A comparison of steroid glucuronlytransferases of rat liver and hepatoma, and effects of phenobarbitone and 3-methylcholanthrene induction.

measured by CO-reduced difference spectroscopy (Omura & Sato, 1964) was retained on this column. The concentrated CM-cellulose eluate was dialyzed to pH7.4, and then applied to a DEAE-Sephadex or QAE [diethyl-(2-hydroxypropyl)aminoethyl]-Sephadex column. Peak I eluted from the DEAE-Sephadex exhibited UTP-glucuronyltransferase activity. UDP-glucuronyltransferase activity towards various substrates was assayed by the following methods : o-aminophenol and p-nitrophenol (Winsnes, 1969) and harmol (Wong, 1969). Protein was assayed by the method of Lowry et al. (1951) and by the biuret method (see Layne, 1957). Specific activity of UDP-glucuronyltransferase towards p-nitrophenol increased to 53nmol/min per mg of protein, resulting in a 10-15-fold purification of the original Lubrol-solubilized microsomal preparation. Enzyme activity towards o-aminophenol was detectable at all stages through to the final step and the specific activity increased also some 10-fold up to 2.3 nmol/min per mg of protein. Harmol was glucuronidated by the (NH4)?S04 fraction, but no conjugate was formed by the peak I eluate from DEAESephadex. When the final active UDP-glucuronyltransferase preparation was analysed by sodium sulphate/polyacrylamide-gel electrophoresis on 5 %gels (0.1 %sodium dodecyl sulphate). Only six polypeptide bands, of molecular-weight range approx. 35 ~ O O O , were visible after staining the gels with Coomassie Blue. This enzyme preparation was stable for at least 1 month at 0°C.