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.
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