Hepatic microsomal ethanol-oxidizing system: Solubilization, isolation, and characterization

Abstract

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD + to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K m for ethanol (7.2 m m, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 m m) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K m for oxygen (8.3 μ m), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H 2O 2-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.