Purification and characterization of an ethanol-induced UDP-glucuronosyltransferase.
A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent Km values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 microM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/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. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.
Hutabarat
R M
RM
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112.
Yost
G S
GS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D000431:Ethanol / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009020:Morphine / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D000431:Ethanol; D013739:Testosterone; D009020:Morphine; D014453:Glucuronosyltransferase