Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes.
Three isoenzymes of UDP-glucuronyltransferase (UDPGT) have been separated and purified from liver microsomes of untreated female rats or female rats pretreated with 3-methylcholanthrene. The UDPGT isoenzymes were purified utilizing Chromatofocusing, column isoelectric focusing, and UDP-hexanolamine Sepharose 4B affinity chromatography. UDPGT activities could also be separated during UDP-hexanolamine affinity chromatography by elution with different UDPGA (UDP-glucuronic acid) concentrations. One isoenzyme exhibits a subunit molecular weight of 56,000 and is capable of conjugating p-nitrophenol, 1-naphthol, and 4-methylumbelliferone. This isoenzyme is inducible by 3-methylcholanthrene treatment and requires high UDPGA concentrations for elution from the UDP-hexanolamine affinity column in contrast to the other UDPGT isoenzymes. A second isoenzyme was purified and displayed a subunit molecular weight of 50,000. This isoenzyme was not induced by 3-methylcholanthrene and was active towards testosterone, the 17-OH position of beta-estradiol, p-nitrophenol, and 1-naphthol. A third isoenzyme was also purified and exhibited a subunit molecular weight of 52,000. This isoenzyme conjugated androsterone and etiocholanolone and was not induced by 3-methylcholanthrene treatment. This study reports the purification of two separate and distinct rat liver UDPGT isoenzymes capable of conjugating p-nitrophenol, only one of which is inducible by 3-methylcholanthrene treatment. Also, this is the first report of the purification of a UDPGT isoenzyme active towards the 3-OH position of androgens.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D014453:Glucuronosyltransferase / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000145:classification / Q000302:isolation & purification*; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013379:Substrate Specificity
D007527:Isoenzymes; D008748:Methylcholanthrene; D014453:Glucuronosyltransferase