Isolation and purification of rat liver morphine UDP-glucuronosyltransferase.
A UDP-glucuronosyltransferase (UDPGT) isoenzyme capable of morphine glucuronidation has been purified to apparent homogeneity and partially characterized from hepatic microsomes of female Wistar rats which have low 3 alpha-hydroxysteroid UDPGT. A rapid and sensitive assay was developed to quantify morphine glucuronide formation using 14C-UDP-glucuronic acid and reverse phase C-18 minicolumns whereby radioactive glucuronides were differentially eluted from 14C-UDP-glucuronic acid. Trisacryl-DEAE and chromatofocusing chromatographic procedures were employed to separate and purify morphine UDPGT in the presence of exogenous phosphatidylcholine. The addition of phospholipid was necessary to stabilize UDPGT activities throughout the purification procedures. Morphine UDPGT was isolated to apparent homogeneity and displayed a pl of 7.9 upon chromatofocusing. A monomeric molecular weight of 56,000 was obtained. The purified enzyme reacted with morphine but not with 4-hydroxybiphenyl, p-nitrophenol, testosterone, androsterone, estrone, bilirubin, 4-aminobiphenyl, or alpha-naphthylamine. The MgCl2 requirement for maximal expression of morphine glucuronidation was higher for the purified enzyme than for solubilized and intact microsomes. Codeine competitively inhibits morphine glucuronidation with an apparent Ki of 1.1 mM with the purified morphine UDPGT. 4-Hydroxybiphenyl UDPGT was separated from morphine UDPGT using a chromatofocusing procedure for Emulgen 911-solubilized microsomes. An apparent pl value of 5.5 was obtained for this protein. Based on this work we conclude that morphine and 4-hydroxybiphenyl can react with separate UDPGT isoforms.
D000818:Animals; D002712:Chlorides; D005260:Female; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D002712:Chlorides; D007527:Isoenzymes; D017895:Manganese Compounds; D008345:Manganese; D014453:Glucuronosyltransferase; C025340:manganese chloride