Effect of cycloheximide treatment on the mixed-function oxidase system of rat liver

The effect of cycloheximide treatment on the mixed-function oxidase system of rat liver was studied at 12 and 24 hours after administration of the antibiotic. Protein synthesis was inhibited in the animals for the 24-hour experimental period with the maximal tolerable dose, 2 mg/kg. Cycloheximide treatment caused an alteration in the sedimentation properties of the endoplasmic reticulum. A "fluffy" fraction containing appreciable amount of drug-metabolizing activity, as well as cytochrome P-450, was recovered with the microsomes from the 15,000 x g supernatant of treated animals, but was never observed in control preparations. Also, there was an increase in the microsomal protein level of treated animals that was apparently unrelated to drug-metabolizing activity; therefore, total protein was not a suitable reference standard for expressing the activity of this enzyme system. However, liver weight was relatively constant during the experimental period, and was used as the reference standard in the present study. N-demethylation of ethylmorphine was significantly decreased in the microsomal preparations from drug-treated rats. This represents a true decrease in total enzymatic activity because a comparable depression of demethylase activity was observed in the corresponding liver homogenates. The apparent Michaelis constant, Km, did not change with drug treatment, but V[max] was significantly lower. The non-competitive inhibition was not a consequence of a direct effect of cycloheximide on the drug-metabolizing enzymes, but was probably related to the blockade of protein synthesis. The change in drug-metabolizing activity produced by cycloheximide treatment did not correlate with a change in the NADPH-cytochrome c reductase activity, the level of cytochrome P-450, or the magnitude of the type I or type II binding spectrum. However, the rate of reduction of cytochrome P-450 in the present of ethylmorphine was significantly decreased, and this decrease closely paralleled the effect of drug treatment on the rate of metabolism. The data suggest that the rate-limiting step in the N-demethylation of ethylmorphine in treated animals, as well as in controls, is the cytochrome P-450 reductase activity.