Mixed Function Oxidase Cytochrome P-450 Research Articles

Methoxyflurane acts at the substrate binding site of cytochrome P450 LM 2 to induce a dependence on cytochrome b5

Rabbit cytochrome P450 isozyme 2 requires cytochrome b 5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [ E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541–2546 ]. To determine whether the requirement for cytochrome b 5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM 2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b 5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b 5, we found that methoxyflurane did not create a cytochrome b 5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b 5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b 5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b 5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b 5 dependence of the mixed function oxidase cytochrome P450 LM 2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM 2 and suggest the focus of future studies of this question.

Effects of dantrolene on adrenal cortical function

Adverse effects of dantrolene include abnormal liver function tests, hepatic injury, and alteration of the hepatic mixed function oxidase cytochrome P-450 system. The following study conducted in rats indicates that the effective dose of dantrolene that has been shown to decrease hepatic cytochrome P-450 also causes adrenal enlargement with a marked reduction in serum glucocorticoids. The ratio of adrenal wet weight/body weight was significantly (P < 0.05) increased following 5 days of injection with a minimum dose of 25 mg dantrolene/kg body weight. The minimum effective dose of dantrolene that significantly (P < 0.05) lowered serum glucocorticoids was 50 mg/kg body weight. Serum glucocorticoids were reduced by half following 5 days of treatment with 100 mg dantrolene/kg body weight. The levels of serum glucocorticoids were 80 per cent of control serum levels and the adrenal wet weight/body weight ratio was 85 per cent of control values following 3 days of recovery after 5 days of pre-treatment with 100 mg/kg of dantrolene. It appears that dantrolene acts in a manner similar to diphenylhydantoin to reduce adrenal glucocorticoid secretion, with a possible alteration or reduction in the negative feedback response to the pituitary and resultant enlargement of the adrenals.

Non-competitive and competitive inhibition of mixed function oxidase in rat liver microsomes by metyrapone.

N-demethylation of aminopyrine and ethylmorphine via hepatic microsomal mixed function oxidase cytochrome P-450 from rat is inhibited by metyrapone by two mechanisms: Non-competitive inhibition occurs at low concentrations (Ki about 2·10−6 M); competitive inhibition is obtained at high concentrations (Ki about 1·10−4 M). Lineweaver-Burk kinetics allow to determine the Ki for inhibition at high concentrations. The Ki for inhibition with low concentrations has been obtained by extrapolation. The inhibitor concentrations which yield 50% inhibition have been plotted versus vmax at different protein dilutions.