Acetone-inducible cytochrome P-450: Purification, catalytic activity, and interaction with cytochrome b5

Abstract A procedure was developed for the purification of an acetone-inducible form of cytochrome P -450 ( P -450 ac ) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P -450 ac preparation containing 16.0 to 16.5 nmol P -450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P -450 ac showed an absorption maximum at 394 nm at 25 °C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P -450 reductase and phospholipid, P -450 ac efficiently catalyzed both the demethylation and denitrosation of N -nitrosodimethylamine (NDMA) showing V max values of 23.8 and 2.3 nmol min −1 nmol P -450 −1 , respectively. The catalytic activity of P -450 ac was greatly affected by cytochrome b 5 which decreased the K m values of these reactions by a factor of 10 and increased the V max values. Cytochrome b 5 appeared to interact with P -450 at a molar ratio of 1:1 and an intact cytochrome b 5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b 5 and showed the highest rate. P -450 ac also catalyzed the oxygenation of N -nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b 5 . Cytochrome b 5 did not enhance the P -450 ac -catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P -450 ac appeared to be similar in property to the previously studied rat P -450 et (ethanol-inducible), rat P -450 j (isoniazid-inducible), and rabbit P -450 LM3a (ethanol-inducible). These P -450 species represent a new class of P -450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.