| 6428319 |
Mackenzie PI, Hjelmeland LM, Owens IS: Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver. Arch Biochem Biophys. 1984 Jun;231(2):487-97.
A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo [a] pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo [a] pyrene and 4-nitrophenol, and was designated GTM2 . |
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