title: Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene.
abstract: Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.
authors.affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia.
authors.forename: B G
authors.initials: BG
authors.lastname: Niranjan
authors.forename: H
authors.initials: H
authors.lastname: Raza
authors.forename: R M
authors.initials: RM
authors.lastname: Shayiq
authors.forename: C R
authors.initials: CR
authors.lastname: Jefcoate
authors.forename: N G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D011498:Protein Precursors / Q000096:biosynthesis*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011498:Protein Precursors; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System