Abstract

The effect of cycloheximide treatment on the mixed-function oxidase system of rat liver was studied at 12 and 24 hr after administration of the antibiotic. Cycloheximide caused an alteration in the sedimentation properties of the endoplasmic reticulum. A "fluffy" fraction containing appreciable amounts of drug-metabolizing activity, as well as cytochrome P-450, was recovered with the microsomes from the 15,000 x g supernatant fraction from the livers 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 decrease in enzymatic activity was most the consequence of a cycloheximide-induced change in the sedimentation properties of the cellular particles, because a comparable loss of demethylase activity was observed in the corresponding liver homogenates. The apparent Michaelis constant, K_m, did not change with drug treatment, but V_max was significantly lower. The reduction of demethylase activity did not result directly from the effect of cycloheximide on the drug-metabolizing enzymes, but may reflect a quantitative change in enzyme levels due to the inhibitory effect of cycloheximide on 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 presence 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 is the cytochrome P-450 reductase activity.