (C) 1996 Academic Press, Inc.