The isolation, purification and characterization of an ethanol-induced UDP-glucuronosyltransferase from rabbit hepatic microsomes.

The UDP-glucuronosyltransferase (GT) enzyme from ethanol-induced male New Zealand white rabbit hepatic protein was purified to homogeneity and characterized. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests and amino acid compositions of the ethanol-induced GT and similarly purified GT from control rabbit liver appeared to be different. The apparent Km values for the ethanol induced GT enzyme for 1-naphthol and morphine as substrates were 43 umM and 109 umM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 nmole/min/mg protein and 4.6 nmole/min/mg protein. The increases in catalytic efficiencies. Apparent Vmax/Km for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0 and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. A polyclonal antibody raised against the purified control GT in sheep had 820-fikd selectivity for precipitation of the control protein as compared to the ethanol-induced protein. The ethanol-induced GT IgG inhibited the activity of the purified ethanol-induced protein. The ethanol-induced GT IgG inhibited the activity of the purified ethanol-induced GT toward naphthol and morphine 90% and 92% respectively. Immunoblots of ethanol-induced microsomes indicated that at least 7 days of 10% ethanol treatment required for induction and 11 days after the 14-day induction period were required for the ethanol-induce GT to be undetected. Immunoblots with the control antibody showed the opposite effects of ethanol treatments, i.e., disappearance with ethanol treatment and emergence after ethanol withdrawal. The quantification of the induction process by rocket electrophoresis indicated that there was a steady increase of the ethanol-induced GT in the microsomes from day 7 to day 14 of alcohol treatment ranging from 4.8 ng/µg microsomal protein. These results demonstrate the production of a unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.