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

https://doi.org/10.1016/0003-9861(86)90373-5Get rights and content

Abstract

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac 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-450ac 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-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min−1 nmol P-450−1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (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.

References (48)

  • Y.Y. Tu et al.

    Chem. Biol. Interactions

    (1983)
  • Y.Y. Tu et al.

    Biochem. Biophys. Res. Commun

    (1981)
  • D.Y. Lai et al.

    Life Sci

    (1980)
  • N.A. Lorr et al.

    Toxicol. Appl. Pharmacol

    (1984)
  • Y.Y. Tu et al.

    Arch. Biochem. Biophys

    (1985)
  • C.S. Yang et al.

    Biochem. Biophys. Res. Commun

    (1985)
  • D.R. Koop et al.

    J. Biol. Chem

    (1982)
  • E.T. Morgan et al.

    J. Biol. Chem

    (1982)
  • D.R. Koop et al.

    Arch. Biochem. Biophys

    (1984)
  • K.-C. Cheng et al.

    J. Biol. Chem

    (1982)
  • P.E. Thomas et al.

    Arch. Biochem. Biophys

    (1979)
  • P. Tamburini et al.

    J. Biol. Chem

    (1985)
  • A.Y.H. Lu et al.

    J. Biol. Chem

    (1975)
  • P.E. Thomas et al.

    J. Biol. Chem

    (1976)
  • D.E. Ryan et al.

    J. Biol. Chem

    (1985)
  • S.S. Park et al.

    Biochem. Pharmacol

    (1986)
  • J.Y.L. Chiang

    Arch. Biochem. Biophys

    (1981)
  • A.Y.H. Lu et al.

    Biochem. Biophys. Res. Commun

    (1974)
  • D.E. Ryan et al.

    Arch. Biochem. Biophys

    (1986)
  • K.W. Miller et al.

    Arch. Biochem. Biophys

    (1984)
  • K.L. Kaul et al.

    Arch. Biochem. Biophys

    (1984)
  • J.P. Casazza et al.

    J. Biol. Chem

    (1984)
  • D.R. Koop et al.

    J. Biol. Chem

    (1985)
  • Y.Y. Tu et al.

    Cancer Res

    (1983)
  • Cited by (0)

    Supported by Grants CA-37037 and ES-03938 from the National Institutes of Health. This work was presented in part at the 76th Annual Meeting of the American Society of Biological Chemists in Washington, DC, June 8–12, 1986.

    View full text