Abstract
The mechanism of cytochrome P-450-dependent oxidation of ethanol has been investigated using reconstituted phospholipid vesicles containing purified preparations of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2. Incorporation of cytochrome b5 into the vesicles resulted in a 5-fold enhancement of cytochrome P-450-catalyzed O-dealkylation of 7-ethoxycoumarin, whereas the cytochrome P-450-dependent ethanol oxidation was slightly inhibited. Superoxide dismutase, added in increasing amounts to the vesicles, inhibited the formation of superoxide anions and, in a concomitant manner, also the production of acetaldehyde from ethanol in the system. Also horseradish peroxidase inhibited ethanol oxidation catalyzed by the vesicles; acetaldehyde formation and H2O2 formation decreased in a concomitant manner as the amount of the peroxidase was increased. Externally added hydrogen peroxide markedly stimulated cytochrome P-450-dependent ethanol oxidation, but not until the concentration of H2O2 reached 0.3 mM, whereas the hydroxyl radical scavenger mannitol completely inhibited the cytochrome P-450-dependent acetaldehyde production. Oxidation of ethanol was also accomplished using vesicles containing cytochrome b5 instead of cytochrome P-450 and in other systems regenerating superoxide anions, e.g. the xanthine-xanthine oxidase system and dihydroxyfumarate. The results are consistent with an iron-catalyzed Haber-Weiss mechanism for regeneration of hydroxyl radicals which subsequently react with ethanol, thereby giving the corresponding aldehyde.
Highlights
Tuted phospholipid vesicles containing purified prepa- The reconstitution of an ethanol-oxidizing system from rations of rabbit liver microsomalNADPH-cytochrome various forms of purified cytochrome P-450 and purified
Nally added hydrogen peroxide markedly stimulated A step towards the elucidation of the mechanism behind cytochromeP-450-dependentethanol oxidation,but not microsomal ethanol oxidation was recently taken by Cederuntil the concentration of HzOz reached 0.3 m ~,baum and co-workers [6,7,8,9] who showed that typical hydroxyl whereas the hydroxyl radical scavenger mannitol com- radical scavengers, like mannitol,thiourea,methionaland pletely inhibited the cytochrome P-450-dependent ac- dimethyl sulfoxide, inhibit cytochrome P-450-dependent oxietaldehyde production
These results suggest thatthe cytochrome P-450 LM2-dependent oxidation of ethanol does not work by the same mechanism as ordinary P-450-catalyzed reactions, here examplified by 0-deakylation of 7-ethoxycou
Summary
(2) suggested the presence of a microsomal ethanol-oxidizing system with properties similar to the cytochrome P-450 system. LM-2 and NADPH-cytochrome P-450 reductase were prepared by the cholate gel filtration method [18, 19]. The properties of these ql u 2.5 vesicles have been described before [10]. Ethanol oxidation was detected essentially as described by Lieber and De Carli [2].The incubations were carried out at 37 "C for 20 min in 25-ml stoppered tubes, containing 5-ml tubes with 1 ml of 15 mM semicarbazide in 160 mM potassium. The preparations obtained were tested for reduction by NADPH-cytochrome P-450 reductase by incubations performed with and without excess superoxide dismutase in the presence of the flavoprotein
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