Membrane effects on drug monooxygenation activity in hepatic microsomes.
The temperature dependence of drug monooxygenation in phenobarbital-induced rat liver microsomes has been investigated. With 7-ethoxycoumarin as a substrate the activity of the microsomes could be measured down to 0 degrees C by the increase in fluorescence of the dealkylated reaction product 7-hydroxycoumarin (umbelliferone). Arrhenius plots of the activities at various temperatures between 0 degrees C and 45 degrees C showed a break in the activation energy around 20 degrees C. Addition of deoxycholate or high concentrations of glycerol, known to solubilize membrane-bound enzymes, abolished the break of the activation energy. Cholesterol, incorporated into the microsomal membrane in amounts equimolar to the microsomal phospholipid content led to a decrease of the activation energy at low temperatures and to an increase at higher temperatures, resulting in a loss of the break. The activity of microsomal NADPH-cytochrome c reductase with the water -soluble electron acceptor dichlorophenolindophenol showed no discontinuity in the Arrhenius plot. In addition the cumene hydroperoxide-mediated and cytochrome P-450-dependent O-dealkylation of 7-ethoxycoumarin proceeded without a break in the activation energy. It is concluded that phospholipid phase transitions affect the electron transfer from the reductase to cytochrome P-450.
D000818:Animals; D002151:Calorimetry; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism*; D003640:Dealkylation; D004790:Enzyme Induction / Q000187:drug effects; D005019:Ethyl Ethers / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D003374:Coumarins; D005019:Ethyl Ethers; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D003579:Cytochrome Reductases; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital