A phenobarbital-inducible hepatic mitochondrial cytochrome P-450 immunochemically related to microsomal P-450b

Abstract

We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria.