Purification and characterization of a hepatic mitochondrial cytochrome P-450 active in aflatoxin B1 metabolism

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Abstract
We have previously shown that phenobarbital (PB) increases hepatic mitochondrial cytochrome P-450 (P-450) content and also the ability to metabolize hepatocarcinogen, aflatoxin B1 [Niranjan, B. G., Wilson, N. M., Jefcoate, C. R., and Avadhani, N. G. (1984) J. Biol. Chem. 259, 12495-12501]. In the present study, we have purified a mitochondrial-specific P-450 with an apparent molecular mass of 52 kdaltons (termed P-450mt3) from PB-induced rat liver using a combination of hydrophobic and ion exchange column chromatography procedures. Polyclonal antibody to P-450mt3 failed to cross-react with P-450mt1 and P-450mt2 purified from .beta.-naphthoflavone- (BNF) induced rat liver mitochondria. Furthermore, P-450mt3 shows an N-terminal amino acid sequence (Ala-Ile-Pro-Ala-Ala-Leu-Arg-Thr-Asp) different from those of both P-450mt1 and P-450mt2, as well as microsomal P-450b. The polyclonal antibody to P-450mt3 cross-reacted with a P-450 of comparable size purified from uninduced mitochondria. These two isoforms, however, showed difference with respect to catalytic properties and amino acid composition. In vitro reconstitution experiments show that P-450mt3 can actively metabolize diverse substrates including (dimethylamino)antipyrine, benzphetamine, and aflatoxin B1 but shows a low vitamin D3 25-hydroxylase activity. The mitochondrial P-450 from uninduced livers, on the other hand, shows relatively high [229 pmol min-1 (nmol of P-450)-1] vitamin D3 25-hydroxylase activity but a considerably lower ability for aflatoxin B1 metabolism and no detectable activity for (dimethylamino)antipyrine and benzphetamine metabolism. In an in vitro reconstituted assay system, P-450mt3 showed a 2-fold higher aflatoxin B1-DNA binding activity [2150 pmol (nmol of P-450)-1 h-1] than the major PB-inducible microsomal P-450b [1080 pmol (nmol of P-450)-1 h-1]. Further, P-450mt3 shows an exclusive requirement for mitochondrial-specific ferredoxin and ferredoxin reductase as carriers of electrons from NADPH. Thus, on the basis of the immunological characteristics, N-terminal amino acid sequence properties, and substrate specificity, P-450mt3 is distinctly different from the two BNF-induced forms reported from this laboratory. Further, despite immunological relatedness, P-450mt3 appears to be different from P-450 purified from uninduced hepatic mitochondria.