Purification and characterization of a hepatic mitochondrial cytochrome P-450 active in aflatoxin B1 metabolism.
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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Shayiq
R M
RM
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104.
Avadhani
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NG
D016604:Aflatoxin B1; D000348:Aflatoxins / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D000348:Aflatoxins; D000596:Amino Acids; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D010105:Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase