Purification and identification of rat hepatic cytosolic enzymes responsible for defluorination of methoxyflurane and fluoroacetate.
Enzymes responsible for the defluorination of methoxyflurane (MOF) and fluoroacetate (FAc) were separated and purified from rat liver cytosol. Both hepatic cytosolic enzymes with defluorination activity were labile and addition of 2-mercaptoethanol had little effect on the stability of these enzymes. Glutathione S-transferase (GT) activity of the same cytosolic fractions was stable for at least 11 days. Separation of defluorination and GT enzymatic activities on DEAE-Sephadex A-50 and reduced glutathione-affinity columns revealed that the defluorinations of MOF and FAc were primarily catalyzed by anionic proteins which also exhibited GT activity. Further identification by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed protein bands with pl values of approximately 6.5 and 6.9 and molecular weights of approximately 20,000. However, other proteins that exhibited no GT activity also defluorinated MOF and FAc, but accounted for only 10% of the total defluorination activity present in anionic proteins. Results from a separate purification experiment using a CM-cellulose column also indicated that the enzymes responsible for defluorination coeluted with cationic GTs. Collectively, these cationic enzymes were responsible for about 20% of the recovered cytosolic defluorination activities. The results suggest that the cytosolic defluorinations of both MOF and FAc are primarily the result of a dehalogenation reaction catalyzed by one or more species of rat liver cytosolic GTs.
Wang
S L
SL
Rice
S A
SA
Serra
M T
MT
Gross
B
B
D000818:Animals; D001711:Biotransformation; D003600:Cytosol / Q000201:enzymology; D005463:Fluoroacetates / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008733:Methoxyflurane / Q000378:metabolism*; D051381:Rats
D005463:Fluoroacetates; D008733:Methoxyflurane; D005982:Glutathione Transferase