Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

doi:10.1016/0006-2952(84)90035-2

Biochemical Pharmacology

Volume 33, Issue 23, 1 December 1984, Pages 3743-3753

https://doi.org/10.1016/0006-2952(84)90035-2 Get rights and content

Abstract

We have purified five isozymes of liver microsomal (LM) P-450 from β-naphthoflavonefed rainbow trout. Four forms (LM₃, LM₁, LM_4a and LM_x) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LM_x into two components, LM₂ and LM_4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (M_r), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a λ_max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM_4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm_4b by M_r, λ_max, or activity towards benzo[a]pyrene. Form LM₂ (2% yield) had a specific content of 10.8, a M_r of 54,000, a λ_max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, lm₁ and LM₃. Neither lm₁ nor LM₃ was active towards benzo[a]pyrene. The properties of LM₂, LM_4a and LM_4b, were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM_4a and LM_4b, each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM₂, and LM₂-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM_4a or LM_4b, was inhibited by LM_4b-IgG but not by LM_4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM_4a and LM_4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to α-naphthoflavone, and regioselectivity towards benzo[a]-pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.

References (61)

F.P. Guengerich
Pharm. Ther.
(1979)
K.M. Lewis et al.
Biochem. Pharmac.
(1982)
D.R. Koop et al.
J. biol. Chem.
(1981)
D.E. Ryan et al.
Archs Biochem. Biophys.
(1982)
H.H. Dieter et al.
J. biol. Chem.
(1982)
D.J. Waxman et al.
J. biol. Chem.
(1982)
J.J. Stegeman
R.F. Addison et al.
Comp. Biochem. Physiol.
(1977)
L. Forlin et al.
Comp. Biochem. Physiol.
(1978)
L. Forlin
Toxic. appl. Pharmac.
(1980)

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^∗: Present address: Department of Biochemistry, The Medical College of Wilwaukee, WI 53226.

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Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

Abstract

Pharm. Ther.

Biochem. Pharmac.

J. biol. Chem.

Archs Biochem. Biophys.

J. biol. Chem.

J. biol. Chem.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Toxic. appl. Pharmac.

Comp. Biochem. Physiol.

Toxic. appl. Pharmac.

Comp. Biochem. Physiol.

Aquat. Toxic.

Comp. Biochem. Physiol.

Archs Biochem. Biophys.

Biochim. biophys. Ada

Comp. Biochem. Physiol.

Archs Biochem. Biophys.

Analyt. Biochem.

Chem. Biol. Interact.

Analyt. Biochem.

J. biol. Chem.

Biochem. biophys. Res. Commun.

J. biol. Chem.

Meth. Enzym.

J. biol. Chem.

J. biol. Chem.

J. biol. Chem.

J. biol. Chem.