Purification and characterization of a morphine UDP-glucuronyltransferase isoform from untreated rat liver.
A morphine UDP-glucuronyltransferase was purified from liver microsomes of untreated Sprague-Dawley rats. A new gel, omega(beta-carboxypropionylamino)octyl Sepharose 4B, was prepared by coupling monomethylsuccinate with omega-aminooctyl Sepharose 4B and this was used as an efficient tool for the separation of microsomal enzymes. Emulgen 911 solubilized microsomes were applied to a column packed with the gel and eluted at pH 7.4 while increasing KCl concentration in a stepwise manner. An isoform was further purified with UDP-hexanolamine Sepharose 4B gel. The purified UDP-glucuronyltransferase (morphine UGT of untreated rat, morphine UGTUT) exhibited a molecular weight of 52000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and was capable of glucuronidating the 3-hydroxyl group of morphine. The isoform catalyzed to a small extent the glucuronidation of 4-hydroxybiphenyl; however, no glucuronidation activity towards androsterone, testosterone, bilirubin, 4-nitrophenol and the 6-hydroxyl group of morphine was observed. The difference in properties, compared with morphine UGT (molecular weight 56000) which was purified previously from phenobarbital-treated rats, is discussed.
Ishii
Y
Y
Faculty of Pharmaceutical Sciences, Kyushu University 62, Fukuoka, Japan.
Oguri
K
K
Yoshimura
H
H
D000818:Animals; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000302:isolation & purification; D066298:In Vitro Techniques; D007475:Ion Exchange Resins; D007527:Isoenzymes / Q000032:analysis* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009020:Morphine / Q000378:metabolism*; D011506:Proteins / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012685:Sepharose; D013379:Substrate Specificity
D007475:Ion Exchange Resins; D007527:Isoenzymes; D011506:Proteins; D009020:Morphine; D012685:Sepharose; D014453:Glucuronosyltransferase