Critical evaluation of 7-ethoxycoumarin O-deethylase activity measurement in intact isolated rat hepatocytes.
In the determination of 7-ethoxycoumarin O-deethylase activity in intact isolated rat hepatocytes various factors influence the assay, including: the decay of 7-ethoxycoumarin fluorescence which is temperature and pH dependent; the measured fluorescence which has to be adjusted for the inner filter effect; glucose addition to the medium which influences the activity; all organic solvents which inhibit the enzymic activity, with dimethylformamide provoking the smallest effect (partial competitive inhibition); the enzymic reaction which is inhibited by the product of reaction; and the presence of bovine serum albumin in the medium which affects the enzymic activity. Biphasic kinetics are observed for the O-deethylation of ethoxycoumarin in intact isolated rat hepatocytes. For the high-affinity component, Km and Vmax values are 5 microM and 43 pmol/min X 10(6) cells and for the low-affinity component are 414 microM and 915 pmol/min X 10(6) cells. Addition of the substrate in dimethylformamide or omitting bovine serum albumin from the medium cause important changes in these kinetic parameters.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012997:Solvents; D013050:Spectrometry, Fluorescence
D012997:Solvents; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D005947:Glucose